ES2761583T3 - Brassica plant comprising a mutant indehiscent allele - Google Patents

Abstract

A partial defective mutant allele of an IND gene, wherein the IND gene comprises a nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising at least 90% sequence identity to the SEQ ID NO: 1, SEQ ID NO: 3 from nucleotide at position 46 to nucleotide at position 633, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7; (b) a nucleic acid molecule that encodes an amino acid sequence comprising at least 90% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4 from amino acid at position 16 to amino acid at position 210, or SEQ ID NO: 4, wherein said partial defective mutant allele comprises one or more missense mutations in the nucleic acid sequence of said IND gene that results in the production of an IND protein in the that at least one amino acid selected from the amino acid in a position corresponding to position 124 of SEQ ID NO: 2, the amino acid in a position corresponding to position 146 of SEQ ID NO: 2, the amino acid in a position corresponding to position 159 of SEQ ID NO: 2, the amino acid at a position corresponding to position 136 of SEQ ID NO: 4, the amino acid at a position corresponding to position 139 of SEQ ID NO: 4 or the amino acid at a position corresponding to position 142 of SEQ ID NO: 4, is substituted with another amino acid.

Description

DESCRIPTION

Brassica plant comprising a mutant indehiscent allele

Field of the Invention

The present invention relates to the field of agriculture, more specifically to the use of molecular biology techniques to alter dehiscent seed plants, particularly of the Brassicaceae family, in particular the Brassica species, and / or accelerate the reproduction of such seed plants. dehiscent. More specifically, the invention relates to improved methods and means for reducing seed shelling or delaying seed shelling until after harvest, in plants such as Brassicaceae plants, particularly Brassicaceae plants grown for seed production, to while maintaining an agronomically relevant threshing capacity of the pods. Procedures are also described to identify molecular markers associated with delayed seed shelling in a dehiscent seed plant population. Procedures and means for increasing yield are also provided, particularly grain and seed yield. The yield increase phenotype may be separate from the delayed or reduced seed fragmentation phenotype.

Background of the Invention

The siliques or pods of Brassica plants release their seeds through a procedure called fruit dehiscence. A silicone consists of two carpels joined margin to margin. The suture between the margins forms a thick nerve, called the replum. As the pod maturity approaches, the two leaflets progressively separate from the replum, along designated lines of pod weakness, ultimately resulting in shelling of the seeds that were attached at reply. The dehiscence zone defines the exact location of leaflet dissociation.

Seed shedding (also known as "seed shelling" or "pod shelling") by mature pods before or during crop harvest is a universal phenomenon with crops developing dehiscent dried fruit. Premature seed shelling results in reduced seed recovery, posing a problem in crops that grow primarily for seeds, such as oil-producing Brassica plants, particularly rapeseed. Another problem related to premature shelling of seeds is an increase in voluntary growth in the subsequent year of cultivation. In rapeseed, productivity losses related to pod shelling are, on average, 20% (Child et al., 1998, J Exp Bot 49: 829-838), but can reach up to 50%, depending of meteorological conditions (MacLeod, 1981, Harvesting in Oilseed Rape, for example, 107-120, Cambridge Agricultural Publishing, Cambridge).

Current commercial varieties of rapeseed are extremely susceptible to shelling. There is little variation for shelled resistance within B. napusexistent cultivation programs, but resistant lines have been found within diploid progenitors of B. napus (B. oleracea and B. rapa) as well as other members of the Brassica genus, mainly B. júncea, B. carinata and B. nigra. Kadkol et al., (1986, Aust. J. Botany 34 (5): 595-601) disclose a higher resistance to shelling in certain records of B. campestris that was associated with the absence of a separation layer in the region of union of the valves of the silicone to the replum. Prakash and Chopra (1988, Plant breeding 101: 167-168) describe the introgression of shelling resistance in Brassica napus from Brassica júncea by non-homologous recombination. Spence et al. (1996, J of Microscopy 181: 195-203) describe that some lines of Brassica júncea show a reduced tendency to shed compared to lines of Brassica napus. Morgan et al., 1998 (Fields Crop Research 58, 153-165) describe genetic variation for pod shelling resistance between rapeseed lines developed from synthetic B. napus and conclude that the lines that required a lot of energy to open their Pods appeared to have increased vascularity in the dehiscence zone and reduced cell wall degradation in the dehiscence zone. They also discovered a significant negative correlation between the length of the pod spur and the force required to cause shelling of the pods. Child and Huttly (1999, Proc 10th Int. Rapeseed Congress) describe the variation in pod maturation in an irradiation-induced mutant B. napus and a population of its parent crop varieties, Jet Neuf, in which the wild type and more resistant mutants showed a great lignification of groups of cells throughout the dehiscence zone, and in which vascular traces located near the internal edge of the dehiscence zone in the mutant were described, to help ensure the leaflets. Child et al. (2003, J Exp Botany 54 (389): 1919-1930) further describe the association between increased resistance to shelling of pods and changes in vascular structure in pods of a resynthesized Brassica napus line. However, traditional cultivation procedures have not been successful in introducing shelled resistance in rapeseed cultivar varieties, without interfering with other desirable traits such as early flowering, maturation, and resistance to blackfoot (Prakash and Chopra, 1990, Genetical Research 56: 1-2).

Several genes, which promote or inhibit pod dehiscence, have been identified in Arabidopsis thaliana through mutant analysis: combined mutants in both SHATTERPROOF1 (SHP1; initially known as ACL1) and SHATTERPROOF2 (SHP2; initially known as AGL5) they result in indehiscent siliques (ie siliques that remain closed with maturation in Arabidopsis thaliana) (Liljegren et al., 2000, Nature 404, 766-770). Similarly, mutants in the INDEHISCEn T gene (called iNd 1) in Arabidopsis thaliana (Liljegren et al., 2004, Cell 116: 843-853; PCT publication WO 01/79517), as well as in ALCATRAZ (known as ALC; Rajani et al., 2001, Current Biology 11, 1914-1922) interfered with the dehiscence of the pods, leading to resistance to shelling of the pods. Constitutive expression of FRUITFUL (FUL), a repressor of SHP and IND, in Arabidopsis thaliana, also resulted in indehiscent silicuas (Ferrandiz et al., 2000, Science, 289, 436-438). These transcription factors are believed to form a nonlinear transcriptional network that controls the identity of the leaflet margin and the shelling of the sheaths. Liljegren et al., (2004, Cell 116: 843 853) further describe that IND, an atypical helix-loop-helix (bHLH) gene, directs leaflet margin differentiation in the separating and lignified layers in Arabidopsis thaliana. The lignified cell layer adjacent to the separation layer together with the endocarp layer b (a single layer of lignified cells in each leaflet) produce a spring-like tension in the drying fruit, which contributes to its opening. Lignification of the leaflet endocarp layer b requires the activities of IND, SHP, ALC and FUL, a MADS domain transcription factor that is expressed throughout the leaflets (Liljegren et al., 2004, cited above; Mandel and Yanofsky, 1995, Plant Cell 7, 1763-1771). FUL and REPLUMLESS (RPL), a homeodomain transcription factor that is expressed in the replum (Roeder et al., 2003, Curr Biol 13, 1630-1635), have been found to set the boundaries of genes conferring identity of the leaflet margin (Gu et al., 1998, Development 125, 1509-1517; Ferrandiz et al., 2000, Science, 289, 436-438; Roeder et al., 2003, cited above). Finally, it was identified that FILAMENTOUS FLOWER (FIL) and YABBY3 (YAB3), two transcription factors of the YABBY family (Sawa et al., 1999, Genes Dev 13, 1079-1088; Siegfried et al., 1999, Development 126, 4117-4128), and JAGGED (JAG), a C2H2 zinc finger transcription factor (Dinneny et al., 2004, Development 131, 1101-1110; Ohno et al., 2004, Development 131, 1111-1122), they contribute redundantly to the proper development of the leaflet and leaflet margin by promoting expression of FUL and SHP in a region-specific manner (Dinneny et al., 2005, Development 132, 4687-4696). Genes have also been identified for a number of hydrolytic enzymes, such as endopolygalacturonases, which play a role, during pod dehiscence, in the programmed breakage of the dehiscence zone in pods of Brassica plants (see, for example, WO 97/13865; Petersen et al., Plant. Mol. Biol., 1996, 31: 517-527).

Liljegren et al., (2004, Cell 116: 843-853) describe five mutant IND alleles from Arabidopsis. Lignified cells in the dehiscence zone are absent or present in plants comprising these mutant alleles, depending on the severity of the mutations (severe ind mutants do not contain lignified cells in the region that corresponds to the inner part of the leaflet margin in wild type plants), but in all cases, the silicone is indehiscent. Wu et al. (2006), Plant 224, 971-979) describe a sixth mutant allele of IND from Arabidopsis. The plants that comprise this mutant allele do not show lignified cells at the junctions of the leaflet margin and the replum, they contain fewer cells in a region of seven cell layers, which seems to encompass the usually known dehiscence zone and the edge of the replum in wild plants, and show incomplete cytokinesis in this layer.

Documents US 2005/0120417 and US 2007/0006336 describe the identification and isolation of two IND1 orthologs from Brassica napus.

WO99 / 00503, WO01 / 79517 and WO0159122 describe down-regulation of the expression of the Arabidopsis ALC, IND, AGL1 and AGL5 genes, and their orthologs, using gene silencing techniques (such as antisense suppression or cosuppression) and mutagenesis.

Vancanneyt et al., 2002 (XIII International Conference on Arabidopsis Research, Seville, Spain, June 28-July 2; 2002) reported that FUL expression of A. thaliana under the control of a CaMV 35S promoter in Rapeseed resulted in a number of transformants resistant to pod shelling. The sheaths of such lines resistant to shelling of the sheaths had no dehiscence zone and the opening of the sheaths could only be achieved by random fracturing of the leaflets applying considerable pressure.

Vancanneyt et al., 2002 (XIII International Conference on Arabidopsis Research, Seville, Spain, June 28-July 2; 2002) also reported that the silencing of the IND gene in Arabidopsis thaliana using so-called dsRNA silencing techniques gave as a result almost complete resistance to shelling of the pods. Ninety-eight percent of the Arabidopsis transgenic lines developed silicuas, which did not open along the leaflet suture and could only be opened by applying considerable pressure to the leaflets.

It is important to note that although seed threshing is a major problem in rapeseed cultivation, which can be solved by developing lines resistant to seed shelling, ultimately, seed separation from pods is still necessary. In normal agricultural practice this is accomplished by threshing the pods using a combine harvester. The threshing of the pods by a combined harvester must be total and must cause minimal damage to the seeds thus released. However, as pod strength increases, the more intense action required to thresh them causes an unacceptable level of damage to the seed. Pods of Brassicaceae plants resistant to pod shelling should thus not be so strong that they cannot be threshed in a combine harvester (Bruce et al., 2001, J. Agric. Engng Res. 80, 343 350).

WO 2004/113542 describes that gene silencing of moderate dsRNA from genes involved in the development of the zone of dehiscence and margins of the leaflet leaflet in Brassicaceae plants allows the isolation of transgenic lines with a greater resistance to pod shelling and less seed shelling, whose pods, however, can still be opened throughout the dehiscence zone applying limited physical forces.

WO09 / 068313 (which claims priority over European patent application EP 07023052) discloses Brassica plants that comprise at least two IND genes, in particular Brassica napus plants, characterized in that they comprise three mutant IND alleles completely defective in their genome and where the resistance to shelling of plant pods is significantly increased compared to the resistance to shelling of pods of a plant that does not comprise mutant IND alleles, but where the plant preferably maintains an agronomically threshing capacity relevant of the pods.

The inventions described below in the different embodiments, examples and claims provide additional improved methods and means for modulating dehiscence properties in dehiscent seed plants. More specifically, the present invention discloses other improved methods and means of reducing seed shelling or delaying seed shelling until after harvest, in plants such as Brassicaceae plants, particularly Brassicaceae plants grown for seed production, at the same time while maintaining an agronomically relevant threshing capacity of the pods. In particular, the present application discloses Brassica plants that comprise at least two IND genes, in particular Brassica napus plants, characterized in that they comprise two partial defective mutant IND alleles in their genome or two partial and two complete mutant IND alleles and in those in which the resistance to shelling of plant pods is significantly increased compared to the resistance to shelling of pods of a plant that does not comprise mutant IND alleles, but in which the plant preferably maintains an agronomically relevant threshing capacity of the pods. Procedures and means for increasing yield are also provided, particularly grain and seed yield. The yield increase phenotype may be separate from the delayed or reduced seed fragmentation phenotype.

Summary of the invention

The inventors have discovered that Brassica napus plants with a pod shelling phenotype similar to that of the Brassica plants described in WO09 / 068313 (claiming priority over European patent application EP 07023052) can be obtained, i.e. which combine increased pod shelling resistance with agronomically relevant threshing capacity of pods by combining two partial defective mutant IND alleles with or without two defective complete mutant IND alleles rather than combining three complete defective mutant IND alleles.

Therefore, herein is described a Brassica plant comprising at least two IND genes, or a cell, part, seed or progeny thereof, characterized in that it comprises at least two partially defective mutant IND alleles in its genome. The IND genes can be IND-A1 or IND-C1 genes. IND genes may comprise a nucleic acid molecule selected from the group consisting of: a nucleic acid molecule comprising at least 90% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3 of the nucleotide in position 46 with the nucleotide at position 633, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7; and a nucleic acid molecule encoding an amino acid sequence comprising at least 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4 for the amino acid at position 16 with the amino acid at position 21 or SEQ ID NO: 4. Partially defective mutant IND alleles may be mutant IND alleles of the IND-C1 gene. The partial defective mutant IND alleles can be selected from the group consisting of ind-a1-EMS06, ind-a1-EMS09, ind-a1-EMS13, ind-c1-EMS04, ind-c1-EMS08, and ind-c1 -EMS09. The plant may further comprise at least one complete defective mutant IND allele in its genome. Complete defective mutant IND alleles may be a mutant IND allele of the IND-C1 gene. The full length mutant IND allele can be selected from the group consisting of ind-a1-EMS01, ind-a1-EMS05, ind-c1-EMS01, and ind-c1-EMS03. The plant may be homozygous for the partial and / or complete defective mutant IND allele. The plant can produce a significantly reduced amount of functional IND protein compared to the amount of functional IND protein produced by a corresponding plant that does not comprise mutant IND alleles. Shelling of the plant seed can be significantly reduced or delayed compared to shelling of the seed of a corresponding plant that does not comprise mutant IND alleles. The plant can maintain an agronomically relevant threshing capacity of the pods. The plant may be a plant of a Brassica crop species, preferably Brassica napus, Brassica juncea, Brassica carinata, Brassica rapa or Brassica oleracea. The plant may be a plant of a species of Brassica oil seed, preferably Brassica napus, Brassica juncea or Brassica rapa.

Also disclosed is a plant or cell, part, seed, or progeny thereof, comprising at least one partially defective mutant allele of an IND gene in its genome, wherein the IND gene comprises a nucleic acid molecule selected from the group consisting of: a nucleic acid molecule comprising at least 90% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3 of the nucleotide at position 46 with the nucleotide at position 633, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7; and a nucleic acid molecule encoding an amino acid sequence comprising at least 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4 for the amino acid at position 16 with the amino acid at position 21 or SEQ ID NO: 4. The partial defective mutant IND allele may be selected from the group consisting of ind-a1-EMS06, ind-a1-EMS09, ind-a1EMS13, ind-c1-EMS04, ind-c1-EMS08 and ind-c1-EMS09. The mutant IND allele can be derived from a plant of a Brassica species. The plant may be a plant of a species of Brassica.

A seed pod obtainable from the plants described herein is also described.

In one aspect, a partial defective mutant allele of an IND gene is provided, wherein the IND gene comprises a nucleic acid molecule selected from the group consisting of: a nucleic acid molecule comprising at least 90% identity of sequence with SEQ ID NO: 1, SEQ ID NO: 3 of the nucleotide at position 46 with the nucleotide at position 633, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7; and a nucleic acid molecule encoding an amino acid sequence comprising at least 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4 for the amino acid at position 16 with the amino acid at position 21 or SEQ ID NO: 4. In one embodiment, the mutant allele is selected from the group consisting of ind-a1 -EMS06, ind-a1-EMS09, ind-a1-EMS13, ind-c1-EMS04, ind-c1- EMS08 and ind-c1-EMS09. In another embodiment, the mutant allele is derived from a plant of a Brassica species, preferably from a Brassica crop species or a Brassica oilseed species. In a still further aspect, a mutant IND protein encoded by the mutant IND alleles of the invention is provided.

Even in a further aspect, a method is provided for identifying a mutant IND allele according to the invention in a biological sample comprising determining the presence of a specific region of mutant IND in a nucleic acid present in the biological sample. In one embodiment, the method further comprises subjecting the biological sample to a polymerase chain reaction assay using a set of at least two primers, said set being selected from the group consisting of: a set of primers, wherein one of said primers specifically recognizes the 5 'flanking region of the mutant IND allele and the other of said primers specifically recognizes the 3' flanking region of the mutant IND allele, respectively; a set of primers, wherein one of said primers specifically recognizes the 5 'or 3' flanking region of the mutant IND allele and the other of said primers specifically recognizes the mutation region of the mutant IND allele; and a set of primers, wherein one of said primers specifically recognizes the 5 'or 3' flanking region of the mutant IND allele and the other of said primers specifically recognizes the junction region between the 3 'or 5' flanking region. and the mutation region of the mutant IND allele, respectively. The primer that specifically recognizes the 5 'or 3' flanking region of the mutant IND allele may consist of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the 5 'or 3' flanking region of the mutant IND allele or of the complement of the The same, respectively, or the primer that specifically recognizes the mutation region of the mutant IND allele may consist of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the mutation sequence of the mutant IND allele or the complementary thereof, or the primer specifically recognizing the junction region between the 5 'or 3' flanking region and the mutation region of the mutant IND allele may consist of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from a sequence spanning the junction region between the flanking region 5 'or 3' and the mutation region of the mutant IND allele or the complementary thereof, in which said 17 to 200 nuc Consecutive leotides are not derived exclusively from the mutation or flanking sequences. The primer that specifically recognizes the 5 'or 3' flanking region of the mutant IND allele may comprise at its 3 'end a nucleotide sequence of at least 17 consecutive nucleotides selected from the 5' or 3 'flanking region of the mutant IND allele. or the complementary thereof, respectively, or the primer that specifically recognizes the mutation region of the mutant IND allele may comprise at its 3 'end a nucleotide sequence of at least 17 consecutive nucleotides selected from the mutant IND allele mutation sequence or of the complement thereof, or the primer that specifically recognizes the junction region between the 5 'or 3' flanking region and the mutation region of the mutant IND allele may comprise at its 3 'end a nucleotide sequence of at least 17 consecutive nucleotides selected from a sequence spanning the junction region between the 5 'or 3' flanking region and the mutation region of the mutant IND allele or c complementary thereof, in which said 17 consecutive nucleotides located in 3 'do not derive exclusively from the mutation or the flanking sequences. In yet another embodiment, the method further comprises subjecting the biological sample to a hybridization assay using a set of specific probes comprising at least one specific probe, said set being selected from the group consisting of: a set of specific probes, in the that one of said probes specifically recognizes the 5 'flanking region of the mutant IND allele and the other of said sodas specifically recognizes the 3' flanking region of the mutant IND allele; a set of specific probes, wherein one of said probes specifically recognizes the 5 'or 3' flanking region of the mutant IND allele and the other of said probes specifically recognizes the mutation region of the mutant IND allele; a set of specific probes, in which one of said probes specifically recognizes the 5 'or 3' flanking region of the mutant IND allele and the other of said probes specifically recognizes the junction region between the 3 'or 5' flanking region and the mutation region of the mutant IND allele, respectively; and a specific probe that specifically recognizes the junction region between the 5 'or 3' flanking region and the mutation region of the mutant IND allele. The probe that specifically recognizes the 5 'or 3' flanking region of the mutant IND allele may consist of a nucleotide sequence of 13 to 1,000 consecutive nucleotides selected from the 5 'or 3' flanking region of the mutant IND allele or the complementary one of the The same, respectively, or a sequence that has a sequence identity of at least 80% with it, or the probe that specifically recognizes the mutation region of the mutant IND allele may consist of a nucleotide sequence of 13 to 1,000 consecutive nucleotides selected from the mutant sequence of the mutant IND allele or the complementary thereof, or a sequence having a sequence identity of at least one 80% the same, or the probe that specifically recognizes the junction region between the 5 'or 3' flanking region and the mutation region of the mutant IND allele may consist of a nucleotide sequence of 13 to 1,000 consecutive nucleotides selected from a sequence encompassing the junction region between the 5 'or 3' flanking region and the mutation region of the mutant IND allele or the complementary thereof, respectively, in which said 13 to 1,000 consecutive nucleotides are not exclusively derived from the mutation or the flanking sequences or a sequence having a sequence identity of at least 80% with it. The probe that specifically recognizes the 5 'or 3' flanking region of the mutant IND allele can comprise a nucleotide sequence of at least 13 consecutive nucleotides selected from the 5 'or 3' flanking region of the mutant IND allele or its complement. , respectively, or the probe that specifically recognizes the mutation region of the mutant IND allele can comprise a nucleotide sequence of at least 13 consecutive nucleotides selected from the mutant sequence of the mutant IND allele or complementary thereof, or the probe that recognizes specifically the junction region between the 5 'or 3' flanking region and the mutation region of the mutant IND allele may comprise a nucleotide sequence of at least 13 consecutive nucleotides selected from a sequence spanning the junction region between the flanking region 5 'or 3' and the mutation region of the mutant or complementary IND allele, respectively, in which said s at least 13 consecutive nucleotides are not derived exclusively from the mutation or flanking sequences. In another particular embodiment, the 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 929 or from 931 to 1622 or complementary thereof, respectively; said mutation region has the nucleotide sequence of nucleotide 930 of SEQ ID NO: 5 or complementary thereto; and said binding region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 930 or from 930 to 1622 or complementary thereof, respectively; the 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 995 or 997 to 1622 or complementary thereof, respectively; said mutation region has the nucleotide sequence of nucleotide 996 of SEQ ID NO: 5 or complementary thereto; and said binding region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 996 or 996 to 1622 or complementary thereof, respectively; or the 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 1035 or from 1037 to 1622 or complementary thereof, respectively; said mutation region has the nucleotide sequence of nucleotide 1036 of SEQ ID NO: 5 or complementary thereto; and said binding region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 1036 or from 1036 to 1622 or complementary thereof, respectively; or the 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 902 or 904 to 1593 or complementary thereof, respectively; said mutation region has the nucleotide sequence of nucleotide 903 of SEQ ID NO: 7 or complementary thereto; and said binding region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 903 or 903 to 1593 or complementary thereof, respectively; or the 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 910 or from 912 to 1593 or complementary thereof, respectively; said mutation region has the nucleotide sequence of nucleotide 911 of SEQ ID NO: 7 or complementary thereto; and said binding region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 911 or from 911 to 1593 or complementary thereof, respectively; or the 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 919 or from 921 to 1593 or complementary thereof, respectively; said mutation region has the nucleotide sequence of nucleotide 920 of SEQ ID NO: 7 or complementary thereto; and said binding region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 920 or from 920 to 1593 or complementary thereof, respectively. The set of probes can be selected from the group consisting of: a set of probes comprising a probe comprising the sequence of SEQ ID NO: 11 and / or a probe comprising the sequence of SEQ ID NO: 12; a set of probes comprising a probe comprising the sequence of SEQ ID NO: 14 and / or a probe comprising the sequence of SEQ ID NO: 15; a set of probes comprising a probe comprising the sequence of SEQ ID NO: 17 and / or a probe comprising the sequence of SEQ ID NO: 18; a probe set comprising a probe comprising the sequence of SEQ ID NO: 20 and / or a probe comprising the sequence of SEQ ID NO: 21; a set of probes comprising a probe comprising the sequence of SEQ ID NO: 23 and / or a probe comprising the sequence of SEQ ID NO: 24; and a set of probes comprising a probe comprising the sequence of SEQ ID NO: 26 and / or a probe comprising the sequence of SEQ ID NO: 27.

In yet another aspect, a method is provided for determining the zygosity status of a mutant IND allele according to the invention in a plant or a cell, part, seed or progeny thereof, which determines the presence of a mutant and / or or a corresponding wild-type IND specific region in the genomic DNA of said plant, or a cell, part, seed or progeny thereof. In one embodiment, the method further comprises subjecting the genomic DNA of said plant, or a cell, part, seed, or progeny thereof, to a polymerase chain reaction assay using a set of at least two or at least three primers, wherein at least two of said primers specifically recognize the wild-type IND allele, said at least two primers being selected from the group consisting of: a first primer that specifically recognizes the 5 'or 3' flanking region of the allele Mutant and wild-type IND, and a second primer that specifically recognizes the 3 'or 5' flanking region of the mutant and wild-type IND allele, respectively; a first primer that specifically recognizes the 5 'or 3' flanking region of the mutant and wild-type IND allele, and a second primer that specifically recognizes the mutation region of the wild-type IND allele; and a first primer that specifically recognizes the 5 'or 3' flanking region of the mutant and wild-type IND allele, and a second primer that specifically recognizes the binding region between the 3 'or 5' flanking region and the region of allele mutation Wild-type mutant IND, respectively; and wherein at least two of said primers specifically recognize the mutant IND allele, said at least two primers being selected from the group consisting of: the first primer that specifically recognizes the 5 'or 3' flanking region of the mutant IND allele and wild-type, and a second primer that specifically recognizes the 3 'or 5' flanking region of the mutant and wild-type IND allele, respectively; the first primer that specifically recognizes the 5 'or 3' flanking region of the mutant and wild-type IND allele mutant, and a third primer that specifically recognizes the mutation region of the mutant IND allele; and the first primer that specifically recognizes the 5 'or 3' flanking region of the mutant and wild-type IND allele, and a third primer that specifically recognizes the binding region between the 3 'or 5' flanking region and the region of mutation of the mutant IND allele, respectively. The primer that specifically recognizes the 5 'or 3' flanking region of the mutant and wild-type IND allele may consist of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the 5 'or 3' flanking region of the mutant IND allele. and wild type or complementary thereof, respectively; or primers that specifically recognize the mutation region of the wild-type or mutant IND allele may consist of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the mutant or wild-type IND allele mutation sequence or the complement thereof , respectively; or primers that specifically recognize the junction region between the 5 'or 3' flanking region and the mutation region of the mutant or wild-type IND allele, may consist of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from a sequence encompassing the junction region between the 5 'or 3' flanking region and the mutation region of the mutant or wild IND allele or the complement thereof, respectively, in which said 17 to 200 consecutive nucleotides are not derived exclusively from the region of mutation or flanking sequences. The primer that specifically recognizes the 5 'or 3' flanking region of the wild type and mutant IND allele may comprise at its 3 'end a nucleotide sequence of 17 consecutive nucleotides selected from the 5' or 3 'flanking region of the allele Mutant and wild-type or complementary IND thereof, respectively; or primers that specifically recognize the mutant region of the wild-type or mutant IND allele may comprise at its 3 'end a nucleotide sequence of 17 consecutive nucleotides selected from the mutant or wild-type IND or complementary mutation sequence thereof, respectively; or primers that specifically recognize the junction region between the 5 'or 3' flanking region and the mutation region of the mutant or wild-type IND allele may comprise at their 3 'end a nucleotide sequence of 17 consecutive nucleotides selected from a sequence spanning the junction region between the 5 'or 3' flanking region and the mutation region of the mutant or wild-type IND allele or the complement thereof, respectively, wherein said 17 consecutive 3 'nucleotides are located they do not derive exclusively from the mutation site or region or from the flanking sequences. In still a further embodiment, the method further comprises subjecting the genomic DNA of said plant, or a cell, part, seed, or progeny thereof, to a hybridization assay using a set of at least two specific probes, wherein the at least one of said specific probes specifically recognizes the wild-type IND allele, said at least one probe selected from the group consisting of: a first probe that specifically recognizes the 5 'or 3' flanking region of the mutant and wild-type iNd allele , and a second probe that specifically recognizes the 3 'or 5' flanking region of the mutant and wild-type IND allele, respectively; a first probe that specifically recognizes the 5 'or 3' flanking region of the mutant and wild type IND allele, and a second probe that specifically recognizes the mutation region of the wild type IND allele; a first probe that specifically recognizes the 5 'or 3' flanking region of the mutant and wild-type IND allele, and a second probe that specifically recognizes the junction region between the 3 'or 5' flanking region and the mutation region from the wild-type mutant IND allele, respectively; and a probe that specifically recognizes the junction region between the 5 'or 3' flanking region and the mutation region of the mutant IND allele; and wherein at least one of said specific probes specifically recognizes the mutant IND allele, said at least one probe selected from the group consisting of: the first probe that specifically recognizes the 5 'or 3' flanking region of the mutant IND allele and wild-type, and the second probe that specifically recognizes the 3 'or 5' flanking region of the mutant and wild-type IND allele, respectively; the first probe that specifically recognizes the 5 'or 3' flanking region of the mutant wild-type IND allele mutant, and a third probe that specifically recognizes the mutation region of the mutant IND allele; the first probe that specifically recognizes the 5 'or 3' flanking region of the mutant and wild-type IND allele, and a third probe that specifically recognizes the junction region between the 3 'or 5' flanking region and the mutation region of the mutant IND allele; and a probe that specifically recognizes the junction region between the 5 'or 3' flanking region and the mutation region of the mutant IND allele. The probe that specifically recognizes the 5 'or 3' flanking region of the mutant and wild-type IND allele may consist of a nucleotide sequence of 13 to 1,000 consecutive nucleotides selected from the 5 'or 3' flanking region of the mutant IND allele. and wild-type or complementary thereof, respectively, or a sequence having at least 80% sequence identity thereto, or the probe that specifically recognizes the mutation region of the mutant or wild-type IND allele may consist in a nucleotide sequence of 13 to 1,000 consecutive nucleotides selected from the mutant or wild-type IND allele mutation region sequence, respectively, or a sequence having at least 80% sequence identity with it, or the probe that specifically recognizes the junction region between the 5 'or 3' flanking region and the mutation region of the mutant or wild-type IND allele may consist of a sequence nucleotide 13 to 1,000 consecutive nucleotides selected from a sequence spanning the junction region between the 5 'or 3' flanking region and the mutation region of the mutant or wild-type IND allele, respectively, or a sequence having at least 80% sequence identity therewith, wherein said 13 to 1,000 consecutive nucleotides do not derive exclusively from the site or region of mutation or flanking sequences. The probe that specifically recognizes the flanking region 'or 3' sequence of the mutant and wild-type IND allele may comprise a nucleotide sequence of at least 13 consecutive nucleotides selected from the 5 'or 3' flanking region of the mutant and wild-type IND allele or complementary thereto , respectively, or the probe that specifically recognizes the mutant region of the wild-type or mutant IND allele may comprise a nucleotide sequence of at least 13 consecutive nucleotides selected from the mutant or wild-type IND allele mutation sequence of the complementary of the itself, or the probe that specifically recognizes the junction region between the 5 'or 3' flanking region and the mutation region of the mutant or wild-type IND allele may comprise a nucleotide sequence of at least 13 consecutive nucleotides selected from a sequence spanning the junction region between the 5 'or 3' flanking region and the mutant region of the mutant or wild-type IND allele of the complex A component thereof, respectively, wherein said at least 13 consecutive nucleotides are not derived exclusively from the mutation or flanking sequences. The 5 'or 3' flanking region may comprise the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 929 or from 931 to 1,622 or complementary thereof, respectively; said wild-type IND allele mutation region may have the nucleotide sequence of nucleotide 930 of SEQ ID NO: 5 or complementary thereto; said mutation region of the mutant IND allele may have the sequence a or complementary to it; said binding region of the wild-type IND allele may comprise the nucleotide sequence of SEQ ID NO: 5 of nucleotide 1 to 930 or 930 to 1622 or of the complementary thereof, respectively; and said junctional region of the mutant IND allele may comprise the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 929 followed by a or followed by the nucleotide sequence SEQ ID NO: 5 from nucleotide 931 to 1622 or complementary thereof, respectively; or the 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 995 or 997 to 1622 or the complementary thereof, respectively; said wild-type IND allele mutation region may have the nucleotide sequence of nucleotide 996 of SEQ ID NO: 5 or complementary thereto; said mutation region of the mutant IND allele may have the sequence a or complementary to it; and said binding region of the wild-type IND allele may comprise the nucleotide sequence of SEQ ID NO: 5 of nucleotide 1 to 996 or 996 to 1622 or complementary thereto, respectively; and said junctional region of the mutant IND allele may comprise the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 995 followed by a or followed by the nucleotide sequence SEQ ID NO: 5 from nucleotide 997 to 1622 or complementary thereof, respectively; or the 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 1035 or from 1037 to 1622 or complementary thereof, respectively; said wild-type IND allele mutation region may have the nucleotide sequence of nucleotide 1036 of SEQ ID NO: 5 or complementary thereto; said mutation region of the mutant IND allele may have the sequence to the complementary thereof; and said binding region of the wild type IND allele may comprise the nucleotide sequence of SEQ ID NO: 5 of nucleotide 1 to 1036 or 1036 to 1622 or complementary thereof, respectively; and said junctional region of the mutant IND allele may comprise the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 1035 followed by tot followed by the nucleotide sequence SEQ ID NO: 5 from nucleotide 1037 to 1622 or complementary thereof, respectively; or the 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 902 or 904 to 1593 or complementary thereof, respectively; said wild-type IND allele mutation region may have the nucleotide sequence of nucleotide 903 of SEQ ID NO: 7 or complementary thereto; said mutation region of the mutant IND allele may have the sequence to the complementary thereof; and said binding region of the wild type IND allele may comprise the nucleotide sequence of SEQ ID NO: 7 of nucleotide 1 to 903 or 903 to 1593 or of the complementary thereof, respectively; and said junctional region of the mutant IND allele may comprise the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 902 followed by tot followed by the nucleotide sequence SEQ ID NO: 7 from nucleotide 904 to 1593 or complementary thereof, respectively; or the 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 910 or 912 to 1593 or complementary thereof, respectively; said wild-type IND allele mutation region may have the nucleotide sequence of nucleotide 911 of SEQ ID NO: 7 or complementary thereto; said mutation region of the mutant IND allele may have the sequence a or complementary to it; and said binding region of the wild-type IND allele may comprise the nucleotide sequence of SEQ ID NO: 7 of nucleotide 1 to 911 or 911 to 1593 or complementary thereof, respectively; and said junctional region of the mutant IND allele may comprise the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 910 followed by a or followed by the nucleotide sequence SEQ ID NO: 7 from nucleotide 912 to 1593 or complementary thereof, respectively; or the 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 919 or from 921 to 1593 or complementary thereof, respectively; said wild-type IND allele mutation region may have the nucleotide sequence of nucleotide 920 of SEQ ID NO: 7 or complementary thereto; said mutation region of the mutant IND allele may have the sequence to the complementary thereof; and said binding region of the wild-type IND allele may comprise the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 920 or from 920 to 1593 or complementary thereof, respectively; and said junctional region of the mutant IND allele may comprise the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 919 followed by tot followed by the nucleotide sequence SEQ ID NO: 7 from nucleotide 921 to 1593 or complementary thereof, respectively. Suitable is a set of at least three specific probes selected from the group consisting of: a set of probes comprising a probe comprising the sequence of SEQ ID NO: 11, a probe comprising the sequence of SEQ ID NO: 12 and / or a probe comprising the sequence of SEQ ID NO: 13; a set of probes comprising a probe comprising the sequence of SEQ ID NO: 14, a probe comprising the sequence of SEQ ID NO: 15 and / or a probe comprising the sequence of SEQ ID NO: 16; a probe set comprising a probe comprising the sequence of SEQ ID NO: 17, a probe comprising the sequence of SEQ ID NO: 18 and / or a probe comprising the sequence of SEQ ID NO: 19; a set of probes comprising a probe comprising the sequence of SEQ ID NO: 20, a probe comprising the sequence of SEQ ID NO: 21 and / or a probe comprising the sequence of SEQ ID NO: 22; a set of probes comprising a probe comprising the sequence of SEQ ID NO: 23, a probe comprising the sequence of SEQ ID NO: 24 and / or a probe comprising the sequence of SEQ ID NO: 25; and a set of probes comprising a probe comprising the sequence of SEQ ID NO: 26, a probe comprising the sequence of SEQ ID NO: 27 and / or a probe comprising the sequence of SEQ ID NO: 28.

Kits are also provided to identify a mutant IND allele according to the invention in a biological sample and kits to determine the zygosity status of a mutant IND allele according to the invention in a plant or cell, part, seed or progeny of the It comprises primers or probes as described above. Also described are procedures for combining the mutant IND alleles according to the invention in a plant, procedures for transferring the mutant IND alleles according to the invention from one plant to another plant, and procedures for making a (hybrid) plant or seed according to the invention.

In another embodiment of the invention, the mutant IND alleles of the invention are used to increase the yield of seeds or grains harvested from Brassica plants. Yield increase may be a consequence of reduced or delayed seed shelling, but may also be independent of reduced or delayed seed shelling. In particular, Brassica plants are described that comprise at least two IND genes, or a cell, part, seed or progeny thereof, characterized in that these plants comprise two mutant homozygous IND alleles in their genome as described herein.

General definitions

"Increased resistance to shelling of pods" and "reducing resistance to shelling of seeds" as used herein refers to a decreased tendency to seed shelling and / or a delay in timing from the threshing of seeds, particularly until after the harvest, of Brassica plants, whose fruits do not normally ripen synchronously, but sequentially, so that some pods open and they break their seeds before or during the harvest. The shear resistance level of the sheaths is positively correlated and, for example, can be measured by determining the force needed to break the sheaths in the "tensile separation test" (Davies and Bruce, 1997, J Mat Sci 32: 5895- 5899; Morgan et al., 1998, Fields Crop Research 58, 153-165), the number of intact pods remaining after, for example, 20 seconds ("IP20"; Morgan et al., 1998, cited above), 9 , 7 or 17 seconds (Bruce et al., 2002, Biosystems Eng 81 (2): 179-184) in a "random impact test", the half-life of the pod sample (hereinafter also referred to as "LD50" ) in a random impact test, that is, the treatment time required to cause 50% of the pods to open in the pod samples analyzed and the "field score for shelling" (Morgan et al., 1998, above). Bruce et al., 2002 (cited above, Morgan et al., 1998 (cited above and the following examples have described random impact tests (RITs) and algorithms to define the half-life of the pod sample at such RITs. Briefly, a sample of intact mature pods is placed in a closed drum together with steel balls and the drum is agitated vigorously for increasing periods of time (eg 10s, 20s, 40s, 80s) .After each period, Opens the drum and counts the number of broken and damaged sheaths The most accurate estimate of the shelling resistance level for each line is calculated by fitting a linear x linear curve to all available data and estimating the time required to break half of the pods within a sample ("pod sample half-life" or "LD50"). However, it is important that the pods are opened mainly along the dehiscence zone and are not simply sprayed, as can be the case with v indehiscent ainas.

An "agronomically relevant increase in pod shelling resistance," as used herein, refers to an increase in pod shelling resistance in a plant resulting in yield losses related to pod shelling. in the field (before harvest) below those normally observed for that plant in the field. For rapeseed, yield losses related to pod shelling in the field are reported to be approximately 11% for a season with, on average, good growing conditions and approximately 25% for a season with, of medium, poor growing conditions. A positive correlation has been found between these levels of seed loss and the level of seed loss at 9.7 s and the treatment time of 17 s, respectively, in the random impact test described by Bruce et al., 2002 (Biosystems Eng 81 (2): 179-184). Alternatively, to determine if the level of pod shelling resistance in a plant is agronomically relevant, the half-life of the pod sample ("LD50", see above) of the plant can be compared to the half-life of the Pod sample from a plant known to have an average level of pod shelling resistance, such as, for rapeseed, all currently available commercially available rape varieties.

As used herein, "poor shelling of pods or seeds" or "fruit or pod dehiscence" refers to a procedure that takes place on a fruit after seed maturation, whereby valves are detached from the central partition releasing the seeds. The region that breaks (that is, the "dehiscence zone") runs the entire length of the fruit between the valves and the replum (external septum). At maturity, the "dehiscence zone" is essentially a layer of non-lignified cells between a region of lignified cells in the valve and the replum. Shelling occurs due to the combination of loosening of the cell wall in the area dehiscence and the stresses established by the differential mechanical properties of the drying cells in the silicone.

A "fruit" of Brassica, as used herein, refers to an organ of a Brassica plant that develops from a gyneceum composed of fused carpels, which, after fertilization, grows to become a "pod (seed)" or "silicua" containing the developing seeds. A Brassica "pod" ("seed") or "silicua" consists of a fruit wall (carpel) that encloses two lobes separated by the septum. The "dehiscence zones" develop on the margins of the carpel adjacent to the septum and extend along the silicone. The cells in the dehiscence zone eventually begin to degrade and this weakens the contact between the walls or valves of the carpel and the septum. Loss of cell cohesion is confined to cells in the dehiscence zone and is the result of rupture of the medial lamina (Meakin and Roberts, 1990, J Exp Bot 41, 995-1011).

"Dehiscence zones", as used herein, refers to layers of single, parenchymal cells, contained in the sutures located on either side of the bivalve sheath of plants, particularly Brassica plants. The dehiscence zones are located between the edge of the sheath leaflet and a central replum that contains the main vascular bundle of the peduncle or pedicel. The dissociation of the cells in the dehiscence zone takes place during the senescence of the pods and is completed when the pods reach full maturity (Meakin and Roberts, 1990, cited above). Separation of the leaflet may occur. The dehiscence zone contains vascular traces, which pass from the sheath wall to the pedicel (stem) and the replum. The pod degradation procedure is carried out only after external force fractures the delicate vascular threads, allowing the leaflets to separate and the seeds to fall to the ground. This occurs during disturbance of the canopy, for example by contact with the harvester during harvest. Vascular tissue contains thickened lignified cells, which form clusters of colenchymal cells that are adjacent to the conducting cells (Meakin and Roberts, 1990, cited above). This provides stiffness to the fabric and, presumably, some resistance to fracture.

As used herein, "agronomically relevant threshing capacity" refers to the resistance of a pod, particularly a rapeseed pod, to opening along the pod dehiscence zone with concurrent release of the seeds, after the application of physical forces that allow the pods to fully open while preventing damage to the seeds, as used, for example, in a combine harvester. A positive correlation has been found between the half-life of a pod sample ("LD50") in a random impact test and its threshing capacity. The half-lives of the rapeseed sample, as determined in a RIT performed as described in the Examples, corresponding to the agronomically relevant threshing capacity, should not exceed 80 seconds. Typical sample half-life values for commercially available rapeseed control lines are approximately 10 seconds. Therefore, lines with significantly increased pod shelling resistance with agronomically relevant threshing capacity have a pod sample half-life at RIT of between approximately 10 and approximately 80 seconds, between approximately 10 and approximately 70 seconds, between approximately 15 and approximately 70 seconds, between approximately 10 and approximately 60 seconds, between approximately 10 and approximately 50 seconds, between approximately 20 and approximately 60 seconds, between approximately 20 and approximately 50 seconds, between approximately 40 and approximately 60 seconds, approximately 57 seconds.

"Dehiscent seed plant" means a plant that produces a dehiscent nut, which has fruit walls that open to allow the seeds it contains to escape. Dehiscent fruits usually contain several seeds and include known fruits, for example, as legumes, capsules, and silicuas.

"Crop plant" refers to plant species grown as a crop, such as Brassica napus (AACC, 2n = 38), Brassica júncea (AABB, 2n = 36), Brassica carinata (BBCC, 2n = 34), Brassica rapa ( syn. B. campestris) (AA, 2n = 20), Brassica oleracea (CC, 2n = 18) or Brassica nigra (BB, 2n = 16). The definition does not cover weeds, such as Arabidopsis thaliana.

The term "nucleic acid sequence" (or nucleic acid molecule) refers to a DNA or RNA molecule in single-stranded or double-stranded form, particularly a DNA encoding a protein or protein fragment according to the invention. An "endogenous nucleic acid sequence" refers to a nucleic acid sequence within a plant cell, eg, an endogenous allele of an IND gene present in the nuclear genome of a Brassica cell. An "isolated nucleic acid sequence" is used to refer to a nucleic acid sequence that is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.

The term "gene" means a DNA sequence that comprises a region (transcribed region), which is transcribed into an RNA molecule (for example, into a pre-mRNA, which comprises intron sequences, which are then cut and spliced into mature mRNA, or directly into mRNA without intron sequences) in a cell, operably linked to regulatory regions (eg, a promoter). Therefore, a gene may comprise several operably linked sequences, such as a promoter, a 5 'leader sequence comprising, for example, sequences involved in the initiation of translation, a coding region (for proteins) (cDNA or DNA genomic) and a 3 'untranslated sequence comprising, for example, transcription termination sites. "Gen endogenous "is used to differentiate itself from a" foreign gene "," transgene "or" chimeric gene ", and refers to a gene from a plant of a certain plant genus, species or variety, which has not been introduced into that plant by transformation (that is, it is not a "transgene"), but is normally present in plants of that genus, species or variety, or is introduced into that plant from plants of another plant genus, species or variety, in the that is normally present, by normal breeding techniques or by somatic hybridization, for example, by fusion of protoplasts. Similarly, an "endogenous allele" of a gene is not introduced into a plant or plant tissue by plant transformation, but is eg generated by plant mutagenesis and / or selection or obtained by screening natural plant populations.

"Gene expression" or "gene expression" refers to the procedure in which a region of DNA, which is operably linked to the appropriate regulatory regions, particularly a promoter, is transcribed into an RNA molecule. The RNA molecule is then processed (by post-transcriptional procedures) within the cell, for example, by RNA splicing and initiation of translation and translation in an amino acid chain (protein), and termination of translation by termination codons. of the translation. The term "functionally expressed" is used herein to indicate that a functional protein is produced; the term "not functionally expressed" to indicate that a protein with significantly reduced functionality or no functionality (biological activity) is produced or that no protein is produced (see below).

The term "protein" refers to a molecule consisting of a chain of amino acids, without reference to a specific mode of action, size, three-dimensional structure, or origin. A "fragment" or "portion" of a protein may therefore still be called a "protein". An "isolated protein" is used to refer to a protein that is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell. "Amino acids" are the main building blocks of proteins and enzymes. They are incorporated into proteins by transfer RNA according to the genetic code, while ribosomes decode messenger RNA. During and after the final assembly of a protein, the amino acid content dictates the spatial and biochemical properties of the protein or enzyme. The main chain of amino acids determines the primary sequence of a protein, but the nature of the side chains determines the properties of the protein. "Similar amino acids," as used herein, refers to amino acids that have similar amino acid side chains, ie, amino acids that have polar, non-polar, or nearly neutral side chains. "Non-similar amino acids," as used herein, refers to amino acids that have different amino acid side chains, for example, an amino acid with a polar side chain is not similar to an amino acid with a non-polar side chain. Polar side chains generally tend to be present on the surface of a protein where they can interact with the aqueous environment found in cells ("hydrophilic" amino acids). On the other hand, "nonpolar" amino acids tend to reside within the center of the protein where they can interact with similar nonpolar neighbors ("hydrophobic" amino acids). Examples of amino acids having polar side chains are arginine, asparagine, aspartate, cysteine, glutamine, glutamate, histidine, lysine, serine, and threonine (all hydrophilic, except cysteine, which is hydrophobic). Examples of amino acids having nonpolar side chains are alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, and tryptophan (all hydrophobic, except glycine which is neutral).

The term "transcription factor" is used to refer to a protein consisting of at least two discrete domains, a DNA-binding domain and an activation or repression domain, that work together to modulate the rate of transcription initiation to from the target gene promoters (Ptashne, 1988, Nature 335, 683-689). The term "basic helix-loop-helix domain transcription factor (bHLH)" is used to refer to a transcription factor that comprises, in addition to the DNA binding domain of bHLH (Heim et al., 2003, Mol Biol Evol 20, 735-747; Toledo-Ortiz et al., 2003, Plant Cell 15, 1749-1770), domains known to be important for the regulation of gene expression that can be conserved at the amino acid level in related proteins of different species (Quong et al., 1993, Mol Cell Biol 13, 792-800). Transcriptional regulators comprising a bHLH domain bind to DNA through residues in the basic region, whereas the helix-loop-helix domain promotes dimerization, allowing family members to form hetero or homodimers (Murre et al. , 1989, Cell 56, 777-783).

The term "gene / ND" refers herein to a nucleic acid sequence encoding an INDEHISCENT protein (IND), which is a basic transcription factor of the helix-loop-helix domain (bHLH) required for the dispersion of seeds (Liljegren et al., 2004, Cell 116: 843-853).

As used herein, the term "allele (s)" means any one or more alternative forms of a gene at a particular locus. In a diploid (or amphidiploid) cell of an organism, alleles of a given gene are located at a specific location or locus (plural loci) on a chromosome. An allele is present on each chromosome of the pair of homologous chromosomes.

As used herein, the term "homologous chromosomes" means chromosomes that contain information for the same biological characteristics and contain the same genes at the same loci but possibly different alleles of those genes. Homologous chromosomes are chromosomes that become paired during meiosis. "Non-homologous chromosomes", which represent all the biological characteristics of an organism, form a set, and the number of sets in a cell is called ploidy. Diploid organisms contain two sets of non-homologous chromosomes, in which each homologous chromosome is inherited from a parent different. In ampidiploid species, there are essentially two sets of diploid genomes, so the chromosomes of the two genomes are called "homologous chromosomes" (and, similarly, the loci or genes of the two genomes are called homologous genes or loci). A diploid or amphidiploid plant species can comprise a large number of different alleles at a particular locus.

As used herein, the term "heterozygous" means a genetic condition that exists when two different alleles reside at a specific locus, but are positioned individually on corresponding pairs of homologous chromosomes in the cell. Conversely, as used herein, the term "homozygous" means a genetic condition that exists when two identical alleles reside at a specific locus, but are positioned individually on corresponding pairs of homologous chromosomes in the cell.

As used herein, the term "locus" (plural loci) means a specific place or places or a site on a chromosome where, for example, a gene or genetic marker is found. For example, the "IND-A1 locus" refers to the position on the A genome chromosome where the IND-A1 gene (and two IND-A1 alleles) can be found, while the "IND-C1 locus" refers to the position on a chromosome of the C genome where the IND-C1 gene (and two IND-C1 alleles) can be found.

Whenever reference is made to a "plant" or "plants" according to the invention, it is understood that the present document also encompasses parts of plants (cells, tissues or organs, seed pods, seeds, cut parts, such as roots, leaves , flowers, pollen, etc.), progeny of plants that retain the distinctive characteristics of the parents (especially the dehiscence properties of the fruit), such as seeds obtained by self-fertilization or crossing, for example, hybrid seed (obtained by crossing two lines inbred parents), hybrid plants and plant parts derived therefrom, unless otherwise indicated.

A "molecular assay" (or test) refers herein to an assay that indicates (directly or indirectly) the presence or absence of one or more particular IND alleles at one or both IND loci (eg, at one or both IND-A1 or IND-C1 loci). In one embodiment, it allows to determine whether a particular IND allele (wild type or mutant) is homozygous or heterozygous at the locus in any individual plant.

"Wild type" (also spelled "wild type" or "wild type"), as used herein, refers to a typical form of a plant or gene as most commonly occurs in nature. A "wild-type plant" refers to a plant with the most common phenotype of such a plant in the natural population. A "wild type allele" refers to an allele of a gene required to produce the wild type phenotype. In contrast, a "mutant plant" refers to a plant with a rare phenotype different from that plant in the natural population or produced by human intervention, for example, by mutagenesis, and a "mutant allele" refers to an allele of a gene required to produce the mutant phenotype.

As used herein, the term "wild-type IND" (eg, wild-type IND-A1 or wild-type IND-C1) means a naturally occurring IND allele found within plants, particularly plants of Brassicacea, especially Brassica plants, that encode a functional IND protein (eg, a functional IND-A1 or IND-C1, respectively). In contrast, the term "mutant IND" (eg, IND-A1 or IND-C1 mutant), as used herein, refers to an IND allele, which does not encode a functional IND protein, ie, an IND allele encoding a non-functional IND protein (eg, a non-functional IND-A1 or IND-C1, respectively), which, as used herein, refers to an IND protein that has no biological activity or significantly reduced biological activity compared to the corresponding wild-type functional IND protein, or that encodes no IND protein at all. A "complete" or "null" mutant IND allele, as used herein, refers to a mutant IND allele, encoding an IND protein that has no biological activity compared to the wild-type functional IND protein. corresponding or that does not encode any protein at all. Such as a "complete defective mutant IND allele" is, for example, a wild-type IND allele, comprising one or more mutations in its nucleic acid sequence, for example, one or more nonsense or missense mutations. In particular, said complete defective mutant IND allele is a wild-type IND allele, comprising a mutation that preferably results in the production of an IND protein lacking at least one functional domain, such as the activation domain, the DNA binding domain and / or dimerization domain, or lacking at least one amino acid crucial for its function, such as an amino acid crucial for DNA binding, for example, arginine at position 127 in SEQ ID NO: 2 or at position 140 in SEQ ID NO: 4 and the like, or glutamine at position 122 in SEQ ID NO: 2 or at position 135 in SEQ ID NO: 4 and the like, so such that the biological activity of the IND protein is either completely abolished or so that the mutation (s) result, preferably, in the absence of production of an IND protein. A "partial defective" mutant IND allele, as used herein, refers to a mutant IND allele, encoding an IND protein that has significantly reduced biological activity compared to the corresponding wild-type functional IND protein. Such a "partial defective mutant IND allele" is, eg, a wild-type IND allele, comprising one or more mutations in its nucleic acid sequence, eg, one or more missense mutations. In particular, said partial defective mutant IND allele is a wild-type IND allele, comprising a mutation that preferably results in the production of an IND protein in which at least one conserved and / or functional amino acid is replaced by another amino acid, such that biological activity is significantly reduced but not completely eliminated. Such a total or partial defective mutant IND allele may also encode a dominant iNd negative protein, which is capable of negatively affecting the biological activity of other IND proteins within the same cell. Such a dominant negative IND protein may be an IND protein that is still capable of interacting with the same elements as the wild-type IND protein, but blocks some aspect of its function. Examples of dominant negative IND proteins are IND proteins that lack the activation domain and / or dimerization domain or specific amino acid residues crucial for activation and / or dimerization, but still contain the DNA binding domain, so they do not they only reduce or eliminate their own biological activity, but further reduce total IND activity in the cell by competing with wild-type and / or partial defective iNd proteins present in the cell for DNA binding sites. Other examples of dominant negative IND proteins are IND proteins that lack the activation domain and / or DNA binding domain or specific amino acid residues crucial for activation and / or DNA binding, but still contain the dimerization, so that not only does it reduce or eliminate its own biological activity, but it further reduces total IND activity in the cell by producing protein dimers that lack at least one functional domain. The mutant alleles may be nucleic acid sequences encoding the IND protein are designated as "ind" (eg, ind-a1 or ind-c1, respectively) herein. Mutant alleles can be "natural mutant" alleles, which are naturally occurring mutant alleles (eg, produced spontaneously without human application of mutagens) or "induced mutant" alleles, which are induced by human intervention, eg, by mutagenesis.

A "significantly reduced amount of functional IND protein" (eg, functional IND-A1 or IND-C1 protein) refers to a reduction in the amount of a functional IND protein produced by the cell comprising a mutant IND allele in at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% (i.e. the cell does not produce functional IND protein) compared to the amount of functional IND protein produced by the cell that does not comprise the mutant IND allele. This definition encompasses the production of a "non-functional" IND protein (eg, truncated IND protein) that has no biological activity in vivo, reduction in the absolute amount of functional iNd protein (eg, no functional IND protein is produced due to the mutation in the IND gene), the production of an IND protein with significantly reduced biological activity compared to the activity of a functional wild-type IND protein (such as an IND protein in which one or more amino acid residues that they are crucial for the biological activity of the encoded IND protein, as exemplified below, they are replaced by another amino acid residue) and / or the adverse effect of dominant negative IND proteins on other functional and / or partially functional IND proteins.

The term "mutant IND protein" as used herein refers to an IND protein encoded by a mutant IND nucleic acid sequence ("ind allele") whereby the mutation results in IND activity significantly reduced and / or nil in vivo, compared to the activity of the IND protein encoded by a wild type, non-mutant IND sequence ("IND allele").

"Mutagenesis", as used herein, refers to the procedure in which plant cells (eg, a plurality of Brassica seeds or other parts, such as pollen, etc.) are subjected to a technique that induces mutations in the DNA of cells, such as contact with a mutagenic agent, such as a chemical (such as ethylmethylsulfonate (EMS), ethylnitrosourea (ENU), etc.) or ionizing radiation (neutrons (such as in mutagenesis) fast neutron, etc.), alpha rays, gamma rays (such as that supplied by a cobalt 60 source), X-rays, UV radiation, etc.), or a combination of two or more of these. Therefore, the desired mutagenesis of one or more IND alleles can be achieved through the use of chemical means such as the contact of one or more plant tissues with ethylmethylsulfonate (EMS), ethylnitrosourea, etc., through the use of physical means, such as X-rays, etc., or gamma radiation, such as that supplied by a cobalt 60 source. While mutations created by irradiation are usually large deletions or other large lesions, such as translocations or complex rearrangements, mutations created by mutagens Chemicals are often more discrete lesions, such as point mutations. For example, EMS alkylates guanine bases, resulting in poor base pairing: an alkylated guanine will pair with a thymine base, primarily resulting in G / C to A / T transitions. After mutagenesis, Brassica plants are regenerated from the treated cells using known techniques. For example, the resulting Brassica seeds can be planted according to conventional cultivation procedures and after self-pollination the seed is formed in the plants. Alternatively, duplicate haploid seedlings can be removed to immediately form homozygous plants, for example, as described by Coventry et al., (1988, Manual for Microspore Culture Technique for Brassica napus. Dep. Crop Sci. Techn. Bull. OAC Publication 0489 Guelph University, Guelph, Ontario, Canada). The additional seed that is formed as a result of such self-pollination in the present or in a later generation can be harvested and screened for the presence of mutant IND alleles. Various techniques are known for detecting specific mutant alleles, eg Deleteagene ™ (Delete-a-gene; Li et al., 2001, Plant J 27: 235-242) uses polymerase chain reaction (PCR) assays to detecting deletion mutants generated by rapid neutron mutagenesis, TILLING (genome-directed local induced lesions; McCallum et al., 2000, Nat Biotechnol 18: 455-457) identifies point mutations induced by EMS, etc. The following examples describe additional techniques for detecting the presence of specific mutant IND alleles.

As used herein, the term "of unnatural origin" or "cultivated" when used in reference to a plant, means a plant with a genome that has been modified by man. A transgenic plant, for example, is a plant of non-natural origin that contains an exogenous nucleic acid molecule, for example, a chimeric gene that comprises a transcribed region that when transcribed produces a molecule of biologically active RNA capable of reducing the expression of an endogenous gene, such as an IND gene and, therefore, has been genetically modified by man. In addition, a plant that contains a mutation in an endogenous gene, for example, a mutation in an endogenous IND gene, (for example, in a regulatory element or in the coding sequence) as a result of exposure to a mutagenic agent is also considered a non-natural plant, since it has been genetically modified by man. Also, a plant of a particular species, such as Brassica napus, that contains a mutation in an endogenous gene, for example, in an endogenous IND gene, which in nature does not occur in that particular plant species, as a result of, for example, directed breeding procedures, such as marker-assisted breeding and selection or introgression, with a plant of the same or another species, such as Brassica júncea or rapa, of that plant is also considered a plant of non-natural origin . In contrast, a plant that contains only spontaneous or natural mutations, that is, a plant that has not been genetically modified by man, is not a "plant that is not of natural origin" as defined herein and, therefore, it is not included in the invention. A person skilled in the art understands that, while a plant of non-natural origin usually has an altered nucleotide sequence compared to a plant of natural origin, a plant of non-natural origin can also be genetically modified by man without altering its sequence. of nucleotides, for example, by modifying their methylation pattern.

The term "ortholog" of a gene or protein herein refers to the homologous gene or protein found in another species, which has the same function as the gene or protein, but is (generally) divergent in sequence from the time that the species that harbor the genes diverged (that is, the genes evolved from a common ancestor by speciation). Therefore, the orthologs of the Brassica napus IND genes can be identified in other plant species (for example, Brassica júncea, etc.) according to both sequence comparisons (for example, based on percentages of sequence identity over the sequence complete or on specific domains) and / or functional analysis.

A "variety" is used in this document in accordance with the UPOV convention and refers to a grouping of plants within a single botanical taxon of the lowest known range, a grouping that can be defined by the expression of the characteristics resulting from A given genotype, or a combination of genotypes, can be distinguished from any other grouping of plants by the expression of at least one of these characteristics and is considered as a unit with respect to its suitability to propagate unchanged (stable).

The term "comprising" should be interpreted as specifying the presence of the indicated parts, steps or components, but does not exclude the presence of one or more additional parts, steps or components. A plant comprising a certain trait can thus comprise additional traits.

It is understood that when a word is referred to in the singular (eg, plant or root), the plural is also included herein (eg, a plurality of plants, a plurality of roots). Therefore, the reference to an element by the indefinite article "a" or "one" does not exclude the possibility that there is more than one of the elements present, unless the context clearly requires that there be one and only one of The elements. The indefinite article "a" or "one / one" usually means "at least one / one".

For the purposes of the present invention, the "sequence identity" of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences that have identical residues (x100) divided by the number of positions compared. A gap, that is, a position in an alignment where a residue is present in one sequence but not in the other, is considered as a position with non-identical residues. The "optimal alignment" of two sequences is found by aligning the two sequences across the entire length according to the Needleman and Wunsch global alignment algorithm (Needleman and Wunsch, 1970, J Mol Biol 48 (3): 443-53) in The European Molecular Biology Open Software Suite (EMBOSS, Rice et al., 2000, Trends in Genetics 16 (6): 276-277; see, for example, http://www.ebi.ac.uk/emboss/align/ index.html) using the default setting (gap opening penalty = 10 (for nucleotides) / 10 (for proteins) and gap extension penalty = 0.5 (for nucleotides) / 0.5 (for proteins)). For nucleotides, the default scoring matrix used is EDNAFULL, and for proteins, the default scoring matrix is EBLOSUM62.

"Substantially identical" or "essentially similar", as used herein, refers to sequences, which, when optimally aligned as defined above, share at least a certain minimum percentage of sequence identity ( as defined below).

"Stringent hybridization conditions" can be used to identify nucleotide sequences, which are substantially identical to a given nucleotide sequence. Rigorous conditions depend on sequence and will be different under different circumstances. Generally, stringent conditions are selected to be approximately 5 ° C less than the thermal melting point (Tf) for specific sequences at a defined pH and ionic strength. Tf is the temperature (at defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be chosen where the salt concentration is approximately 0.02 molar at pH 7 and the temperature is at least 60 ° C. Decreasing the salt concentration and / or increasing the temperature increases the rigor. The stringent conditions for RNA-DNA hybridizations (Northern blots using a probe of, for example, 100 nt) are, for example, those that include at least one wash in 0.2X SSC at 63 ° C for 20 minutes, or terms equivalents.

"High stringency conditions" can be provided, for example, by hybridization at 65 ° C in an aqueous solution containing 6x SSC (20x SSC contains 3.0M NaCl, 0.3M Na-citrate, pH 7.0) , 5x Denhardt's (100X Denhardt's contains 2% Ficoll, 2% polyvinylpyrrolidone, 2% bovine serum albumin, 0.5% sodium dodecyl sulfate (SDS), and 20 | jg / ml denatured carrier DNA (fish sperm DNA single-stranded, with an average length of 120-3,000 nucleotides) as a non-specific competitor After hybridization, the high stringency wash can be carried out in several stages, with a final wash (approximately 30 minutes) at the hybridization temperature of 0 , 2-0.1X SSC, 0.1% SDS.

"Moderate stringency conditions" refers to conditions equivalent to hybridization in the solution described above but at approximately 60-62 ° C. Rigorous moderate washing can be performed at the hybridization temperature in 1x SSC, 0.1% SDS.

"Low stringency" refers to conditions equivalent to hybridization in the solution described above at approximately 50-52 ° C. Low stringency wash can be performed at the hybridization temperature in 2x SSC, 0.1% SDS. See also Sambrook et al., (1989) and Sambrook and Russell (2001).

"Increased yield harvested" or "Increased seed or grain yield" refers to the largest amount of seeds or grains harvested from a plurality of plants, each comprising mutant IND alleles according to the invention, compared to the amount of seed or grain harvested from a similar number of isogenic plants without the mutant IND alleles. Yield is generally expressed in units of volume of seed harvested per unit area, such as bushels / acre or kg / ha. The yield increase is generally expressed as a percentage, so the yield of the reference or control plant is called 100% and the yield of the plants as described herein is expressed in% with respect to the yield of the plant. of control. The observed yield increases in the Brassica plants, as described herein, ranged from at least 101% to at least 124% and further yield increases are expected to be feasible. The performance increase can also vary from 104% to 108% or from 105% to 110%.

Detailed description

As described in WO09 / 068313 (which claims the priority of European patent application EP 07023052), it was previously discovered that Brassica napus plants, which are homozygous for a complete defective ind allele in only one of its two IND genes , i.e. IND-A1 or IND-C1, did not show a significant increase in pod shelling resistance compared to Brassica napus plants that do not comprise mutant IND alleles, whereas in Brassica napus plants, which were homozygous for a full effective ind allele in both IND genes, pod shelling resistance increased significantly, but the pod shelling resistance level was too high to maintain an agronomically relevant threshing capacity. In contrast, pod shelling resistance was significantly increased in Brassica napus plants comprising three complete defective ind alleles of the two Brassica napus IND genes, at a level whereby plants maintain agronomically relevant threshing capacity of the pods.

The inventors surprisingly discovered that the Brassica napus plants with a pod shelling phenotype similar to the Brassica plants described in WO09 / 068313 (which claims the priority of European patent application EP 07023052), i.e. combining Increased pod shelling resistance with agronomically relevant threshing capacity of pods can also be obtained by combining two partial defective IND mutant alleles with two complete defective mutant IND alleles rather than combining three complete defective mutant IND alleles. It was further found that mutations in the IND-C1 gene resulted in a stronger increase in resistance to shelling of pods than mutations in the IND-A1 gene. A stronger increase in pod shelling resistance in Brassica napus plants was, for example, observed when the two complete defective mutant IND alleles were complete defective mutant IND alleles of the IND-C1 gene and the two partial defective mutant IND alleles were When partial defective mutant IND alleles of the IND-A1 gene, when the two complete defective mutant IND alleles were from the IND-A1 gene and the partial defective mutant IND alleles were from the IND-C1 gene. Surprisingly, Brassica napus plants that combine increased pod shelling resistance with agronomically relevant threshing ability of pods can also be obtained by introducing two partially defective mutant IND alleles, in particular the IND-C1 gene, alone.

Therefore, a Brassica plant comprising at least two IND genes is described herein, in particular a Brassica napus plant comprising an IND-A1 gene and / or an IND-C1 gene, characterized in that it comprises two alleles IND partial defective mutants in their genome, in particular of an IND-A1 gene and / or an IND-C1 gene, preferably of an IND-C1 gene, is described herein, whereby the alle alleles result in a significantly reduced amount of functional IND protein of the type encoded by the wild-type equivalent of these mutant alleles, and therefore a significantly reduced overall amount of functional iNd proteins produced in plant cells, specifically in developing seed pods , in vivo.

The Brassica plant may further comprise two complete defective mutant IND alleles in its genome, in particular an IND-C1 gene and / or an IND-A1 gene, respectively, preferably an IND-C1 gene, such as those described in WO09 / 068313 (claiming priority from European patent application EP 07023052), for example, ind-a1-ems01, ind-a1-ems05, ind-c1-ems01 or ind-c1-ems03 and the like.

It is believed that by combining enough copies of specific partial defective mutant IND alleles with sufficient copies of specific complete and / or wild type mutant IND alleles in a plant, particularly a Brassica plant, it is possible to adjust the amount and / or the type of functional IND proteins produced, which in turn influences the dehiscence properties of the plant fruit. Therefore, the absolute and relative amount of IND proteins can be adjusted in such a way as to provide plants that produce enough IND proteins to allow for agronomically relevant threshing ability of seed pods while reducing seed shelling before or during Harvest.

Also described herein is a plant, in particular a Brassica plant, comprising at least one partially defective mutant IND allele, encoding a partially functional IND protein, such as those described below, eg, ind-a1- ems06, ind-a1- ems09, ind-a1-ems13, ind-c1-ems04, ind-c1-ems08 or indc1-ems09, and the like, while the remaining alleles may be partial defective, complete defective IND alleles and / or wild type.

This document describes a Brassica plant comprising at least two IND genes, in particular a Brassica napus plant, comprising two partial ind defective alleles and complete ind defective alleles of the two IND genes in that Brassica plant, in particular of the Brassica napus IND-A1 and / or IND-C1 genes, preferably the IND-C l gene, so that n <2 (for example, n = 0, 1 or 2), so that at least one allele produces at least one functional IND protein.

We describe a single homozygous IND mutant (n = 2, i.e., homozygous for a partially defective mutant allele of an IND gene), and / or a homozygous IND double mutant- (n = 4, i.e., homozygous for a mutant allele complete and / or partial defective of two IND genes) of a Brassica species plant comprising at least two IND genes, in particular of Brassica napus, so that the mutant alleles are mutant alleles of the two IND genes in that plant Brassica, in particular of the IND-A1 and / or IND-C1 genes. Such mutant plants can be used for breeding purposes.

This document describes a Brassica napus plant with a homozygous single partial defective IND mutant, in which the plant genotype can be described as ind-a1P / ind-a1P, IND-C1 / IND-C1, or iND- A1 / IND-A1, ind-c1P / ind-c1P. A homozygous double partial mutant Brassica napus plant is described herein, in which the plant genotype can be described as ind-a1P / ind-a1P, ind-c1P / ind-c1P. Herein we describe a homozygous full and partial double IND mutant Brassica napus plant, in which the plant genotype can be described as either ind-a1F / ind-a1F, ind-c1P / ind-c1P or ind- a1P / ind-a1P, ind-c1F / ind-c1F.

In addition, novel nucleic acid sequences of partial defective IND mutant genes / alleles of Brassica species, as well as partial defective mutant IND proteins, are provided herein. Methods of generating and combining partial defective mutant IND alleles in Brassica plants, as well as Brassica plants and parts of plants comprising specific combinations of complete and partial defective mutant IND alleles in their genome, are also described so as to reduce the Shelling of seeds is reduced in these plants. The use of these plants to transfer partial defective mutant iNd alleles to other plants is also described herein, as well as the plant products of any of the plants described. In addition, kits and procedures for marker assisted selection (MAS) are provided to combine or detect IND genes and / or alleles. Each of the embodiments of the invention is described in detail hereinafter.

The Brassica plants described herein that exhibit reduced or delayed shelled seeds have an increased yield of the harvested seed. However, it was observed that not only the seed yield harvested from Brassica plants comprising only the ind-c1-09 allele in the homozygous state (showing an observable reduced or delayed seed sheltering phenotype), but also can describe the yield of seeds harvested from other Brassica plants that comprise the two mutant IND alleles in a homozygous state, that is, in which the genotype of the plant can be described as ind-a1P / ind-a1P, IND-C1 / IND -C1, or IND- A1 / IND-A1, ind-c1P / ind-c1P also increased significantly, compared to isogenic Brassica plants that do not comprise mutant IND alleles, despite the absence of a shelling phenotype of Observable reduced or delayed seed in Brassica plants comprising the mutant IND alleles. Brassica plants comprising at least two IND genes are also described, in which at least two alleles produce a functional IND protein, plants that have a higher seed yield. It will be clear that the two mutant alleles at the IND-A locus or at the IND-C locus can be the same mutant allele or a different mutant allele.

Nucleic acid sequencing according to the invention

Partial defective mutant ind nucleic acid sequences are provided encoding partially IND proteins. functional, i.e. IND proteins with significantly reduced biological activity (i.e. IND nucleic acid sequences comprising one or more mutations, resulting in significantly reduced biological activity of the encoded IND protein) of Brassicaceae IND genes, particularly of Brassica species, especially of Brassica napus, but also of other species of Brassica crops. For example, Brassica species comprising an A and / or C genome may comprise alleles of IND-A1 or IND-C1 genes, which are essentially similar to the partial defective mutant IND alleles of the present invention and which can be identified and combined on one floor. Furthermore, mutagenesis methods can be used to generate mutations in wild-type IND alleles, thereby generating mutant ind alleles essentially similar to the partial defective mutant IND alleles of the present invention for use in accordance with the invention. Because specific IND alleles are preferably combined in a plant by crossing and selecting, the ind nucleic acid sequences can be provided within a plant (i.e. endogenously), eg, a Brassica plant, preferably a Brassica plant It can be crossed with Brassica napus or it can be used to make a "synthetic" plant of Brassica napus. Hybridization between different Brassica species is described in the art, for example, as referenced in Snowdon (2007, Chromosome research 15: 85 95). Interspecific hybridization can, for example, be used to transfer genes from, for example, the C genome in B. napus (AACC) to the C genome in B. carinata (BBCC), or even from, for example, the C genome in B napus (AACC) to the B genome in B. juncea (AABB) (due to the sporadic event of illegitimate recombination between its C and B genomes). The "resynthesized" or "synthetic" Brassica napus lines can be produced by crossing the original ancestors, B. oleracea (CC) and B. rapa (AA). Barriers of incompatibility between species and between genera can be successfully overcome in crosses between Brassica crop species and their relatives, for example, using embryo rescue techniques or protoplast fusion (see, for example, Snowdon, above).

However, ind isolated nucleic acid sequences (eg, isolated from the plant by cloning or synthetically made by DNA synthesis), as well as their variants and fragments of any of these, are also provided herein as these may used to determine which sequence is endogenously present in a plant or part of the plant, whether the sequence encodes a functional, partially functional, non-functional protein, or no protein (eg, by expression in a recombinant host cell as described below) and for the selection and transfer of specific alleles from one plant to another, to generate a plant having the desired combination of partial and / or complete defective mutant IND alleles.

Novel wild type IND-A1 and IND-C1 partial defective mutant IND nucleic acid sequences have been isolated from Brassica napus. Wild type IND sequences as described in WO09 / 068313 (which claims priority over European patent application EP 07023052) are represented in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 from the sequence listing, while the novel ind mutant sequences of partial defects of these sequences, and of sequences essentially similar to these, are described herein below and in the Examples, with reference to the sequences Wild type IND. The genomic DNA encoding the Brassica napus IND protein does not comprise any introns.

The "IND-A1 nucleic acid sequences" or the "IND-A1 variant nucleic acid sequences" according to the invention are nucleic acid sequences encoding an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 2 or nucleic acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, 99% or 100% of sequence identity with SEQ ID NO: 1 or SEQ ID NO: 5 . These nucleic acid sequences may also be referred to as "essentially similar" or "essentially identical" to the IND sequences provided in the sequence listing.

The "IND-C1 nucleic acid sequences" or the "IND-C1 variant nucleic acid sequences" according to the invention are nucleic acid sequences encoding an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 98%, 99% or 100% of sequence identity with SEQ ID NO: 4 (IND-C1-long) or with SEQ ID NO : 4 from amino acid at position 16 to amino acid at position 210 (IND-C1-short) or nucleic acid sequences having at least 80%, at least 85%, at least 90%, at least one 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 3 (IND-C1-long), with SEQ ID NO: 3 from the nucleotide at position 46 up to the nucleotide at position 633 (IND-C1-short) or with SEQ ID NO: 7. These nucleic acid sequences may also be referred to as "essentially similar" or "essentially identical" to the IND sequences provided in the sequence listing.

Therefore, the invention provides novel partial defective mutant nucleic acid sequences of nucleic acid sequences encoding wild-type functional IND-A1 and IND-C1 proteins, including variants and fragments thereof (as defined below). , whereby the nucleic acid sequence mutation preferably results in the insertion of one or more amino acids, their deletion or substitution is deleted or substituted compared to the wild-type IND protein, particularly in the substitution of one or more amino acids and therefore the biological activity of the IND protein is significantly reduced. A significant reduction in the biological activity of the IND protein refers herein to a reduction in the DNA binding activity, dimerization capacity and / or transcriptional activity of the IND protein, such that resistance shelling of the sheath of a plant expressing the mutant IND protein is increased compared to a plant expressing the corresponding wild-type IND protein.

To determine the functionality of a specific IND allele / protein in plants, particularly in Brassica plants, the level of resistance to shelling of pods in plants can be determined by performing macroscopic, microscopic and histological tests on fruits and flowers of plants comprising the specific IND allele / protein and corresponding wild-type plants analogous to tests performed on Arabidopsis fruits and flowers as described in Liljegren et al (2004, cited above) or as described in the Examples below. Briefly, changes in pod shelling resistance can be evaluated and / or measured, for example, by macroscopic tests, such as inspection of seed pods with the naked eye to assess, for example, the presence or absence of the margins of the leaflet, the length of the spur of the pods, etc .; a Manual Impact Test (MIT) to compare the level of resistance to shelling of pods between different mutant IND lines and the corresponding wild type lines by evaluating the ease of opening of the pods by twisting the pods gently; a random impact test (RIT) to compare the threshing capacity of seed pods of plants from different mutant IND lines and the corresponding wild-type lines, respectively, measuring the half-life of the pod samples from these lines; and / or by microscopic tests to examine, for example, whether the cells in the leaflet margin and the dehiscence zone of the seed pods and how they are affected by mutations in IND. Once the dimerization partner of the IND protein (for example, the IND protein itself in case its operation depends on the formation of a homodimer or another protein if its operation depends on the formation of a heterodimer) and / or the gene (s) whose transcription is regulated by the IND protein is identified and characterized, the functionality of a specific IND protein / allele may alternatively be evaluated by recombinant DNA techniques as known in the art, for example, co-expressing both partners of the dimer in a host cell (eg, a bacterium, such as E. coli) and evaluating whether dimers can still form, whether dimers can still bind to the bHLH binding site of regulated genes, and / or or if the transcription of these genes is still regulated by this binding.

Both isolated and endogenous nucleic acid sequences are provided herein. Fragments of the mutant IND sequences and mutant IND variant nucleic acid sequences defined above are also provided for use as primers or probes and as kit components according to another aspect of the invention (see below). A "fragment" of an ind nucleic acid sequence or a variant thereof (as defined) can be of various lengths, such as at least 10, 12, 15, 18, 20, 50, 100, 200, 500, 600 contiguous nucleotides of the IND or ind sequence (or of the variant sequence).

Nucleic acid sequences encoding functional IND proteins

The nucleic acid sequences represented in the sequence listing encode functional wild-type IND proteins from Brassica napus. Therefore, these sequences are endogenous to the Brassica napus plants from which they were isolated. Other Brassica crop species, varieties, breeding lines, or wild records can be screened for other IND alleles, which encode the same IND proteins or variants thereof. For example, nucleic acid hybridization techniques (eg, Southern blot analysis, using, for example, stringent hybridization conditions) or PCR-based techniques can be used to identify endogenous IND alleles to other Brassica plants, such as various varieties of Brassica napus, lines or registers, although plants, organs and tissues of Brassica juncea (especially IND alleles in the A genome), Brassica carinata (especially iNd alleles in the C genome) and Brassica rapa (can also be screened) genome A) and Brassica oleracea (genome C) to detect other wild-type IND alleles. To screen such plants, organs, or plant tissues for the presence of IND alleles, the IND nucleic acid sequences provided in the sequence listing, or variants or fragments of any of these, can be used. For example, complete sequences or fragments can be used as probes or primers. For example, specific or degenerate primers can be used to amplify nucleic acid sequences encoding IND proteins from the genomic DNA of the plant, organ, or plant tissue. These IND nucleic acid sequences can be isolated and sequenced using standard molecular biology techniques. Bioinformatic analysis can be used to characterize the allele (s), for example, to determine which IND allele the sequence corresponds to and which IND protein or variant protein is encoded by the sequence.

Whether a nucleic acid sequence encodes a functional IND protein can be analyzed by recombinant DNA techniques as known in the art, for example, by a genetic complementation test using, for example, an Arabidopsis plant, which is homozygous for an allele. complete defective ind mutant or a Brassica napus plant, which is homozygous for a complete defective ind mutant allele of both the IND-A1 gene and the IND-C1 gene.

Furthermore, it is understood that IND nucleic acid sequences and their variants (or fragments of any of these) can be identified in silico, by selecting nucleic acid databases for essentially similar sequences. Similarly, a nucleic acid sequence can be chemically synthesized. Fragments of nucleic acid molecules according to the invention are also provided, which are described below. Fragments include nucleic acid sequences that encode only the bHLH domain, or smaller fragments that comprise part of the bHLH domain, such as the basic domain or the HLH domain, etc.

Nucleic acid sequences encoding mutant IND proteins

The invention provides nucleic acid sequences comprising one or more nucleotide deletions, insertions or substitutions relative to the wild-type IND nucleic acid sequences represented in SEQ ID NO: 1,3, 5 and 7 of the sequence listing, wherein the mutation (s) in the nucleic acid sequence result in significantly reduced biological activity, i.e. partial defective biological activity, of the encoded IND protein relative to the type IND protein wild as well as fragments of such mutant nucleic acid molecules. Such mutant nucleic acid sequences (called indP sequences) can be generated and / or identified using various known procedures, as described below. Again, such nucleic acid molecules are provided both endogenously and in isolation.

Basically any mutation in the wild-type IND nucleic acid sequences resulting in an IND protein comprising at least one amino acid insertion, Deletion and / or substitution, deletion and / or substitution relative to the IND type protein Wild can lead to significantly reduced or no biological activity. However, it is understood that certain mutations in the IND protein are more likely to result in a complete abolition of the biological activity of the IND protein, such as mutations whereby significant portions of the functional domains, such as the binding domain DNA ("b"), dimerization domain ("HLH") and / or transcriptional regulatory domains are missing, or by which certain crucial amino acid residues within these domains, such as the Gln (Q amino acids) ), Ala (A) and Arg (R) at positions 5, 9 and 13 or the basic amino acid residues (in particular the Arg (R) residues) at positions 10 and 12 of the consensus domain sequence bHLH defined by Heim et al., (2003, Mol Biol Evol 20, 735-747; corresponding to positions 123, 127 and 131, and 128 and 130, respectively, in SEQ ID NO: 10, see Table 1) are missing or are substituted, preferably by non-similar or non-conservative amino acids, while others mutations in the IND protein are more likely to produce a significant reduction in the biological activity of the IND protein, such as mutations that lead to specific amino acid substitutions, eg, the conserved amino acids listed in Table 1, causing DNA binding less efficient, less efficient dimerization, and / or less efficient regulation of transcription without completely abolishing the biological activity of the encoded IND protein. WO09 / 068313 (claiming priority over European patent application EP 07023052) describes, for example, complete defective mutant IND alleles, in particular ind-a1-ems01, ind-c1-EMS01 and ind-c1-EMS03, which they comprise a nonsense mutation resulting in the production of truncated IND proteins lacking the bHLH domain and complete defective mutant IND alleles, in particular ind-a1-ems05, encoding a mutant IND protein in which the Arg conserved at the position 10 of the consensus bHLH domain is replaced by an aromatic His, whereas the present invention describes partial defective mutant IND alleles, in particular, for example, ind-c1-ems09, encoding a mutant IND protein in which the Ala conserved in the Position 9 of the bHLH consensus domain is replaced by a Thr and indc1-ems04, which encodes a mutant IND protein in which the Arg conserved at position 12 of the bHLH consensus domain is replaced by a Cys.

Nucleic acid molecules can comprise one or more mutations, such as:

- a "nonsense mutation", which is a change in the nucleic acid sequence that results in the substitution of one amino acid for another amino acid;

- a "nonsense mutation" or "TERMINATION codon mutation", which is a change in the nucleic acid sequence that results in the introduction of a premature TERMINATION codon and, therefore, the termination of translation ( resulting in a truncated protein); the plant genes contain the translation termination codons "TGA" (UGA in to Rn), "TAA" (UAA in RNA) and "TAG" (UAG in RNA); thus any substitution, insertion, deletion of nucleotides that results in one of these codons being in the mature mRNA being translated (in-frame) will terminate the translation;

- an "insertion mutation" of one or more amino acids, because one or more codons have been added in the nucleic acid coding sequence;

- a "deletion mutation" of one or more amino acids, because one or more codons have been removed in the nucleic acid coding sequence;

- a "frame shift mutation", which results in the nucleic acid sequence being translated into a different reading frame downstream of the mutation. A frame change mutation can have several causes, such as the insertion, deletion, or duplication of one or more nucleotides.

Table 1 indicates the length of the Arabidopsis IND protein in SEQ ID NO: 10, the DNA encoding the Arabidopsis IND in SEQ ID NO: 9 and the Brassica napus IND-A1 and IND-C1 proteins in s Eq ID NO: 2 and 6 and SEQ ID NO: 4 and 8, respectively; the position of the bHLH domains in the IND-A1 and IND-C1 proteins of Brassica napus as a function of the indicated position of the pfam domain PF00010, smart domain SM00353, prosite domain PS50888 and superfam domain G3D.4.10.280.10 or SSF47459 of the protein Arabidopsis IND according to The Arabidopsis Information Resource (TAIR) database (http://www.arabidopsis.org/; (http://www.arabidopsis.org/; locus At4 g00120.1; SEQ ID NO : 10); the position of the bHLH domains and conserved amino acids in the IND-A1 and IND-C1 proteins of Brassica napus as a function of the indicated position of the bHLH domain and the conserved amino acids in the IND protein of Arabidopsis according to Heim et al ., (2003, Mol Biol Evol 20, 735-747), according to Toledo-Ortiz et al., (2003, Plant Cell 15: 1749-1770), and according to Liljegren et al., (2004, Cell , 116, 843-853); as further described in WO09 / 068313 (which claims priority over European patent application EP 07023052).

Table 1 IND proteins: regions and positions of amino acids (AA)

continuation

Optimal alignment of the Arabidopsis IND (SEQ ID NO: 9) and amino acid (SEQ ID NO: 10) nucleic acid sequences with the IND nucleic acid sequences, in particular the Brassica IND nucleic acid sequences (SEQ ID NO: 1 and 3) and amino acids (SEQ ID NO: 2 and 4) of the present invention, allows determining the positions of the corresponding conserved domains and amino acids in these Brassica sequences (see Table 1 for the IND sequences from Brassica of SEQ ID NO: 1 to 4).

Thus, in one embodiment, partial defective mutant IND nucleic acid sequences are provided that comprise one or more of any of the types of mutations described above. Partial ind defective sequences may comprise one or more stop codon (nonsense) mutations, one or more nonsense mutations and / or one or more frame shift mutations are provided. Any of the above mutant nucleic acid sequences are provided per se (in isolation), such as plants and plant parts that comprise such sequences endogenously. The most preferred ind alleles are described in the tables below herein and the seed deposits of Brassica napus seeds comprising one or more ind alleles have been deposited as indicated.

A nonsense mutation in an IND allele, as used herein, is a mutation in an IND allele whereby one or more translation stop codons are introduced into the coding DNA and the corresponding mRNA sequence of the Corresponding wild type IND allele. The translation stop codons are TGA (UGA in the mRNA), TAA (UAA) and TAG (UAG). Therefore, any mutation (deletion, insertion, or substitution) that leads to the generation of an in-frame termination codon in the coding sequence will result in termination of translation and truncation of the amino acid chain. A partial defective mutant IND allele is described comprising a nonsense mutation in which an in-frame stop codon is introduced into the IND codon sequence by a single nucleotide substitution, such as the CAG to TAG, TGG mutation to TAG, TGG to TGA or CAA to TAA. A partial defective mutant IND allele is described comprising a nonsense mutation in which an in-frame termination codon is introduced into the IND codon sequence by double nucleotide substitutions, such as the CAG to TAA, TGG to TAA mutation , or CGG to TAG or TGA. A partial defective mutant IND allele is described comprising a nonsense mutation in which an in-frame stop codon is introduced into the IND codon sequence by triple nucleotide substitutions, such as the CGG to TAA mutation. The truncated protein lacks the amino acids encoded by the encoding DNA downstream of the mutation (i.e. the C-terminal part of the IND protein) and maintains the amino acids encoded by the encoding DNA upstream of the mutation (i.e. the N-terminal part of the IND protein). A partial defective mutant IND allele is described comprising a nonsense mutation present anywhere against the conserved Leu moiety of the H2 domain (at position 56 in the bHLH consensus domain sequence as described by Heim et al., 2003, see Table 1), so that at least the rest of the preserved Leu is missing. The more truncated the mutant IND protein is compared to the wild-type IND protein, the more the truncation can result in significantly reduced activity of the mutant iNd protein. It is believed that in order for the mutant IND protein to retain some biological activity, it should at least comprise the DNA-binding domain (b). A partial defective mutant IND allele is described comprising a nonsense mutation resulting in a truncated protein of less than about 170 amino acids (lacking the conserved Leu), less than about 150 amino acids (without the H2 domain), less than about 145 amino acids (without the L and H2 domains), or less than about 130 amino acids (without the HLH domain) (see Table 1).

The following tables herein describe a range of possible nonsense mutations in the Brassica napus IND sequences suitable for the invention:

Table 2a Possible termination codon mutations in IND-A1 (SEQ ID NO: 1) Position of Wild Type Position ^ codon Wild Type ^ amino acid

25 74 tgg ^ tag TRP ^ TERMINATION

75 tgg ^ tga TRP ^ TERMINATION

74 + 75 tgg ^ taa TRP ^ TERMINATION

(continuation)

aminoácido aminoácidos nucleótidos mutante mutaWild Type Position Position ^ Codon Type

amino acid nucleotide mutant mutant

57 169 cag ^ tag GLN> TERMINATION 169 + 171 cag ^ taa GLN> TERMINATION

91 271 caa ^ taa GLN> TERMINATION

98 292 cag ^ tag GLN> TERMINATION 292 + 294 cag ^ taa GLN> TERMINATION

122 364 cag ^ tag GLN> TERMINATION 364 + 366 cag ^ taa GLN> TERMINATION

128 382 + 383 cgg ^ tag ARG> TERMINATION 382 + 384 cgg ^ tga ARG> TERMINATION 382 + 383 + 384 cgg ^ taa ARG> TERMINATION

138 412 + 413 cgg ^ tag ARG> TERMINATION 412 + 414 cgg ^ tga ARG> TERMINATION 412 + 413 + 414 cgg ^ taa ARG> TERMINATION

168 502 + 503 cgg ^ tag ARG> TERMINATION 502 + 504 cgg ^ tga ARG> TERMINATION 502 + 503 + 504 cgg ^ taa ARG> TERMINATION

169 505 cag ^ tag GLN> TERMINATION 505 + 507 cag ^ taa GLN> TERMINATION

181 542 tgg ^ tag TRP • TERMINATION

543 tgg ^ tga TRP • TERMINATION 542 + 543 tgg ^ taa TRP • TERMINATION

Table 2b Possible END-codon mutations in IND-C1 (SEQ ID NO: 3) Position of Wild Type Position ^ codon Wild Type ^ amino acid mutant mutant nucleotide amino acids

41 122 tgg ^ tag TRP ^ TERMINATION

123 tgg ^ tga TRP ^ TERMINATION 122 + 123 tgg ^ taa TRP ^ TERMINATION

50 148 caa ^ taa GLN ^ TERMINATION

73 271 cag ^ tag GLN ^ TERMINATION 271 + 272 cag ^ taa GLN ^ TERMINATION

104 310 caa ^ taa GLN ^ TERMINATION

111 331 cag ^ tag GLN ^ TERMINATION 331 + 333 cag ^ taa GLN ^ TERMINATION

135 403 cag ^ tag GLN ^ TERMINATION 403 + 405 cag ^ taa GLN ^ TERMINATION

141 421 + 422 cgg ^ tag ARG ^ TERMINATION 421 + 423 cgg ^ tga ARG ^ TERMINATION 421 + 422 + 423 cgg ^ taa ARG ^ TERMINATION

151 451 + 452 cgg ^ tag ARG ^ TERMINATION 451 + 453 cgg ^ tga ARG ^ TERMINATION 451 + 452 + 453 cgg ^ taa ARG ^ TERMINATION

181 541 + 542 cgg ^ tag ARG ^ TERMINATION 541 + 543 cgg ^ tga ARG ^ TERMINATION 541 + 542 + 543 cgg ^ taa ARG ^ TERMINATION

182 544 cag ^ tag GLN ^ TERMINATION 544 + 546 cag ^ taa GLN ^ TERMINATION

187 559 cag ^ tag GLN ^ TERMINATION 559 + 561 cag ^ taa GLN ^ TERMINATION

191 571 cag ^ tag GLN ^ TERMINATION 571 + 573 cag ^ taa GLN ^ TERMINATION

Obviously, mutations are not limited to those shown in the preceding tables and it is understood that analogous TERMINATION mutations may be present in ind alleles other than those represented in the sequence listing and mentioned in the preceding tables.

A missense mutation in an IND allele, as used herein, is any mutation (deletion, insertion, or substitution) in an IND allele whereby one or more codons are changed in the coding DNA and the sequence of Corresponding mRNA from the corresponding wild-type IND allele, resulting in the replacement of one or more amino acids in the wild-type IND protein by one or more amino acids in the mutant IND protein. In one embodiment, a partial defective mutant IND allele is provided comprising a nonsense mutation resulting in a substitution of a valine moiety (Val) at position 124 of the IND protein in SEQ ID NO: 2 or a sequence essentially similar thereto, in particular for a methionine (Met) moiety, such as the ind-a1-EMS06 allele (Table 3a). In another embodiment, a partial defective mutant IND allele is provided comprising a nonsense mutation resulting in a substitution of a glycine residue (Gly) at position 146 of the IND protein in SEQ ID NO: 2 or a sequence essentially similar to it, in particular by a serine residue (Ser), such as the ind-a1-EMS09 allele (Table 3a). In yet another embodiment, a partial defective mutant IND allele is provided comprising a nonsense mutation resulting in a substitution of an alanine residue (Ala) at position 159 of the IND protein in SEQ ID NO: 2 or a sequence essentially similar thereto, in particular by a valine moiety (Val), such as the ind-a1 -EMS13 allele (Table 3a). In yet another embodiment, a partial defective mutant IND allele is provided comprising a nonsense mutation resulting in a substitution of a threonine residue (Thr) at position 136 of the IND protein in SEQ ID NO: 4 or a sequence essentially similar thereto, in particular by a methionine (Met) residue, such as the ind-c1-EMS08 allele (Table 3b). In a further embodiment, a partial defective mutant IND allele is provided comprising a nonsense mutation resulting in a substitution of an alanine residue (Ala) at position 139 of the IND protein in SEQ ID NO: 4 or a sequence essentially similar thereto, in particular by a threonine residue (Thr), such as the ind-c1-EMS09 allele (Table 3b). In yet a further embodiment, a partial defective mutant IND allele is provided comprising a nonsense mutation resulting in a substitution of an arginine (Arg) residue at position 142 of the IND protein in SEQ ID NO: 4 or a sequence essentially similar thereto, in particular by a cysteine residue (Cys), such as the ind-c1-EMS04 allele (Table 3b). Reference seed comprising the alleles ind-a1-EMS06, ind-a1-EMS09, ind-a1-EMS13, ind-c1-EMS08, ind-c1-EMS09 and ind-c1-EMS04 have been deposited in the NCIMB Limited (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, Scotland, AB21 9YA, UK) on July 7, 2008, with accession number NCIMB 41570, NCIMB 41571, NCIMB 41573, NCIMB 41574 and NCIMB 41575 , respectively.

Table 3a: Nonsense mutations in IND-A1

Position of Wild Type ^ Wild Type Position ^ Amino Acid Number Name SEQ ID: Nucleoti Two Mutant Codon Mutant Amino Acid Allele Deposit

124 370 930 gtg ^ atg VAL ^ MET ind-a1- NCIMB EMS06 41570 146 436 996 ggc ^ agc GLY ^ SER ind-a1- NCIMB EMS09 41571 159 * 476 1036 gcc ^ gtc ALA ^ VAL ind-a1- NCIMB EMS13 41572

Table 3b: Nonsense mutations in IND-C1

Position Position c e Wild type ^ Wild type ^ amino acid Number Name nucleotide amino acids s mutant codon mutant allele SEQ reservoir

136 407 903 acg ^ atg THR ^ MET ind-c1-EMS08 NCIMB 41573 139 * 415 911 gct ^ act ALA ^ THR ind-c1-EMS09 NCIMB 41574 142 * 424 920 cgt ^ tgt ARG ^ CYS ind-c1-EMS04 NCIMB 41575

In another embodiment, a partial defective mutant IND allele is provided comprising a nonsense mutation encoding an IND protein in which one or more of the conserved amino acids indicated above or in Table 1 is / are substituted, such as mutant IND alleles Partial defective is ind-a1-EMS13, ind-c1-EMS04, and ind-c1-EMS09 (indicated with * in Table 3). As described in Heim et al., (2003, Mol Biol Evol 20, 735-747), Toledo-Ortiz et al., (2003, Plant Cell 15: 1749-1770), Liljegren et al., (2004, Cell , 116, 843-853) and WO09 / 068313 (claiming priority over European patent application EP 07023052), some of the conserved amino acids are more crucial to the biological activity of the IND protein than others. Thus, for example, nonsense mutations that result in the replacement of, for example, amino acids at position 5, 9 (for example, ind-c1 -EMS09) and 13 or at positions 10 (for example, ind -a1-EMS05) and 12 (for example, ind-c1-EMS04) of the consensus bHLH domain sequence defined by Heim et al., (cited above) are more likely to result in significantly reduced activity, due to a reduced ability to bind to target DNA of the IND protein. Similarly, nonsense mutations that result in the replacement of, for example, amino acids at positions 16, 20, 23, 27 at helixl or at positions 36, 39, 43, 49 (eg, ind-a1 -EMS13), 53 and 56 in helix2 of the bHLH consensus domain sequence defined by Heim et al., (Cited above) are more likely to result in significantly reduced activity, due to reduced dimerization capacity, of the protein IND.

A partial defective mutant IND allele comprising a usable nonsense mutation is an IND allele comprising a nonsense mutation corresponding to the nonsense mutation in the Arabidopsis partial defective ind-1 alleles (Liljegren et al., 2004, cited above) (see Table 1).

A frame shift mutation in an IND allele, as used herein, is a mutation (deletion, insertion, duplication, and the like) in an IND allele that results in translation of the nucleic acid sequence into a different framework downstream of the mutation.

Amino acid sequences according to the invention

Partially defective mutant IND amino acid sequences (i.e., IND amino acid sequences comprising one or more mutations, resulting in significantly reduced biological activity of the IND protein) of Brassicaceae, particularly of Brassica species, especially Brassica, are provided. napus, but also from other species of Brassica crops. For example, Brassica species comprising an A and / or C genome can encode different IND-A1 or IND-C1 amino acids, which are essentially similar to the new partial defective mutant IND proteins of the present invention. Furthermore, mutagenesis methods can be used to generate mutations in wild-type IND alleles, thereby generating mutant alleles that can encode additional mutant IND proteins, which are essentially similar to the partial defective mutant IND proteins of the present invention. In one embodiment, the mutant IND amino acid sequences are provided within a Brassica plant (ie, endogenously). However, isolated IND amino acid sequences (eg, isolated from the plant or made synthetically), as well as their variants and fragments of any of these, are provided herein.

The amino acid sequences, which are essentially similar to the new partial defective mutant IND proteins of the present invention, can be obtained by replacing amino acids in the partial defective amino acid sequences of the present invention with other amino acids that have similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break a-helical structures or sheet structures p). Conservative substitution tables are well known in the art (see, eg, Creighton (1984) Proteins. W.H. Freeman and Company and Table 4 of the present patent application).

Table 4 Ems of Conserved Amino Acid Substitutions

Novel partial defective mutant IND amino acid sequences have been isolated from wild-type IND-A1 and IND-C1 proteins from Brassica napus. Wild-type IND sequences as described in WO09 / 068313 (which claims priority over European patent application EP 07023052) are represented in SEQ ID NO: 2 and SEQ ID NO: 4, while the new IND sequences Partial defective mutants of these sequences, and sequences essentially similar to these, are described herein below and in the Examples, with reference to wild-type IND sequences. As described above, the Brassica napus wild-type IND proteins are approximately 185-210 amino acids in length and comprise a number of structural and functional domains.

The "IND-A1 amino acid sequences" or the "IND-A1 variant amino acid sequences" according to the invention are amino acid sequences having at least 75%, at least 80%, at least 85%, at least one 90%, at least 95%, 98%, 99% or 100% of sequence identity with SEQ ID NO: 2. These amino acid sequences may also be referred to as "essentially similar" or "essentially identical" to the IND sequences provided in the sequence listing.

The "IND-C1 amino acid sequences" or the "IND-C1 variant amino acid sequences" according to the invention are amino acid sequences having at least 75%, at least 80%, at least 85%, at least one 90%, at least 95%, 96%, 97%, 98%, 99% or 100% of sequence identity with SEQ ID NO: 4 (IND-C1-long) or with SEQ ID NO: 4 from amino acid at position 16 to amino acid at position 210 (IND-C1-short). These amino acid sequences may also be referred to as "essentially similar" or "essentially identical" to the IND sequences provided in the sequence listing.

Thus, the invention provides new partial defective mutant sequences of amino acid sequences. of wild-type functional IND-A1 and IND-C1 proteins, including variants and fragments thereof (as defined below), whereby mutation in the amino acid sequence preferably results in a significant reduction in biological activity of the IND protein compared to the biological activity of the corresponding wild-type IND protein. A significant reduction in the biological activity of the IND protein refers herein to a reduction in the DNA binding activity, dimerization capacity and / or transcriptional activity of the IND protein, such that resistance The shelling of the pod of a plant expressing the mutant IND protein is increased compared to a plant expressing the corresponding wild-type IND protein compared to the shelling resistance of the pod of a corresponding wild-type plant.

Both isolated and endogenous amino acid sequences are provided herein. Fragments of the IND amino acid sequences and the IND variant amino acid sequences defined above are also provided. A "fragment" of an IND amino acid sequence or a variant thereof (as defined) can be of various lengths, such as at least 10, 12, 15, 18, 20, 50, 100, 150, 175, 180 contiguous amino acids of the IND sequence (or of the variant sequence).

Amino acid sequences of functional IND proteins

The amino acid sequences represented in the sequence listing are Brassica napus wild-type functional IND proteins. Therefore, these sequences are endogenous to the Brassica napus plants from which they were isolated. Other Brassica crop species, varieties, breeding lines or wild records can be selected for other functional IND proteins with the same amino acid sequences or variants thereof, as described above.

Furthermore, it is understood that IND amino acid sequences and their variants (or fragments of any of these) can be identified in silico, by selecting amino acid databases for essentially similar sequences. Fragments of amino acid molecules according to the invention are also provided. Fragments include amino acid sequences from the bHLH domain, or smaller fragments that comprise part of the bHLH domain, such as the basic domain or the HLH domain, etc.

Amino acid sequences of mutant IND proteins

The invention provides nucleic acid sequences comprising one or more amino acid deletions, insertions or substitutions relative to the wild-type IND amino acid sequences represented in SEQ ID NO: 2 and 4 of the sequence listing, in which (s) ) mutation (s) in the amino acid sequence result in significantly reduced biological activity, i.e. partial defective biological activity, of the IND protein encoded relative to the wild-type protein, as well as fragments of such molecules of mutant amino acids. Such mutant amino acid sequences can be generated and / or identified using various known procedures, as described above. Again, such amino acid molecules are provided both endogenously and in isolation.

Basically as described above, any mutation in the wild type IND amino acid sequences resulting in an IND protein comprising at least one amino acid insertion, deletion and / or substitution relative to the wild type IND protein it can lead to significantly reduced or no biological activity. However, it is understood that certain mutations in the IND protein are more likely to result in a complete abolition of the biological activity of the IND protein, such as mutations leading to truncated proteins, whereby significant portions of the functional domains are missing , such as the DNA binding domain ("b"), the dimerization domain ("HLH") and / or amino acids that are important in the regulation of transcription (see Table 1), or mutations by which certain crucial amino acid residues within these domains, such as the amino acids Gln (Q), Ala (A) and Arg (R) at positions 5, 9 and 13 or the basic amino acid residues (in particular the Arg (R) residues )) at positions 10 and 12 of the bHLH consensus domain sequence defined by Heim et al., (cited above; corresponding to positions 123, 127 and 131, and 128 and 130, respectively, in SEQ ID NO: 10 , see Table 1) missing or substituted, pr efferent by non-similar or non-conservative amino acids, while other mutations of the protein are more likely to produce a significant reduction in the biological activity of the IND protein, such as mutations that lead to specific amino acid substitutions, for example, conserved amino acids indicated in Table 1, causing less efficient DNA binding, less efficient dimerization, and / or less efficient regulation of transcription without completely abolishing the biological activity of the encoded IND protein.

Partially defective mutant IND proteins are described that comprise one or more deletion or insertion mutations, whereby the deletion (s) or insertion (s) result in a mutant protein having significantly reduced activity in vivo. Such mutant IND proteins are IND proteins in which at least 1, at least 2, 3, 4, 5, 10, 20, 30, 50, 100, 100, 150, 175, 180 or more amino acids are deleted or inserted in comparison with the wild-type IND protein, whereby the deletion (s) or insert (s) result in a mutant protein having significantly reduced activity in vivo.

Partially defective mutant IND proteins are described which are truncated whereby truncation results in a mutant protein having significantly in vivo activity. Such truncated IND proteins are IND proteins that lack functional domains in the C-terminal part of the corresponding wild-type IND protein and that maintain the N-terminal part of the corresponding wild-type IND protein. Therefore, a partial defective mutant IND protein is described which comprises the N-terminal part of the wild-type IND protein corresponding to, but not including, the conserved Leu residue of the H2 domain (at position 56 in the consensus sequence of the bHLH domain as described by Heim et al., 2003, see above). The more truncated the mutant protein is compared to the wild-type protein, the more the truncation can result in significantly reduced activity of the mutant IND protein. It is believed that in order for the mutant IND protein to retain some biological activity, it should at least comprise the DNA-binding domain (b). A partial defective mutant IND protein is described comprising the N-terminal part of the corresponding wild-type IND protein that lacks part or all of the second H domain, and / or lacks part or all of the L domain, and / or lacks part or all of the first domain H (see Table 1).

In yet another embodiment, partial defective mutant IND proteins comprising one or more substitution mutations are provided, whereby the substitution (s) result in a mutant protein having significantly reduced activity in vivo. In one embodiment, a partial defective mutant IND protein is provided comprising a substitution mutation resulting in a substitution of a valine moiety (Val) at position 124 of the IND protein in SEQ ID NO: 2 or a sequence essentially similar thereto, in particular by a methionine (Met) residue, such as the partial defective mutant IND protein encoded by the ind-a1-EMS06 allele (Table 3a). In another embodiment, a partial defective mutant IND protein is provided comprising a substitution mutation resulting in a substitution of a glycine residue (Gly) at position 146 of the IND protein in SEQ ID NO: 2 or a sequence essentially similar thereto, in particular by a serine residue (Ser), such as the partial defective mutant IND protein encoded by the ind-a1-EMS09 allele (Table 3a). In yet another embodiment, a partial defective mutant IND protein is provided comprising a substitution mutation resulting in a substitution of an alanine residue (Ala) at position 159 of the IND protein in SEQ ID NO: 2 or a sequence essentially similar thereto, in particular by a valine moiety (Val), such as the partial defective mutant IND protein encoded by the ind-a1-EMS13 allele (Table 3a). In yet another embodiment, a partial defective mutant IND protein is provided comprising a substitution mutation resulting in a substitution of a threonine residue (Thr) at position 136 of the IND protein in SEQ ID NO: 4 or a sequence essentially similar thereto, in particular by a methionine residue (Met), such as the partial defective mutant IND protein encoded by the ind-c1-EMS08 allele (Table 3b). In a further embodiment, a partial defective mutant IND protein is provided comprising a substitution mutation resulting in a substitution of an alanine residue (Ala) at position 139 of the IND protein in SEQ ID NO: 4 or a sequence essentially similar thereto, in particular by a threonine (Thr) residue, such as the partial defective mutant IND protein encoded by the ind-c1-EMS09 allele (Table 3b). In still a further embodiment, a partial defective mutant IND protein is provided comprising a substitution mutation resulting in a substitution of an arginine (Arg) residue at position 142 of the iNd protein in SEQ ID NO: 4 or a sequence essentially similar thereto, in particular by a cysteine residue (Cys), such as the partial defective mutant IND protein encoded by the ind-c1-EMS04 allele (Table 3b).

In another embodiment, a partial defective mutant IND protein is provided comprising a substitution mutation resulting in the replacement of conserved amino acid residues as noted above or in Table 1, such as the partial defective mutant IND protein encoded by ind-a1-EMS13, indc1-EMS04 or ind-c1-EMS09 (indicated with * in Table 3).

Methods according to the invention

Procedures are described for generating and selecting dehiscent seed plants and cells, parts, seeds and offspring thereof, containing at least one partial allele and / or at least one complete ind defective allele. In particular, procedures are described for generating and selecting Brassica plants comprising at least two IND genes, in particular Brassica napus plants and cells, parts, seeds and offspring thereof, containing at least one partial defective ind allele and / or at least complete at least one of the at least two different IND loci in the genome, for example at least one of the two different loci of the IND-A1 and IND-C1 gene from Brassica, and to distinguish between the presence of ind alleles complete defects, partial defective ind alleles, and wild-type IND alleles in a dehiscent seed plant or part of the plant. Therefore, procedures are described (such as mutagenesis and / or marker assisted selection) to generate and / or identify partial ind defective alleles and / or complete ind defective alleles or dehiscent seed plants or plant parts, comprising said alleles ind and to combine an adequate number of partial defective ind alleles and / or complete ind defective alleles and / or different types of partial defective ind alleles and / or complete ind defective alleles in a single dehiscent seed plant to alter the dehiscence properties of the fruit of the plants, in particular to reduce seed threshing or delay seed threshing until after harvest, while maintaining agronomically relevant threshing capacity of pods.

The complete and partial defective mutant alleles according to the invention can be generated (eg mutagenesis-induced) and / or identified using a variety of methods, which are conventional in the art, eg using PCR-based methods to amplify part or all of of genomic DNA or cDNA.

After mutagenesis, plants are either grown from the treated seeds or regenerated from the treated cells using known techniques. For example, mutagenized seeds can be planted according to conventional cultivation procedures and after self-pollination seeds are formed in plants. Alternatively, duplicate haploid seedlings can be extracted from treated microspore or pollen cells to immediately form homozygous plants, for example, as described by Coventry et al., (1988, Manual for Microspore Culture Technique for Brassica napus. Dep. Crop Sci. Techn. Bull. OAC Publication 0489. Univ. Of Guelph, Guelph, Ontario, Canada). Additional seed that is formed as a result of such self-pollination in the current generation or in a subsequent generation can be harvested and screened for the presence of mutant IND alleles, using techniques that are conventional in the art, eg, reaction-based techniques. polymerase chain (PCR) (amplification of ind alleles) or hybridization-based techniques, eg, Southern blot analysis, BAC library screening, and the like, and / or direct sequencing of ind alleles. To detect the presence of point mutations (called single nucleotide polymorphisms or SNPs) in mutant IND alleles, standard SNP detection methods can be used in the art, for example, techniques based on oligoligation, techniques based on single base extension or techniques based on differences in restriction sites, such as ARADO.

As described above, mutagenization (spontaneous and induced) of a specific wild-type IND allele results in the presence of one or more deleted, inserted or substituted nucleotides (hereinafter called "mutation region") in the allele Resulting mutant IND. The mutant IND allele can thus be characterized by the location and configuration of one or more deleted, inserted or substituted nucleotides in the wild-type IND allele. The site in the wild-type IND allele where one or more nucleotides, respectively, have been inserted, deleted or replaced herein, is also referred to as a "region or mutation sequence". A "5 'or 3' flanking region or sequence" as used herein refers to a region or DNA sequence in the mutant IND allele (or the corresponding wild type) of at least 20 bp, preferably at least 50 bp, at least 750 bp, at least 1500 bp, and up to 5000 bp of DNA other than DNA containing the one or more deleted, inserted, or substituted nucleotides, preferably DNA from the mutant IND allele (or the corresponding wild type) found immediately upstream and contiguous with (flanking region or 5 '"sequence) or immediately downstream and contiguous with (" 3' flanking region or sequence) the mutation region in the mutant IND allele (or in the wild-type IND allele A "binding region" as used herein refers to a region of DNA in the mutant IND allele (or the corresponding wild type) where the mutation region and the 5 'or 3' flanking region are linked together. A "sequence cia spanning the junction region between the mutation region and the 5 'or 3' flanking region "thus comprises a mutation sequence as well as the flanking sequence contiguous thereto.

The tools developed to identify a specific mutant IND allele or the plant or plant material comprising a specific mutant IND allele, or products comprising plant material comprising a specific mutant IND allele are based on the specific genomic characteristics of the specific mutant IND allele in comparison to the genomic characteristics of the corresponding wild-type IND allele, such as a genomic region-specific restriction map comprising the mutation region, molecular markers, or the sequence of the flanking and / or mutation regions.

Once a specific mutant IND allele has been sequenced, primers and probes can be developed that specifically recognize a sequence within the 5 'flanking, 3' flanking and / or mutational regions of the mutant IND allele in nucleic acid ( DNA or RNA) from a sample using a molecular biology technique. For example, a PCR procedure can be developed to identify the mutant IND allele in biological samples (such as plant samples, plant material, or products comprising plant material). Such PCR relies on at least two specific "primers": one recognizes a sequence within the 5 'or 3' flanking region of the mutant IND allele and the other recognizes a sequence within the 3 'or 5' flanking region of the IND allele mutant, respectively; or one that recognizes a sequence within the 5 'or 3' flanking region of the mutant IND allele and the other that recognizes a sequence within the mutation region of the mutant IND allele; or one that recognizes a sequence within the 5 'or 3' flanking region of the mutant IND allele and the other recognizes a sequence that spans the junction region between the 3 'or 5' flanking region and the mutation region of the mutant IND allele specific (as described below), respectively.

The primers preferably have a sequence of between 15 and 35 nucleotides that under optimized PCR conditions "specifically recognize" a sequence within the 5 'or 3' flanking region, a sequence within the mutation region, or a sequence encompassing the junction region between the flanking and 3 'or 5' mutation regions of the specific mutant IND allele, such that a specific fragment ("mutant IND specific fragment" or discriminating amplicon) is amplified from a nucleic acid sample comprising the specific mutant IND allele. This means that only the directed mutant IND allele, and no other sequences in the plant genome, are amplified under optimized PCR conditions.

The suitable PCR primers for the invention can be the following:

- oligonucleotides ranging in length from 17 nt to approximately 200 nt, comprising a nucleotide sequence of at least 17 consecutive nucleotides, preferably 20 consecutive nucleotides selected from the 5 'or 3' flanking sequence of a specific mutant or complementary IND allele of the itself (i.e., for example, the 5 'or 3' sequence flanking one or more deleted, inserted or substituted nucleotides in the mutant iNd alleles of the invention, such as the 5 'or 3' sequence flanking nonsense mutations , missense or frame shift described above or the 5 'or 3' sequence flanking the END codon mutations indicated in the Tables above or the substitution mutations indicated above or complementary thereto (primers that recognize flanking sequences 5 '); or

- oligonucleotides ranging in length from 17 nt to approximately 200 nt, comprising a nucleotide sequence of at least 17 consecutive nucleotides, preferably 20 nucleotides selected from or complementary to the sequence of the mutation region of a specific mutant IND allele. (ie, for example, the nucleotide sequence inserted or substituted in the IND genes of the invention or the complementary thereof (primers that recognize mutation sequences).

Primers can, of course, be longer than the 17 consecutive nucleotides mentioned, and can, for example, be 18, 19, 20, 21, 30, 35, 50, 75, 100, 150, 200 nt long or even plus. The primers may consist entirely of a nucleotide sequence selected from the mentioned nucleotide sequences of flanking and mutation sequences. However, the nucleotide sequence of the primers at their 5 'end (ie outside the 17 consecutive nucleotides located 3') is less crucial. Thus, the 5 'sequence of the primers may consist of a nucleotide sequence selected from the flanking or mutation sequences, as appropriate, but may contain several (eg, 1,2, 5, 10) mismatches. The 5 'sequence of the primers may even consist entirely of a nucleotide sequence unrelated to the flanking or mutation sequences, such as, for example, a nucleotide sequence representing restriction enzyme recognition sites. Said unrelated sequences or flanking Dn sequences with mismatches should preferably not exceed 100, more preferably not more than 50 or even 25 nucleotides.

Furthermore, suitable primers may comprise or consist of a nucleotide sequence spanning the junction region between flanking and mutation sequences (i.e., for example, the junction region between a 5 'or 3' sequence flanking one or more nucleotides deleted, inserted or substituted in the mutant IND alleles of the invention and the sequence of one or more nucleotides inserted or substituted or the sequence 3 'or 5', respectively, flanking one or more nucleotides deleted, such as the region of junction between a 5 'or 3' sequence flanking nonsense, missense, or frame shift mutations in the IND genes of the invention described above and the sequence of nonsense, nonsense, or frame shift mutations or the junction region between a 5 'or 3' sequence flanking a possible TERMINATION codon mutation as outlined in the Tables above or substitution mutations in and the sequence of the potential END codon mutation or substitution mutations, respectively), provided that the nucleotide sequence is not derived exclusively from the mutation region or the flanking regions.

It will also be immediately clear to the person skilled in the art that properly selected PCR primer pairs should also not comprise sequences complementary to each other.

For purposes of the present invention, the "complementary to a nucleotide sequence depicted in SEQ ID No: X" is the nucleotide sequence that can be derived from the depicted nucleotide sequence by replacing nucleotides through their complementary nucleotide accordingly with the Chargaff rules (A ^ T; G ^ C) and reading the sequence in the 5 'to 3' direction, that is, in the opposite direction to the represented nucleotide sequence.

Examples of primers suitable for identifying specific mutant IND alleles are described in the examples.

As used herein, "the nucleotide sequence of SEQ ID No. Z from position X to position Y" indicates the nucleotide sequence that includes both nucleotide endpoints.

Preferably, the amplified fragment is between 50 and 1,000 nucleotides in length, such as between 50 and 500 nucleotides in length, or between 100 and 350 nucleotides in length. Specific primers can have a sequence that is between 80 and 100% identical to a sequence within the 5 'or 3' flanking region, to a sequence within the mutation region, or to a sequence spanning the binding region between the flanking and 3 'or 5' mutation regions of the specific mutant IND allele, provided the mismatches still allow specific identification of the specific mutant IND allele with these primers under optimized PCR conditions. However, the range of allowable mismatches can be easily determined experimentally and is known to one of skill in the art.

Detection and / or identification of a "mutant IND specific fragment" can occur in several ways, for example, by estimating size after gel or capillary electrophoresis or by fluorescence-based detection procedures. Mutant IND specific fragments can also be directly sequenced. Other sequence specific procedures for detecting amplified DNA fragments are also known in the art.

Standard PCR protocols are described in the art, such as in the "PCR Applications Manual" (Roche Molecular Biochemicals, 2nd edition, 1999) and other references. Optimal PCR conditions, including the sequence of specific primers, are specified in a "PCR identification protocol" for each specific mutant IND allele. However, it is understood that a number of parameters in the PCR identification protocol may need to be adjusted to specific laboratory conditions, and may be slightly modified to obtain similar results. For example, the use of a different procedure for DNA preparation may require adjustment of, for example, the amount of primers, polymerase, MgCh concentration, or the hybridization conditions used. Similarly, selection of other primers may dictate other optimal conditions for the PCR identification protocol. However, these adjustments will be apparent to a person skilled in the art, and are further detailed in current PCR application manuals, such as the one cited above.

Examples describe PCR identification protocols to identify specific mutant IND alleles.

Alternatively, specific primers can be used to amplify a specific fragment of mutant IND that can be used as a "specific probe" to identify a specific mutant IND allele in biological samples. Contact with the nucleic acid of a biological sample, with the probe, under conditions that allow hybridization of the probe with its corresponding fragment in the nucleic acid, results in the formation of a nucleic acid / probe hybrid. The formation of this hybrid can be detected (eg, nucleic acid labeling or probe), whereby the formation of this hybrid indicates the presence of the specific mutant IND allele. Such identification procedures based on hybridization with a specific probe (either in a solid phase vehicle or in solution) have been described in the art. The specific probe is preferably a sequence that, under optimized conditions, specifically hybridizes to a region within the 5 'or 3' flanking region and / or within the mutation region of the specific mutant IND allele (hereinafter " specific region of mutant IND "). Preferably, the specific probe comprises a sequence of between 10 and 1000 bp, 5th and 600 bp, between 100 and 500 bp, between 150 and 350 bp, which is at least 80%, preferably between 80 and 85%, more preferably between 85 and 90%, especially preferably between 90 and 95%, most preferably between 95% and 1003 identical (or complementary) to the nucleotide sequence of a specific region. Preferably, the specific probe will comprise a sequence of from about 13 to about 100 contiguous nucleotides identical (or complementary) to a specific region of the specific mutant IND allele.

Specific probes suitable for the invention may be as follows:

- oligonucleotides ranging in length from 13 nt to approximately 1000 nt, comprising a nucleotide sequence of at least 13 consecutive nucleotides selected from the 5 'or 3' flanking sequence of a specific mutant IND allele or its complement (i.e. , eg, the 5 'or 3' sequence flanking one or more deleted, inserted or substituted nucleotides in the mutant IND alleles of the invention, such as the 5 'or 3' sequence flanking nonsense mutations, sense mutations frame shift or frame shift described above or the 5 'or 3' sequence flanking the possible END codon mutations indicated in the Tables above or the substitution mutations indicated above), or a sequence that has at least 80% sequence identity (probes that recognize 5 'flanking sequences); or

- oligonucleotides ranging in length from 13 nt to approximately 1000 nt, comprising a nucleotide sequence of at least 13 consecutive nucleotides selected from or complementary to the mutation sequence of a specific mutant IND allele (i.e., for example, the nucleotide sequence inserted or substituted in the IND genes of the invention, or the complementary thereof), or a sequence that has at least 80% sequence identity with them (probes that recognize mutation sequences).

Probes may consist entirely of a nucleotide sequence selected from the mentioned nucleotide sequences of flanking and mutation sequences. However, the nucleotide sequence of the probes at their 5 'or 3' ends is less crucial. Thus, the 5 'or 3' sequences of the probes may consist of a nucleotide sequence selected from the flanking or mutation sequences, as appropriate, but may consist of a nucleotide sequence unrelated to the flanking or mutation sequences. . Said unrelated sequences preferably should not have a length greater than 50, more preferably not greater than 25 or even not greater than 20 or 15 nucleotides.

Furthermore, suitable probes may comprise or consist of a nucleotide sequence spanning the junction region between the flanking and mutation sequences (i.e., for example, the junction region between a 5 'or 3' sequence that flanks one or more deleted, inserted or substituted nucleotides in the mutant IND alleles of the invention and the sequence of one or more inserted or substituted nucleotides or the 3 'or 5' sequence, respectively, flanking one or more deleted nucleotides, such as the junction region between a 5 'or 3' sequence flanking nonsense, missense, or frame shift mutations in the IND genes of the invention described above and the sequence of nonsense, missense, or frame shift mutations frame, or the junction region between a 5 'or 3' sequence flanking a possible END codon mutation as outlined in the Tables above or substitution mutations indicated adas above and the sequence of the potential codon TERMINATION or substitution mutation, respectively), provided that the mentioned nucleotide sequence does not derive exclusively from the mutation region or from the flanking regions.

Examples describe specific probes suitable for identifying specific mutant IND alleles.

Detection and / or identification of a "specific mutant IND region" that hybridizes to a specific probe can occur in several ways, for example, by estimating size after gel electrophoresis or by fluorescence-based detection procedures. Other sequence-specific methods for detecting a "mutant IND specific region" that hybridizes with a specific probe are also known in the art.

Alternatively, plants or plant parts that comprise one or more ind mutant alleles can be generated and identified using other procedures, such as the "Delete-a-gene ™" method, which uses PCR to detect deletion mutants generated by mutagenesis. neutron scans (reviewed by Li and Zhang, 2002, Funct Integr Genomics 2: 254-258), using the TILLING (Targeting Induced Local Lesions IN Genomes) procedure that identifies point mutations induced by EMS using denaturing high performance liquid chromatography (DHPLC) to detect base pair changes by heteroduplex analysis (McCallum et al., 2000, Nat Biotech 18: 455 and McCallum et al., 2000, Plant Physiol. 123, 439-442) , etc. As mentioned, TILLING uses high throughput screening for mutations (eg using Cel 1 cleavage of mutant wild-type DNA heteroduplex and detection using a sequencing gel system). Therefore, the use of TILLING to identify plants or plant parts comprising one or more ind mutant alleles and methods for generating and identifying such plants, plant organs, tissues and seeds are encompassed herein. Therefore, a procedure is described that comprises the steps of mutagenizing plant seeds (for example, EMS mutagenesis), grouping of plant or DNA individuals, PCR amplification of a region of interest, heteroduplex formation and high-yield detection, identification of the mutant plant, sequencing of the mutant PCR product. It is understood that other methods of mutagenesis and selection can also be used to generate such mutant plants.

Instead of inducing mutations in IND alleles, natural (spontaneous) mutant alleles can be identified by procedures known in the art. For example, ECOTILLING (Henikoff et al., 2004, Plant Physiology 135 (2): 630-6) can be used to detect a plurality of plants or plant parts with respect to the presence of natural mutant ind alleles. Regarding the previous mutagenesis techniques, Brassica species that comprise an A and / or C genome are preferably selected, so that the ind identified allele can subsequently be introduced into other Brassica species, such as Brassica napus, by crossing (interbreeding or intraspecific) and selection. At ECOTILLING, natural polymorphisms in breeding lines or related species are selected using the TILLING methodology described above, in which individuals or groups of plants are used for PCR amplification of the ind target, heteroduplex formation, and high throughput analysis . This can be followed by selecting individual plants that have a required mutation that can be used later in a breeding program to incorporate the desired mutant allele.

The identified mutant alleles can be sequenced and the sequence can be compared to the wild type allele to identify the mutation (s). Optionally, the functionality can be tested as stated above. Using this approach, a plurality of ind mutant alleles (and Brassica plants comprising one or more of these) can be identified. The desired mutant alleles can be combined with the desired wild-type alleles by breeding and selection procedures as described below. Finally, a single plant is generated comprising the desired number of mutant inds and the desired number of wild-type IND alleles.

Oligonucleotides suitable as PCR primers or specific probes for detection of a specific mutant IND allele can also be used to develop procedures for determining the zygosity status of the specific mutant IND allele.

A PCR-based assay can be developed to determine the presence of a corresponding specific mutant wild type and / or mutant IND allele:

To determine the zygosity status of a specific mutant IND allele, two primers that specifically recognize the wild-type IND allele can be designed such that they are directed toward each other and have the mutation region located between the primers. These primers can be primers that specifically recognize the 5 'and 3' flanking sequences, respectively. This set of primers enables simultaneous diagnostic PCR amplification of the mutant as well as the corresponding wild-type IND allele.

Alternatively, to determine the zygosity status of a specific mutant IND allele, two primers that specifically recognize the wild-type IND allele can be designed such that they are directed toward each other and that one of them specifically recognizes the mutation region. . These primers can be primers that specifically recognize the sequence of the 5 'or 3' flanking region and the mutation region of the wild-type IND allele, respectively. This primer set, along with a third primer that specifically recognizes the sequence of the mutation region in the mutant IND allele allows simultaneous diagnostic PCR amplification of the mutant IND gene as well as the wild-type IND gene.

Alternatively, to determine the zygosity status of a specific mutant IND allele, two primers that specifically recognize the wild-type IND allele can be designed such that they are directed toward each other and that one of them specifically recognizes the binding region. between the 5 'or 3' flanking region and the mutation region. These primers can be primers that specifically recognize the 5 'or 3' flanking sequence and the junction region between the mutation region and the 3 'or 5' flanking region of the wild-type IND allele, respectively. This set of primers, together with a third primer that specifically recognizes the junction region between the mutation region and the 3 'or 5' flanking region of the mutant IND allele, respectively, allow simultaneous diagnostic PCR amplification of the mutant IND gene. , as well as the wild type IND gene.

Alternatively, the zygosity status of a specific mutant IND allele can be determined using alternative primer sets that specifically recognize wildtype and mutant iNd alleles.

If the plant is homozygous for the mutant IND gene or the corresponding wild-type IND gene, the PCR diagnostic assays described above will result in a single typical PCR product, preferably typical in length, for either the mutant or the IND allele. wild type. If the plant is heterozygous for the mutant IND allele, two specific PCR products will appear, reflecting both the amplification of the mutant and the wild type IND allele.

Identification of IND wild type and mutant specific PCR products can occur by estimation of size after gel or capillary electrophoresis (eg, for mutant IND alleles comprising a number of inserted or deleted nucleotides resulting in a size difference between the wild type amplified fragments and the mutant IND allele, such that said fragments can be visibly separated in a gel); by evaluating the presence or absence of the two different fragments after gel or capillary electrophoresis, whereby the diagnostic PCR amplification of the mutant IND allele can optionally be performed separately from the PCR diagnostic amplification of the IND allele wild type; by direct sequencing of the amplified fragments; or by fluorescence-based detection procedures.

Examples of suitable primers for determining the zygosity of specific mutant IND alleles are described in the Examples.

Alternatively, to determine the zygosity status of a specific mutant IND allele, a PCR-based assay can be developed to determine the presence of a specific mutant and / or corresponding wild-type IND allele:

To determine the zygosity status of a specific mutant IND allele, two specific probes that recognize the wild-type IND allele can be designed such that one of them specifically recognizes a sequence within the wild-type IND allele and that the mutation region it is located between the sequences recognized by the probes. These probes can be probes that specifically recognize the 5 'and 3' flanking sequences, respectively. The use of one or, preferably, the use of one or both probes, as well as the corresponding wild-type IND allele.

Alternatively, to determine the zygosity status of a specific mutant IND allele, two specific probes that recognize the wild type IND allele can be designed such that one of them specifically recognizes a sequence within the upstream IND wild type allele and downstream of the mutation region, preferably upstream of the mutation region, and one of them specifically recognizes the mutation region. These probes can be probes that specifically recognize the sequence of the 5 'or 3' flanking region, preferably the 5 'flanking region and the wild-type IND allele mutation region, respectively. The use of one or, preferably, both probes, optionally, together with a third probe that specifically recognizes the sequence of the mutation region in the mutant IND allele, allows for diagnostic hybridization of the mutant and the wild-type IND gene.

Alternatively, to determine the zygosity status of a specific mutant IND allele, a specific probe that recognizes the wild-type IND allele can be designed such that the probe specifically recognizes the junction region between the 5 'or 3' flanking region. , preferably the 5 'flanking region and the wild-type IND allele mutation region. This probe, optionally, together with a second probe that specifically recognizes the junction region between the 5 'or 3' flanking region, preferably the 5 'flanking region and the mutant region of the mutant IND allele, allows for diagnostic hybridization of the mutant and of the wild-type IND gene.

Alternatively, the zygosity status of a specific mutant IND allele can be determined using alternative probe sets that specifically recognize wild type and mutant IND alleles.

If the plant is homozygous for the mutant IND gene or the corresponding wild-type IND gene, the diagnostic hybridization assays described above will result in a single specific hybridization product, such as one or more hybridization (restriction) DNA fragments, typical, preferably typical in length, neither for the mutant or the wild-type IND allele. If the plant is heterozygous for the mutant IND allele, two products of specific hybridization, reflecting both the mutant hybridization and the wild-type IND allele.

Identification of IND wild type and mutant specific PCR products can occur by estimation of size after gel or capillary electrophoresis (eg, for mutant IND alleles comprising a number of inserted or deleted nucleotides resulting in a size difference between wild-type hybridizing (restriction) DNA fragments and the mutant IND allele, such that such fragments can be visibly separated on a gel); by evaluating the presence or absence of the two different specific hybridization products after gel or capillary electrophoresis, whereby the diagnostic hybridization of the mutant iNd allele can optionally be performed separately from the diagnostic hybridization of the type IND allele wild; by direct sequencing of the DNA hybridization fragments (restriction); or by fluorescence-based detection procedures.

Examples of suitable probes to determine the zygosity of specific mutant IND alleles are described in the examples.

Likewise, detection procedures specific for a specific mutant IND allele that differ from PCR-based amplification or hybridization procedures can also be developed using the specific sequence information of the specific mutant IND allele provided herein. Such alternative detection methods include linear signal amplification detection methods based on invasive cleavage of particular nucleic acid structures, also known as Invader ™ technology, (as described, for example, in US Patent 5,985,557. "Invasive Cleavage of Nucleic Acids", 6,001,567 "Detection of Nucleic Acid sequences by Invader Directed Cleavage), RT-PCR-based detection procedures, such as Taqman, or other detection procedures, such as SNPlex, Single Base Extension (SBE ), and the like Briefly, in Invader ™ technology, the target mutation sequence may, for example, hybridize to a first labeled nucleic acid oligonucleotide comprising the nucleotide sequence of the mutation sequence or a sequence spanning the region of junction between the 5 'flanking region and the mutation region and with a second nucleic acid oligonucleotide comprising the sequence 3 'flanking immediately downstream and adjacent to the mutation sequence, in which the first and second oligonucleotides overlap by at least one nucleotide. The duplex or triplex structure produced by this hybridization allows selective cleavage of the probe with an enzyme (Cleavase®) that leaves the target sequence intact. The cleaved labeled probe is subsequently detected, potentially through an intermediate step that results in further signal amplification.

A "kit", as used herein, refers to a set of reagents for the purpose of carrying out the method of the invention, more particularly, the identification of a specific mutant IND allele in biological samples or the determination of the zygosity state of plant material comprising a specific mutant IND allele. More particularly, a preferred embodiment of the kit of the invention comprises at least two specific primers, as described above, for the identification of a specific mutant IND allele, or at least two or three specific primers for the determination of zygosity status. Optionally, the kit may further comprise any other reagents described herein in the PCR identification protocol. Alternatively, in accordance with another embodiment of the present invention, the kit may comprise at least one specific probe, which hybridizes specifically to nucleic acid from biological samples to identify the presence of a specific mutant IND allele therein, for identification of a specific mutant IND allele, or at least two or three specific probes for zygosity determination. The kit may further comprise any other reagents (such as, but not limited to, hybridization buffer, tag) for identification of a mutant IND allele. specific in biological samples, as described above, for the identification of a specific mutant IND allele, or at least two or three specific probes for the determination of zygosity status. Optionally, the kit may further comprise any other reagent (such as, but not limited to, buffer, hybridization marker) for the identification of a specific mutant IND allele in biological samples, using the specific probe.

The kit of the invention can be used and its components can be specifically adjusted, for quality control purposes (eg purity of seed lots), detection of the presence or absence of a specific mutant IND allele in plant material or material that includes or derives from plant material, such as, among others, food or food products.

By the term "primer" as used herein, we mean any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template dependent procedure, such as PCR. Primers are generally 10 to 30 nucleotide oligonucleotides, but longer sequences can be used. Primers can be provided in double-stranded form, although single-stranded form is preferred. Probes can be used as primers, but are designed to bind to target DNA or RNA and do not have to be used in an amplification procedure.

The term "recognize" as used herein when referring to specific primers, refers to the fact that specific primers specifically hybridize to a nucleic acid sequence on a specific mutant IND allele under the conditions set forth in the procedure. (such as the conditions of the PCR identification protocol), so the specificity is determined by the presence of positive controls and negatives.

The term "hybridization", as used herein when referring to specific probes, refers to the fact that the probe binds to a specific region in the nucleic acid sequence of a specific mutant IND allele under restricted conditions. standard. Standard stringency conditions as used herein refer to the hybridization conditions described herein or to the conventional hybridization conditions described by Sambrook et al., 1989 (Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, NY) which, for example, can comprise the following steps: 1) immobilize fragments of plant genomic DNA or DNA from the BAC library in a filter, 2) prehybridize the filter for 1 to 2 hours at 65 ° C in 6X SSC, 5X Denhardt's Reagent, 0.5% SDS and 20 | jg / ml denatured carrier DNA, 3) add labeled hybridization probe, 4) incubate for 16-24 hours , 5) wash the filter once for 30 minutes. at 68 ° C in 6X SSC, 0.1% SDS, 6) wash the filter three times (twice for 30 minutes in 30 ml and once for 10 minutes in 500 ml) at 68 ° C in 2 X SSC, 0.1% SDS, and 7) expose the filter for 4 to 48 hours to X-ray film at -70 ° C.

As used herein, a "biological sample" is a sample of a plant, plant material, or product that comprises plant material. The term "plant" is intended to encompass plant tissues, at any stage of maturity, as well as any cells, tissues, or organs taken or derived from such plants, including, without limitation, any seed, leaf, stem, flower, root, cell individual, gamete, cell culture, tissue culture or protoplast. "Plant material" as used herein refers to material that is obtained or derived from a plant. Products that comprise plant material refer to food, feed or other products that are produced using plant material or that may be contaminated by plant material. It is understood that, in the context of the present invention, such biological samples are analyzed for the presence of specific nucleic acids for a specific mutant IND allele, implying the presence of nucleic acids in the samples. Therefore, the methods mentioned herein for identifying a specific mutant IND allele in biological samples, relate to the identification in biological samples of nucleic acids comprising the specific mutant IND allele.

Also described herein is the combination of specific IND alleles in a plant, the transfer of one or more specific mutant IND alleles from one plant to another plant, plants comprising one or more specific mutant IND alleles, the progeny obtained from these plants and for planting cells, plant parts and plant seeds derived from these plants.

A procedure is described for combining an adequate number of partial defective ind alleles and / or complete defective ind alleles and / or different types of partial defective alleles and / or complete defective ind alleles in a single dehiscent seed plant to alter the properties of Dehiscence of the fruit of the plant, in particular to reduce seed threshing or delay seed threshing until after harvest, while maintaining an agronomically relevant threshing capacity of the pods.

In one aspect, a method is provided to alter the dehiscence properties of the fruit, in particular to reduce seed shelling or delay seed shelling until after harvest, while maintaining agronomically relevant threshing capacity at the same time. from pods, from a Brassica plant comprising at least two IND genes, comprising the stages of:

- generate and / or select a Brassica plant comprising at least two IND genes, in which at least two alleles of the at least two IND genes are partial defective ind alleles, as described above, - select a plant with properties altered dehiscence of the fruit, in particular a plant in which the threshing of seeds is reduced or delayed until after harvest, while the pods maintain an agronomically relevant threshing capacity at the same time

wherein said partial defective alleles are generated by mutagenesis.

The Brassica plant that comprises at least two IND genes can be a Brassica napus plant that comprises an IND-A1 gene and an IND-C1 gene. The at least two partial ind defective alleles may be partial defective alleles of the IND-C1 gene.

The method may further comprise the step of generating and / or selecting a Brassica plant comprising at least two IND genes, in which at least two additional alleles of the at least two IND genes are complete defective ind alleles, as has been previously described. The Brassica plant that comprises at least two IND genes can be a Brassica napus plant that comprises an IND-A1 gene and an IND-C1 gene. The at least two partial ind defective alleles may be partial ind defective alleles of the IND-A1 gene and the at least two complete ind defective alleles are complete ind defective alleles of the IND-C1 gene.

Also described is a procedure for making a hybrid Brassica plant or seed that comprises at least two IND genes, in particular a hybrid plant or seed of Brassica napus, in which the dehiscence properties of the fruit of the plant or of the Plant grown from seed are disturbed, in particular where seed shelling is reduced or delayed until after harvest, while the pods maintain at the same time an agronomically relevant threshing capacity, comprising the stages of:

- generating and / or identifying a first plant comprising a first partial defective allele in the homozygous state and a second plant comprising a second partial defective allele in the homozygous state, as described above,

crossing the first and second plants and collecting hybrid F1 seeds from the cross that comprise two partial defective ind alleles of the at least two IND genes.

The first plant may additionally comprise a first complete ind defective allele in the homozygous state and the second plant may additionally comprise a second complete ind defective allele in the homozygous state, as described above, and F1 hybrid seeds comprising two ind defective alleles are collected partial and two complete ind defective alleles of the at least two IND genes.

The possibility of using parent plants comprising an inactivated partial and / or complete allele in the homozygous state to produce hybrid seeds from which plants can be grown showing reduced or delayed seed shelling, while maintaining them at the same time an agronomically relevant threshing capacity of the pods provides advantages over the use of a mother plant comprising two complete ind defective alleles removed in the homozygous state and a mother plant comprising a complete ind defective allele state as described in WO09 / 068313 (claiming priority over European patent application EP 07023052), since both mother plants produce pods that show an agronomically relevant threshing capacity, while the pods of the parent plant comprising two complete ind defective alleles in a homozygous state produce tubular pods from which it is difficult to harvest the seed ace.

The first and second partial defective ind alleles may be the same, so that F1 hybrid seeds are homozygous for a partial defective ind allele. The first and second complete defective ind alleles may be the same, so that F1 hybrid seeds are homozygous for a complete defective ind allele.

Complete defective ind alleles (i.e. IND alleles whose functional expression is completely abolished), such as those described in WO09 / 068313 (which claims priority of European patent application EP 07023052), and / or partial defective ind alleles (i.e. IND alleles whose functional expression is partially abolished) according to the invention, can be combined according to standard culture techniques.

Partial and / or complete ind defective alleles can, for example, be combined with a single dehiscent seed plant by

(a) generate and / or identify two or more plants, each of which comprises one or more partial defective alleles and / or total defective alleles, as described above for the partial and in WO09 / 068313 ( claiming the priority of European patent application Ep 07023052), for complete defective ind alleles,

(b) crossing a first plant comprising one or more selected partial and / or complete ind defective alleles with a second plant comprising one or more other selected partial and / or complete ind defective alleles, collecting F1 seeds from the cross and, optionally, identifying an F1 plant comprising one or more selected partial and / or complete ind defective alleles from the first plant with one or more selected partial and / or complete ind defective alleles and ind from the second plant, as described above,

(c) optionally, repeat step (b) until obtaining an F1 plant that comprises all partial and / or complete defective alleles,

d) optionally,

- identify an F1 plant, which is homozygous or heterozygous for a selected partial and / or complete ind defective allele by determining the zygosity status of the mutant IND alleles, as described above for the partial and in WO09 / 068313 (which claims the priority of European patent application EP 07023052), for complete defective ind alleles, or

- generate plants that are homozygous for one or more of the alleles indicating partial and / or total elimination selected by one of the following steps:

- extraction of duplicate haploid plants from treated microspore or pollen cells of F1 plants comprising one or more partial and / or complete ind defective alleles, as described above, - self-fertilization of F1 plants comprising the one or more ind alleles Partial and / or complete defectives selected for one or more generations (y), collect the F1 Sy seeds from the self-fertilizations and identify the F1 Sy plants, which are homozygous for the one or more partial and / or complete defective ind alleles, such as described above.

Partial and / or complete ind defective alleles can, for example, be transferred from one dehiscent seed plant to another by

(a) generate and / or identify a first plant comprising one or more selected partial or complete ind defective alleles, as described above (wherein the first plant is homozygous or heterozygous for the one or more partial ind defective alleles and / or complete), as described above (in which the first plant is homozygous or heterozygous for the one or more partial and / or total ind defective alleles), (b) crossing the first plant comprising the one or more partial and / or complete ind defective alleles with a second plant that does not comprise the one or more partial and / or complete ind defective alleles, collect F1 seeds from the cross (where the seeds are heterozygous for a partial and / or complete ind defective allele if the first plant was homozygous for that partial and / or complete ind defective allele and in which half of the seeds are heterozygous and half of the seeds are azigas for, that is, they do not understand, a partial and / or complete ind defective allele if the first plant was heterozygous for that partial and defective ind allele and / or complete, and optionally identifying F1 plants comprising one or more selected partial or complete ind defective alleles, as described above,

(c) backcross F1 plants comprising one or more selected partial and / or complete ind defective alleles with the second plant that does not comprise one or more selected partial and / or complete ind defective alleles for one or more generations (x), collect BCx seeds from the crosses and identify in each generation the BCx plants comprising one or more selected partial or complete ind defective alleles, as described above,

d) optionally, generate Bcx plants that are homozygous for one or more of the partial and / or complete ind defective alleles selected by one of the following steps:

- extracting duplicate haploid plants from treated microspore or pollen cells from BCx plants comprising one or more partial and / or complete ind defective alleles, as described above, - self-fertilize BCx plants comprising one or more partial ind defective alleles and / or complete for one or more generations (y), collect BCx Sy seeds from the self-fertilizations and identify BCx Sy plants, which are homozygous for the one or more desired partial and / or complete ind defective alleles, as has been previously described.

The first and second dehiscent seed plants may be Brassicaceae plants, particularly Brassica plants, especially Brassica napus plants or plants from other Brassica crop species. Alternatively, the first plant may be a Brassicaceae plant, particularly a Brassica plant, especially a Brassica napus plant or a plant from another Brassica crop species, and the second plant may be a plant from a Brassicaceae breeding line. , particularly from a Brassica culture line, especially from a Brassica napus breeding line or from a breeding line of another Brassica crop species. "Breeding line", as used herein, is a plant line, preferably homozygous, that is distinguished from other plant lines by a preferred genotype and / or phenotype that is used to produce hybrid offspring.

SEQUENCES

SEQ ID NO: 1: DNA encoding the IND-A1 gene encoding a wild-type IND-A1 protein from Brassica napus.

SEQ ID NO: 2: Wild-type IND-A1 protein encoded by SEQ ID NO: 1.

SEQ ID NO: 3: DNA encoding the IND-C1 gene encoding a wild-type IND-C1 protein from Brassica napus.

SEQ ID NO: 4: Wild-type IND-C1 protein encoded by SEQ ID NO: 3.

SEQ ID NO: 5: Genomic DNA of the IND-A1 gene encoding a wild-type IND-A1 protein from Brassica napus. SEQ ID NO: 6: Wild-type IND-A1 protein encoded by SEQ ID NO: 5.

SEQ ID NO: 7: Genomic DNA of the IND-C1 gene encoding a wild-type IND-C1 protein from Brassica napus. SEQ ID NO: 8: Wild-type IND-C1 protein encoded by SEQ ID NO: 7.

SEQ ID NO: 9: DNA encoding the Arabidopsis IND1 gene.

SEQ ID NO: 10: Arabidopsis IND1 protein encoded by SEQ ID NO: 9.

SEQ ID NO: 11: Oligonucleotide for the detection of IND-A1-EMS06 and -WT.

SEQ ID NO: 12: Oligonucleotide for the detection of IND-A1-EMS06.

SEQ ID NO: 13: Oligonucleotide for the detection of IND-A1-WT.

SEQ ID NO: 14: Oligonucleotide for the detection of IND-A1-EMS09 and -WT.

SEQ ID NO: 15: Oligonucleotide for the detection of IND-A1-EMS09.

SEQ ID NO: 16: Oligonucleotide for the detection of IND-A1-WT.

SEQ ID NO: 17: Oligonucleotide for the detection of IND-A1-EMS13 and -WT.

SEQ ID NO: 18: Oligonucleotide for the detection of IND-A1-EMS13.

SEQ ID NO: 19: Oligonucleotide for the detection of IND-A1-WT.

SEQ ID NO: 20: Oligonucleotide for the detection of IND-C1-EMS04 and -WT.

SEQ ID NO: 21: Oligonucleotide for the detection of IND-C1-EMS04.

SEQ ID NO: 22: Oligonucleotide for the detection of IND-C1-WT.

SEQ ID NO: 23: Oligonucleotide for detection of IND-C1-EMS08 and -WT.

SEQ ID NO: 24: Oligonucleotide for the detection of IND-C1-EMS08.

SEQ ID NO: 25: Oligonucleotide for the detection of IND-C1-WT.

SEQ ID NO: 26: Oligonucleotide for the detection of IND-C1-EMS09 and -WT.

SEQ ID NO: 27: Oligonucleotide for the detection of IND-C1-EMS09.

SEQ ID NO: 28: Oligonucleotide for the detection of IND-C1-WT.

Unless otherwise indicated in the Examples, all recombinant DNA techniques are carried out according to standard molecular biology techniques as described in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, in volumes 1 and 2 of Ausubel et al., (1994) Current Protocols in Molecular Biology, Current Protocols, USA. and in volumes I and II of Brown (1998) Molecular Biology LabFax, second edition, Academic Press (United Kingdom). Standard materials and procedures for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK. Standard materials and procedures for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany. Standard procedures for AFLP analysis are described in Vos et al., (1995, NAR 23: 4407-4414) and in published EP patent application EP 534858.

Examples

Example 1 - Generation and Isolation of Partial Defective Mutant IND (alleles)

Mutations were generated in the IND genes represented in SEQ ID NO: 1 or 3 and 5 or 7 of the sequence listing and identified as follows:

- 30,000 seeds from an elite oilseed rapeseed breeding line (M0 seeds) were pre-soaked for two hours in wet filter paper in deionized or distilled water. Half of the seeds were exposed to 0.8% EMS and half to 1% EMS (Sigma: M0880) and incubated for 4 hours.

- The mutagenized seeds (M1 seeds) were rinsed 3 times and dried in a fume hood overnight. 30,000 M1 plants were grown in the soil and self-fertilized to generate M2 seeds. M2 seeds were harvested for each individual M1 plant.

- 4,800 M2 plants, derived from different M1 plants, were grown twice and DNA samples were prepared from leaf samples from each individual M2 plant according to the CTAB procedure (Doyle and Doyle, 1987, Phytochemistry Bulletin 19:11 -fifteen).

- DNA samples were analyzed for the presence of point mutations in IND genes that cause amino acid substitution in IND proteins, particularly in the bHLH domain of IND proteins, by direct sequencing using standard sequencing techniques (Agowa) and sequence analysis to detect the presence of point mutations using NovoSNP software (VIB Antwerp). - The partial defective mutant IND (ind) alleles indicated in Tables 3a and b above were thus identified.

In conclusion, the examples above show how partial defective mutant IND alleles can be generated and isolated. Also, plant material comprising such mutant alleles can be used to combine selected mutant IND alleles in a plant, as described in the following examples.

Example 2 - Identification of a Brassica plant comprising a partial defective IND mutant allele of Brassica

Brassica plants comprising the mutations in the IND genes identified in Example 1 were identified as follows:

- For each mutant IND gene identified in the DNA sample of an M2 plant, at least 50 M2 plants derived from the same M1 plant were grown as the M2 plant comprising the IND mutation and DNA samples were prepared from samples of leaves of each individual M2 plant.

- DNA samples were selected to detect the presence of the point IND mutation identified as described above in Example 1.

- Heterozygous and homozygous M2 plants (as determined by electropherograms) comprising the same mutation were self-fertilized and M3 seeds were harvested.

Example 3 - Analysis of the dehiscence properties of the fruit of Brassica plants comprising a partial and / or complete defective mutant Brassica IND gene

To determine the correlation between the presence of partial and / or complete defective mutant IND genes in Brassica plants and the dehiscence properties of the fruit of the Brassica plants, the dehiscence properties of the fruit of the Brassica napus plants comprising a Partially defective mutant IND gene in the homozygous state alone or a complete and partial defective mutant IND gene in the homozygous state were analyzed in the greenhouse and in the field and compared with the dehiscence properties of the fruit of Brassica napus plants comprising 2 to 4 complete defective ind alleles as described in WO09 / 068313 (which claims the priority of European patent application EP 07023052) as follows:

- To examine whether the margins of the fruit leaflet and the dehiscence properties of the seed pods were affected and how they were affected by the presence of partial and / or total defective mutant IND genes, the ind fruit was compared with the wild type fruit using the following macroscopic tests: (a) Inspection of seed pods and plants in general with the naked eye to determine the differences in the phenotype of pods and plants caused by the presence of different partial and / or complete defective mutant IND genes. Determination of the phenotype of the pods: When the pods were fully cultivated and filled, just before yellowing, the degree of sharpness of the area delineating the leaflet and the spur was evaluated in the area where both valves no longer touch ( at the distal end of the sheath) of 5 random sheaths (from different plants if there are several plants per line available) and a score of 1 to 5: 1 was attributed for clear indentation and a fine, sharp area separating the leaflet and the spur; 2 for some indentation and a transparent, although more blurred, area that separates the leaflet from the spur; 3 for leaflets and spur that are still well observable as two different tissues but with a very smooth transition between them; 4 for leaflets and spur that are barely observable as different tissues; 5 for a completely smooth transition between the leaflets and the spur without any clear differentiation between the two types of tissues, that is, the smaller the indentation between the leaflet and the spur at the distal end of the sheaths, the higher the score. A score of 1 (acute indentation between the leaflet and the peak) corresponds to a wild-type phenotype of the pods, more specifically, a pod phenotype sensitive to pod shelling; a score of 2 to 4 (more gradual transition between leaflet and beak) corresponds to a pod shelling resistant pod phenotype, in which seed shelving is significantly reduced or delayed while maintaining threshing ability agronomically relevant of the pods, so that the pods can still be opened along the dehiscence zone by applying limited physical forces; and a score of 5 (without indentation between the leaflet and the spur) corresponds to a pod phenotype resistant to pod shelling, in which seed shelving is reduced or delayed to a degree that no longer allows a agronomically relevant threshing of the pods, so that the pods cannot be opened along the dehiscence zone by applying limited physical forces.

(b) Manual Impact Test (MIT) to determine the increase in resistance to shelling of the sheath caused by the presence of different partial and / or complete defective mutant IND genes: The level of resistance to shelling of the sheath of the lines from Brassica napus comprising a partial defective mutant IND gene in the homozygous state alone or a partial and complete mutant IND gene mutant in the homozygous state and from the Brassica lines comprising the corresponding wild-type IND alleles were compared in a semiquantitative way determining the forces required to open closed mature pods by manually applying torque to the pods. A score of 1 to 5 was attributed to the shelling resistance of the pods based on this physical strength: 1 for pods that open fully along the dehiscence zone with the least twist, 2-4 for pods that open only at the base of the dehiscence zone and need stronger torque to fully open and 5 for sheaths that can only be crushed and do not open along the dehiscence zone.

(c) Random Impact Test (RIT) to determine the increase in resistance to shelling of the sheath caused by the presence of different partial and / or complete defective mutant IND genes: The level of resistance to shelling of the sheath of the lines from Brassica napus comprising a partial defective mutant IND gene in the homozygous state alone or a partial and complete mutant mutant IND gene in the homozygous state and from the Brassica lines comprising the corresponding wild-type IND alleles were quantitatively compared by determining the half-life of pod samples from both lines according to Bruce et al., (2002, cited above,). More specifically, two replicate samples of 20 intact mature pods from each line were subjected to a RIT. 20 pods were placed along with six 12.5mm diameter steel balls in a 20cm diameter cylindrical container with its vertical axis. The container was then subjected to a simple harmonic movement with a frequency of 4.98 Hz and a stroke of 51 mm in the horizontal plane. The pods, in which the strength was checked before the test, were shaken for cumulative times of 10, 20, 40 and, if more than 50% of the pods remain intact, 80 s. The drum was opened after each period and the number of closed pods was counted. The pods were examined and classified as "closed" if the dehiscence zone of both leaflets was still closed. Therefore, the pods were classified as "open" if one or both leaflets had been separated, so that the seed had been released. If most of the pods were broken or damaged without opening the dehiscence zone, the sample was marked as "uncountable" (indicated with * in Table 5b). To give each point equal weight, the data was uniformly separated into the independent variable, time, by adding 1 and taking log- ¹⁰ . The percentage of open pods p was transformed using the logit transformation, that is, logit p = loge (p / 100-p). Next, a linear model was fitted to the transformed time and percentage data, and was used to estimate the half-life.

(d) Field tests to determine the relationship between pod shelling resistance, threshing capacity, and yield and the presence of certain mutant IND alleles in plants: The shelling resistance level of pods, the ability to Threshing and yield of the Brassica lines comprising the mutant IND alleles and the Brassica lines comprising the corresponding wild-type IND alleles were compared semi-quantitatively by determining and comparing the level of seed shelling (SHAT), the capacity of combine harvester (CHA1) and threshing capacity (CHA2) and quantitatively determining and comparing seed yield per plot after combining (YLDP) and seed yield after straw threshing (YLDS) in the field between plots with ind plants and plots with wild type plants. Parcels were assigned a score of 1-9 to indicate the level of seed destruction in the parcel before harvest: a score of 1 to indicate that virtually all plants on the parcel had been shelled prior to harvest at a score of 9 to indicate that virtually no plants on the plot had been shelled prior to harvest. TO the plots were assigned a score of 1-5 to indicate the level of harvesting capacity of the combined combine in the plot: a score of 1, 3 or 5 to indicate that it was difficult, feasible or easy, respectively, to harvest the plot with a combine harvester. The plots were assigned a score of 1-5 to indicate the level of threshing capacity of the plot: a score of 1, 3 or 5 to indicate that it was difficult, feasible or easy, respectively, to manually harvest the seed left in the straw after the combine harvester. Seed yield per plot after combining (YLDP; expressed in grams per plot) was determined by harvesting the seeds per plot with a combine harvester and weighing the seeds and seed yield after cutting the straw (YLDS; expressed in% by weight of the straw) was determined by manually harvesting the remaining seeds in the straw after the seed harvest with the combine harvester.

- To examine more closely whether the cells in the valve margin of the seed pods are affected by the presence of partial and / or complete defective mutant IND genes, the ind fruit sections were compared with wild type fruit sections by microscopic evaluation of the seed pods:

- Explants: Explants of approximately 3 mm taken from the center and distal ends of the sheaths from a similar stage of development (approximately 35 days after anthesis (DAA), a stage of development that closely corresponds to the onset of visible yellowing of the pericarp ) and size were harvested from plants grown in a greenhouse (three pods for each genotype). A dehiscence zone was dissected from the pods.

- Fixation: Fixation was performed in 100 mM K-phosphate buffer at pH 7 with 10% formalin and 0.25% glutaraldehyde for a total of 4 hours. Vacuum infiltration was performed after 1 and 2 hours for 15 minutes. The fixative was renewed after each vacuum infiltration.

- Dehydration: The specimen was rinsed twice 30 minutes with 100 mM K-phosphate buffer at pH 7. Dehydration was performed with technical ethanol diluted with 0.85% NaCl in water: 60 minutes (') in 50% ethanol 90 'in 70% ethanol, 90' in 80% ethanol, 90 'in 90% ethanol, 90' in 95% ethanol, 90 'in 100% ethanol at room temperature.

- Inclusion: Inclusion was performed with Leica 7022-31731 Historesin or Kulzer Histo-Technik 7100 (Heraeus) inclusion kits, which are three-component resin kits (a basic resin, an activator and a hardener). The three components were used in the proportions recommended by the manufacturer as follows: the sample was incubated for 4 hours in 50% ethanol / 50% basic resin, overnight in 30% ethanol / 70% basic resin (optional: at 4 ° C), for 2 to 4 hours in 100% basic resin, for one day in 100% basic resin after renewal of the basic resin and vacuum infiltration for 20 '(optionally at 4 ° C), for one day in basic activator resin (1%) ("infiltration medium") after vacuum infiltration in this medium for 20 minutes. The sample was washed with hardener activator (1%) basic resin (1 ml in 15 ml) ("embedding medium"). The inclusion was performed in flat inclusion molds (AGAR G3531 flat inclusion molds with cavities of approximately 300 pl: 14 mm long x 6 mm wide x 4 mm deep): 100-125 pl of medium / cavity were added. inclusion, the inclusion medium was polymerized at 55 ° C for about one hour, the tissue was placed in the polymerized inclusion medium (1 explant / well), the wells were filled with inclusion medium, the inclusion medium was polymerized for 3-5 hours at 55 ° C, the molds were cooled, the plastic blocks were removed from the molds and stored at room temperature in a sealed container (eg Eppendorf tube). - Sectioning: The plastic blocks were glued with the flat side on a 1 cm3 perpex block and cut into squares around the sample. 4 pm sections (3 to 4 explants per genotype, approximately 25 sections per explant) were cut with a ralph glass knife (made at position -1 of the Reichert-Jung histoknifemaker using 6mm thick glass rods under a cutting angle of approximately 6 °) on the microtome. The sections were joined on Vectabond-treated glass slides (Vector Laboratories).

- Demonstration of lignin: Eukitt-mounted unstained sections were examined using a microscope equipped for fluorescence (with the Zeiss filter set 02). Lignin shows a bluish transparent fluorescence. - Histology evaluation: unstained sections were visualized using DIC-Normski or autofluorescence (with Zeiss 18 filter - Excitation BP390-420; Emission LP450).

Vegetal material:

Progeny of a plant line comprising a complete defective mutation in the IND-A1 gene (indicated as inda1F), in particular the allele ind-a1-EMS01 described in document WO09 / 068313 (which claims priority over the European patent application EP 07023052) (indicated as ind-a1-01 in Table 5) and a partial defective mutation in the IND-C1 gene (indicated as ind-c1P), in particular the alleles ind-c1-EMS04, -EMS08 and -EMS09 indicated in Table 3b (indicated as ind-c1-04, -08 and -09 in Table 5), with genotype ind-a1F / ind-a1F, And-c1PAnd-c1P (i.e., homozygous double mutant plants) , IND-A1 / IND-A1, ind-c1P / md-c1P (i.e. single homozygous mutant plants) and ND-A1 / IND-A1, IND-C1 / IND-C1 (i.e. wild-type plants) .

Progeny of a plant line comprising a partial defective mutation in the IND-A1 gene (indicated as inda1P), in particular, the alleles ind-a1-EMS06, -EMS09 and -EMS13 alleles indicated in Table 3a (indicated as inda1- 06, -09 and -13 in Table 5) and a complete defective mutation in the IND-C1 gene (indicated as ind-c1F), in particular the ind-c1-MS S0l allele and the ind-c1-EMS03 allele described in WO09 / 068313 (which claims priority over European patent application EP 07023052) (indicated as ind-c1-01 and -03 in Table 5), with genotype ind-a1P / ind-a1P, ind-c1F / ind-c1 F (i.e. plants homozygous double mutant), IND-A1 / IND-A1, ind-c1F / indc1F (i.e. single mutant homozygous plants) and IND-A1 / IND-A1, IND-C1 / IND-C1 (i.e. plants of type wild).

Macroscopic evaluation:

a) Inspection of seed pods and plants with the naked eye.

- Homozygous double mutant IND sister plant pods with genotype ind-a1F / ind-a1F, ind-c1P / ind-c1P or ind-a1Plind-a1P, ind-c1F / ind-c1F showed a phenotype similar to those of plants comprising a complete ind defective allele in the homozygous state and a complete ind defective allele in the heterozygous state described in WO09 / 068313 (claiming priority over European patent application EP 07023052) (genotype: ind-a1F / ind- a1F, IND-C1 / ind-c1Fo IND- A1 // nd-a1F, ind-c1F / ind-c1F, where ind-a1F is a complete defective ind-a1 allele, in particular the ind-a1-EMS01 allele or ind-a1-EMS05 described in WO09 / 068313 (which claims priority over European patent application EP 07023052) and in which ind-c1F is a complete defective ind-c1 allele, in particular the ind-c1- allele. EMS01 or ind-c1-EMS03 described in WO09 / 068313 (claiming priority over European patent application EP 07023052)). More specifically, the sheath leaflet margins of these IND mutant sister plants were generally better defined than in the homozygous double mutant IND sister plants described in WO09 / 068313 (which claims priority over European patent application EP 07023052 ) (which showed a lack of leaflet margin definition, particularly evident at both the proximal and distal end of the fruit, compared to wild type IND sister plant pods), but the marked indentation between the leaflet and the spur at the distal end of pods in wild-type sister plants it was still largely absent in these mutant plants as in double homozygous full defective sister plants, which also showed a more gradual transition between the leaflet and the spur tissue (see also the visual score in Table 5a for greenhouse grown plants and in Table 5b for cultivated plants fords in the field).

- Simple homozygous mutant IND sister plant pods (genotype: IND-A1 / IND-A1, ind-c1P / ind-c1P or ind- a1P / ind-a1P, IND-C1 / IND-C1) showed pod morphology similar to wild-type IND sister plant pods, except for homozygous single mutant IND sister plant pods with genotype IND-A1-EMS01 / IND-A1-EMS01, ind-c1- EMS09 / ind-c1-EMS09, that showed an altered pod morphology similar to the pods of the double homozygous mutant IND sister plants with genotype ind-a1F / ind-a1F, indc1P / ind-c1P or ind-a1P / ind-a1P, ind-c1F / ind- c1F (see also visual score in Table 5a for greenhouse grown plants and Table 5b for field grown plants). It was also observed that the presence of the ind-c1-EMS09 allele in a heterozygous state in plants (genotype: IND-Al / IND-A1, IND-C1 / ind-c1-EMs09) was sufficient to cause a similar altered sheath morphology to the pods of the twin sister plants homozygous double mutants with genotype ind-a1F / ind-a1F, ind-c1P / ind-c1P or ind-a1P / ind-a1P, ind-c1F / ind-c1F. It is believed that the ind-c1-EMS09 allele, which comprises a substitution mutation in a conserved amino acid of the basic DNA binding domain, could produce a dominant negative IND protein that is still capable of forming dimers, but is not capable of binding to the bHLH binding site of the regulated gene (s).

b) Random impact test:

- Table 5 shows that the LD50 value was, in general, higher for the plant pods that comprise a complete defective ind-c1 allele in the homozygous state and a partial defective ind-a1 allele in the homozygous state than for the pods of plants comprising a partially defective ind-a1 allele in the homozygous state indicating that mutations in the IND-C 1 allele could have a stronger effect on shelling resistance than pod mutations in the IND-A1 allele.

Table 5a

Genotype Visual score LD50 Lower corrected 95 Upper corrected pods 0 sec% 95%

(continuation)

Genotype Visual score LD50 Corrected lower 95 Corrected upper of the pods (0 (sec)% 95%

10

IND-A1-09 / IND-A1 -09, IND-C1- 0 9.36 2.6 1.7

01 / IND-C1-01

ind-a1 -09 / ind-a1 -09, IND-C1-01 / IND- 1 9.05 2.83 2.15

C1-01

ind-a1-09 / ind-a1-09, ind-c1-01 / ind- 8 52.03 8.96 14.03 c1-01

IND-A1-09 / IND-A1 -09, IND-C1- 0 8,44 2,74 2,26

03 / IND-C1-03

ind-a1 -09 / ind-a1 -09, IND-C1-03 / IND- 0 9.05 2.83 2.15

C1-03

ind-a1-09 / ind-a1-09, ind-c1 -03 / ind- 8.5 85.57 23.34 64.64 c1-03

IND-A1-13 / IND-A1-13, IND-C1- 3 12.91 2.4 2.46

01 / IND-C1-01

ind-a1-13 / ind-a1-13, IND-C1-01 / IND- 2 14,2 2,2 2,59

C1-01

ind-a1-13 / ind-a1-13, ind-c1-01 / ind- 7 61.21 9.6 15.18 c1-01

IND-A1-13 / IND-A1-13, IND-C1- 0 8,86 * *

03 / IND-C1-03

ind-a1-13 / ind-a1-13, IND-C1- 0 7.74 3.98 1.54

03 / IND-C1-03

ind-a1-13 / ind-a1-13, ind-c1 -03 / ind- 9 56.68 8.9 13.6 c1-03

IND-A1-01 / IND-A1-01, IND-C1- 0 7,89 2,88 2

04 / IND-C1-04

IND-A1-01 / IND-A1-01, ind-c1-04 / 0 10.91 2.5 2

ind-c1-04

ind-a1-01 / ind-a1-01, ind-c1-04 / ind- 9 37.8 5.77 8.4

c1-04

IND-A1-01 / IND-A1-01, IND-C1- 0 8.94 2.78 2.38

08 / IND-C1-08

IND-A1-01 / IND-A1-01, ind-c1-08 / 0 9.8 2.8 2.1

ind-c1-08

ind-a1-01 / ind-a1-01, ind-c1-08 / ind- 8, 31.81 6.66 10.45 c1-08

IND-A1-01 / IND-A1-01, IND-C1- 0 7.22 3.56 1.82

09 / IND-C1-09

IND-A1-01 / IND-A1-01, ind-c1-09 / 8.5 46.6 7.82 11.48 ind-c1-09

ind-a1-01 / ind-a1-01, ind-c1-09 / ind- 9 90.11 * *

c1-09

Table 5b

continuation

c) Field tests

Table 5c shows the seed shelling level (SHAT), the combine harvester's harvesting capacity (CHA1), the threshing capacity (CHA2), the seed yield per plot after combining (YLDP) and the yield of Seeds after straw threshing (YLDS) determined as described above for field plots with ind plants and wild type plants as indicated. The YieldWTSeg% value represents the YLDP as a percentage of the wild type segregator within a segregating population.

Table 5c

(continuation)

SHAT genotype (1 CHA1 (1 CHA2 (1 YLDP (in YLDS Yield (in%

9) 5) 5) grams per weight WTSeg% of straw) plot)

md-a1-06 / md-a1-06, imd- c1- 8.7 4.4 4.9 2525.1 112 1.1 01 / imd-c1-01

IND-A1-06 / IND-A1 -06, 8.6 4.6 4.9 2102.0 100 0.5

IND-C1-03 / IND-C1-03

md-a1-06Amd-a1-06, IND- 8.7 4.8 5.0 2292.7 109 0.4

C1-03 / IND-C1-03

md-a1-06 / md-a1-06, imd- c1- 8.8 4.1 4.6 2276.2 108 1.3 03 / md-c1-03

IND-A1-09 / IND-A1 -09, 8.3 5.0 4.9 1964.2 100 0.3

IND-C1-01 / IND-C1-01

imd-a1 -09 / imd-a1 -09, IND- 8.0 4.7 5.0 1872.0 95 0.4

C1-01 / IND-C1-01

md-a1-09 / md-a1-09, imd- c1- 9.0 2.7 3.8 2323.3 118 5.7 01Amd-c1-01

IND-A1-09 / IND-A1 -09, 8.6 4.8 5.0 2168.9 100 0.5

IND-C1-03 / IND-C1-03

imd-a1 -09 / imd-a1 -09, IND- 8.3 4.7 5.0 1985.6 92 0.4

C1-03 / IND-C1-03

imd-a1-09 / imd-a1-09, imd- c1- 9.0 1.9 3.6 1726.7 80 13.6 03 / md-c1-03

IND-A1-13 / IND-A1-13, 8.3 4.8 5.0 1977.1 100 0.4

IND-C1-01 / IND-C1-01

imd-a1-13Amd-a1-13, IND- 8.4 4.2 4.9 1929.3 98 0.5

C1-01 / IND-C1-01

imd-a1-13Amd-a1-13, imd- c1- 8.9 3.6 4.7 2445.6 124 2.0 01Amd-c1-01

IND-A1-13 / IND-A1-13, 8.3 4.2 5.0 1885.1 100 0.4

IND-C1-03 / IND-C1-03

im d-al-13 / im d-al-13, IND- 8.3 4.8 5.0 2137.8 113 0.6

C1-03 / IND-C1-03

imd-a1-13Amd-a1-13, imd- c1- 8.9 2.8 3.9 2120.9 113 4.8 03 / md-c1-03

IND-A1-01 / IND-A1-01, 8.8 4.9 4.8 2120.4 100 0.6

IND-C1-04 / IND-C1-04

IND-A1-01 / IND-A1-01, imd- 8.6 4.8 5.0 2136.4 101 0.6 c1-04 / md-c1-04

imd-a1-01 / md-a1-01, imd- c1- 9.0 1.8 2.8 1437.0 68 19.1 04 / md-c1-04

IND-A1-01 / IND-A1-01, 8.0 4.8 5.0 2250.4 100 0.6

IND-C1-08 / IND-C1-08

IND-A1-01 / IND-A1-01, imd- 8.3 4.3 4.9 2131.3 95 0.5 c1-08 / md-c1-08

imd-a1-01 / md-a1-01, imd- c1- 8.9 3.0 4.1 2385.1 106 2.5 08 / md-c1-08

IND-A1-01 / IND-A1-01, 8.7 4.9 5.0 2080.0 100 0.4

IND-C1-09 / IND-C1-09

IND-A1-01 / IND-A1-01, imd- 8.7 4.6 4.6 2447.8 118 1.0 c1-09 / md-c1-09

imd-a1-01 / md-a1-01, imd- c1- 9.0 1.1 1.8 589.6 28 28.4 09 / md-c1-09

Microscopic evaluation:

- Homozygous double mutant IND sister plant pods with genotype imd-a1F / md-a1F, md-c1PAmd-c1P or ind- a1P / md-a1P, ind-c1F / md-c1F, and single mutant IND sister plant pods homozygotes with the genotype IND- A1-EMS01 / IND-A1-EMS01, md-c1-EMS09 / md-c1-EMS09, grown in greenhouse conditions, showed lignification in the distal end of the dehiscence zone and a poor differentiation of cells belonging to the dehiscence zone of neighboring cell types, such as vascular tissue cells and the lignified cell layer normally found on the inner wall of the sheath (i.e., enb cells). In the center of the sheaths, lignification did not occur throughout the entire dehiscence zone, but the sheaths showed only a few additional layers of lignified cells, instead, where the inner wall of the sheath is attached to the septum.

- Homozygous double mutant IND sister plant pods with genotype ind-a1-EMS01 / ind-aA1-EMS01, ind- c1-EMS09 / ind-c1-EMS09 showed both at their distal ends and in the center of the pods, lignification throughout the dehiscence zone and poor differentiation of cells belonging to the dehiscence zone from neighboring cell types, such as vascular tissue cells and the lignified cell layer normally found on the inner wall of the sheath (i.e. enb cells).

Example 4 - Detection and / or transfer of mutant IND genes in Brassica lines (elite)

Mutant IND genes are transferred to Brassica (elite) breeding lines by the following procedure: A plant containing a mutant IND gene (donor plant) is crossed with a Brassica (elite) line (elite parent / recurring parent) or variety lacking the mutant IND gene. The following introgression scheme is used (the mutant IND gene is abbreviated to ind while the wild type is represented as IND):

Initial crossing: ind / ind (donor plant) X IND / IND (elite father)

Plant F1: IND / ind

Cross BC1: IND / ind X IND / IND (recurring parent)

BC1 plants: 50% IND / ind and 50% IND / IND

50% IND / ind are selected using molecular markers (eg AFLP, PCR, Invader ™, and the like; see also below) for the mutant IND allele (ind).

Crossing BC2: IND / ind (plant BC1) X IND / IND (recurring parent)

BC2 plants: 50% IND / ind and 50% IND / IND

50% IND / ind are selected using molecular markers for the mutant IND (ind) allele.

Backcrossing is repeated to plants BC3 to BC6 BC3-6: 50% IND / ind and 50% IND / IND

50% IND / ind are selected using molecular markers for the mutant IND allele (ind). To reduce the number of backlinks (eg, to BC3 instead of BC6), specific molecular markers can be used for the genetic background of the elite parent.

Cross BC3-6 S1: IND / ind X IND / ind

BC3-6 S1 plants: 25% IND / IND and 50% IND / ind and 25% ind / ind

Plants containing ind are selected using molecular markers for the mutant IND allele (ind). Individual BC3-6 S1 plants that are homozygous for the mutant IND allele (ind / ind) are selected using molecular markers for the wild type and mutant IND alleles. These plants are then used for seed production.

To select plants comprising a point mutation in an IND allele, direct sequencing can be used by standard sequencing techniques known in the art, such as those described in Example 1.

Alternatively, PCR assays can be developed to discriminate plants comprising a specific point mutation in an IND allele from plants that do not comprise that specific point mutation. Thus, the following discriminant PCR assays can be performed to detect the presence or absence and zygosity status of the mutant alleles identified in Example 1 (see Table 3a and 3b):

- template DNA:

- Genomic DNA isolated from the leaf material of homozygous or heterozygous mutant Brassica plants (comprising a mutant IND allele, hereinafter referred to as "IND-Xx-EMSXX").

- Wild-type DNA control: Genomic DNA isolated from the foliar material of wild-type Brassica plants (comprising the wild-type equivalent of the mutant IND allele, hereinafter referred to as "IND-Xx-WT").

- Positive DNA control: Genomic DNA isolated from the leaf material of homozygous mutant Brassica plants known to comprise IND-Xx-EMSXX.

- In general, each set of primers consists of a specific primer for both the mutant target gene and the wild-type gene (for example, specific primer for both the IND-A1-EMS06 allele and the IND-A1-WT allele) and a specific primer for nucleotide difference (eg, specific primer for the IND-A1-EMS06 allele or the IND-A1-WT allele). Typically, the last nucleotide in the last primer matches the nucleotide difference, but one (or more) additional target specific nucleotides can be added to enhance hybridization between the primer and its target sequence.

- PCR mix: 2.5 µl x10 PCR buffer (15 mM MgCh), 0.25 µl dNTP (20 mM), 1 µl direct primer (10 | jM), 1 | jl of reverse primer (10 jM), 0.25 | jl of Taq-polymerase (5U / jiI), 19.5 | jl of H ² O Milli-Q, 0.5 | jl of DNA (20-50 ng / jil) = Total volume of 25 jil.

- Thermocycling profile: 4 min at 95 ° C; 30x [1 min at 95 ° C (denaturation) and 1 min at hybridization temperature and 2 min at 72 ° C (elongation)]; 5 min at 72 ° C; cool to 4 ° C. The optimal hybridization temperature can be determined by temperature gradient PCR in which the hybridization temperature can be varied, for example, between 57 ° C and 70 ° C in an MJ Research PTC-200 thermal cycler (Biozym). The optimal hybridization temperature for wild-type IND-specific primers is the temperature at which a transparent PCR fragment of the expected size (as described below) can be detected for the DNA sample from the wild-type Brassica plant. and not for the DNA sample from the mutant Brassica plant. The optimal hybridization temperature for mutant IND-specific primers is the temperature at which a transparent PCR fragment of the expected size (as described below) can be detected for the DNA sample from the mutant Brassica plant and not for the DNA sample from the wild-type Brassica plant.

- After amplification, 5 jil of loading pigment (orange pigment) is added to 15 jil of the PCR samples and the samples are loaded on a 1.5% agarose gel.

- The band patterns obtained after amplification of the genomic DNA of mutant Brassica plants are evaluated as follows:

- Data from DNA samples isolated from the leaf material of mutant Brassica plants should not be accepted within a single PCR cycle and a single PCR mix unless:

- the wild-type DNA control shows the PCR fragment of the size expected for the IND-Xx-WT-specific PCR assay and no PCR fragment of the size expected for the IND-Xx-EMSXX-specific PCR assay

- the positive DNA control shows the PCR fragment of the expected size for the IND-Xx-EMSXX specific PCR assay and no PCR fragment of the expected size for the IND-Xx-WT specific PCR assay

- Lanes that do not show PCR product of the expected size for the IND-Xx-W T specific PCR assay and the PCR fragment of the expected size for the IND-Xx-EMSXX specific PCR assay, indicate that the corresponding plant from which the template genomic DNA was prepared, it is a homozygous mutant for IND-Xx-EMSXX.

- Lanes showing the PCR fragment of the expected size for the IND -Xx-W Ty specific PCR assay and the IND-Xx-EMSXX specific PCR assay, indicate that the corresponding plant from which the DNA was prepared genomic template, is a heterozygous mutant for IND-Xx-EMSXX.

- Lanes showing the PCR fragment of the expected size for the IND -Xx-W T specific PCR assay and no PCR products of the expected size for the IND-Xx-EMSXX specific PCR assay, indicate that the corresponding plant from which the template genomic DNA was prepared, it is a wild-type plant.

As an alternative, Invader ™ (Third Wave Agbio) technology can be used to discriminate plants that comprise a specific point mutation in an IND allele from plants that do not comprise that specific point mutation. The following Invader ™ discriminant probes were developed to detect the presence or absence and zygosity status of the mutant alleles identified in Example 4 (see Table 6:

- Probes specific for the corresponding wild-type mutant or target IND gene (indicated as "5'flaplx" and "5 'flap2-x", respectively) and "invasive" probes that can be used in combination with them are indicated in Table 6. Generally, each set of probes consists of a probe specific for the mutant or wild-type target gene of which the first nucleotide after the 5 'flap sequence matches the nucleotide difference (nucleotide underlined in Table 6). (the so-called "primary probe"; for example, the probe with SEQ ID NO: 12 is specific for IND-A1-EMS06 and the probe with SEQ ID NO: 13 is specific for IND-A1-WT) and a probe specific for nucleotides upstream of the nucleotide difference (the so-called "invader® oligo"; for example, the probe with SEQ ID NO: 11 is specific for nucleotides upstream of the nucleotide difference between IND-A1-EMS06 and IND-A1-WT). The last nucleotide in the last primer can match the nucleotide difference in the mutant (as indicated by the bold nucleotides in Table 6), but other nucleotides can also be used for this last nucleotide as long as the primary probe and invader® oligo they can still form a single base overlay when hybridized to target DNA to generate the specific invasive structure recognized by Cleavase® (Third Wave Agbio) enzymes.

- Invader ™ test procedure and data interpretation are performed as prescribed by the manufacturer (Third Wave Agbio). Briefly, the nucleotide sequences indicated as "flap 1" and "flap2" in Table 6 represent the sequences of the 5 'flap "that are cut from the primary probes in the primary phase of the Invader ™ assay and that are complementary to the sequences in the FRET ™ cassette 1 and 2, respectively, and not complementary to the wild-type or target mutant sequences. If the primary probes are cut in the primary phase and the flapl probe and / or the flap2 probe hybridize to FRET ™ cassettes 1 and 2, respectively, in the secondary phase, a signal is generated indicative of the presence in the sample of the Wild type or corresponding mutant target IND gene, respectively.

Table 6

Claims (17)

1. A partial defective mutant allele of an IND gene, in which the IND gene comprises a nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising at least 90% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3 from nucleotide at position 46 to nucleotide at position 633, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7; (b) a nucleic acid molecule encoding an amino acid sequence comprising at least 90% sequence identity with s Eq ID NO: 2, SEQ ID NO: 4 from the amino acid at position 16 to the amino acid at position 210, or SEQ ID NO: 4, wherein said partial defective mutant allele comprises one or more missense mutations in the nucleic acid sequence of said IND gene resulting in the production of an IND protein in which at least one amino acid selected from the amino acid at a corresponding position to position 124 of SEQ ID NO: 2, the amino acid in a position corresponding to position 146 of SEQ ID NO: 2, the amino acid in a position corresponding to position 159 of SEQ ID NO: 2, the amino acid in a position corresponding to position 136 of SEQ ID nO: 4, the amino acid at a position corresponding to position 139 of SEQ ID NO: 4 or the amino acid at a position corresponding to position 142 of SEQ ID NO: 4, is substituted with another amino acid. 2. A mutant allele according to claim 1, which is selected from the group consisting of: - an IND allele having the sequence of SEQ ID NO: 1 in which the G at position 370 is replaced by A; - an IND allele having the sequence of SEQ ID NO: 1 in which the G at position 436 is replaced by A; - an IND allele having the sequence of SEQ ID NO: 1 where C at position 476 is replaced by T; - an IND allele having the sequence of SEQ ID NO: 3 where C at position 407 is replaced by T; - an IND allele having the sequence of SEQ ID NO: 3 in which the G at position 415 is replaced by A; and - an IND allele having the sequence of SEQ ID NO: 3 in which the C at position 424 is replaced by T. 3. A mutant allele according to claim 1, which is derived from a plant of a Brassica species, preferably a Brassica cultivar species or a Brassica oilseed species. 4. A mutant IND protein encoded by a mutant allele according to any one of claims 1 to 3. 5. A method of identifying a mutant IND allele according to any one of claims 1 to 3, in a biological sample comprising determining the presence of a specific region of mutant IND in a nucleic acid present in the biological sample. 6. A method according to claim 5, further comprising subjecting the biological sample to a polymerase chain reaction assay using a set of at least two primers, said set being selected from the group consisting of: - a set of primers, in which one of said primers specifically recognizes the 5 'flanking region of the mutant IND allele and the other of said primers specifically recognizes the 3' flanking region of the mutant IND allele, respectively, - a set of primers, in which one of said primers specifically recognizes the 5 'or 3' flanking region of the mutant IND allele and the other of said primers specifically recognizes the mutation region of the mutant IND allele, - a set of primers, in which one of said primers specifically recognizes the 5 'or 3' flanking region of the mutant IND allele and the other of said primers specifically recognizes the junction region between the 3 'or 5' flanking region and the mutation region of the mutant IND allele, respectively. 7. A method according to claim 5, further comprising subjecting the biological sample to a hybridization assay using a set of specific probes comprising at least one specific probe, said set being selected from the group consisting of: - a set of specific probes, in which one of said probes specifically recognizes the 5 'flanking region of the mutant IND allele and the other of said sodas specifically recognizes the 3' flanking region of the mutant IND allele, - a set of specific probes, in which one of said probes specifically recognizes the 5 'or 3' flanking region of the mutant IND allele and the other of said probes specifically recognizes the mutation region of the mutant IND allele, - a set of specific probes, in which one of said probes specifically recognizes the 5 'or 3' flanking region of the mutant IND allele and the other of said probes specifically recognizes the junction region between the 3 'or 5 flanking region 'and the mutation region of the mutant IND allele, respectively, - a specific probe that specifically recognizes the junction region between the 5' or 3 'flanking region and the mutation region of the mutant IND allele. 8. A method according to any one of claims 5 to 7, wherein - said 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 929 or from 931 to 1622 or the complementary thereof, respectively; said mutation region has the nucleotide sequence of nucleotide 930 of SEQ ID NO: 5 or complementary thereto; and said binding region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 930 or from 930 to 1622 or the complementary thereof, respectively, or - said 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 995 or from 997 to 1622 or the complementary thereof, respectively; said mutation region has the nucleotide sequence of nucleotide 996 of SEQ ID NO: 5 or complementary thereto; and said binding region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 996 or 996 to 1622 or the complementary thereof, respectively or - said 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 1035 or from 1037 to 1622 or the complementary thereof, respectively; said mutation region has the nucleotide sequence of nucleotide 1036 of SEQ ID NO: 5 or complementary thereto; and said binding region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 1036 or from 1036 to 1622 or the complementary thereof, respectively or - said 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 902 or 904 to 1593 or the complementary thereof, respectively; said mutation region has the nucleotide sequence of nucleotide 903 of SEQ ID NO: 7 or complementary thereto; and said binding region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 903 or from 903 to 1593 or the complementary thereof, respectively or - said 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 910 or from 912 to 1593 or the complementary thereof, respectively; said mutation region has the nucleotide sequence of nucleotide 911 of SEQ ID NO: 7 or complementary thereto; and said binding region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 911 or from 911 to 1593 or the complementary thereof, respectively or - said 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 919 or from 921 to 1593 or the complementary thereof, respectively; said mutation region has the nucleotide sequence of nucleotide 920 of SEQ ID NO: 7 or complementary thereto; and said binding region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 920 or from 920 to 1593 or complementary thereof, respectively. 9. A method for determining the zygosity status of a mutant IND allele according to any one of claims 1 to 3 in a plant or cell, part, seed or progeny thereof, comprising determining the presence of a mutant and / or a corresponding wild-type IND specific region in the genomic DNA of said plant, or a cell, part, seed or progeny thereof. 10. A method according to claim 9, wherein the method further comprises subjecting the genomic DNA of said plant, or a cell, part, seed or progeny thereof, to a polymerase chain reaction assay using a set of at least two or at least three primers, in which at least two of said primers specifically recognize the wild type IND allele, said at least two primers being selected from the group consisting of: - a first primer that specifically recognizes the 5 'or 3' flanking region of the mutant and wild type IND allele, and a second primer that specifically recognizes the 3 'or 5' flanking region of the mutant and wild type IND allele, respectively, - a first primer that specifically recognizes the 5 'or 3' flanking region of the mutant and wild-type IND allele, and a second primer that specifically recognizes the mutation region of the wild-type IND allele, - a first primer that specifically recognizes the 5 'or 3' flanking region of the mutant and wild-type IND allele, and a second primer that specifically recognizes the junction region between the 3 'or 5' flanking region and the mutation region of the mutant-type IND allele wild, respectively, and wherein at least two of said primers specifically recognize the mutant IND allele, said at least two primers being selected from the group consisting of: - the first primer that specifically recognizes the 5 'or 3' flanking region of the mutant and wild type IND allele, and a second primer that specifically recognizes the 3 'or 5' flanking region of the mutant and wild type IND allele, respectively, - the first primer that specifically recognizes the 5 'or 3' flanking region of the mutant and wild-type IND allele mutant, and a third primer that specifically recognizes the mutation region of the mutant IND allele, - the first primer that specifically recognizes the 5 'or 3' flanking region of the mutant and wild-type IND allele, and a third primer that specifically recognizes the junction region between the 3 'or 5' flanking region and the mutation region of the mutant IND allele, respectively . 11. A method according to claim 9, wherein the method further comprises subjecting the genomic DNA of said plant, or a cell, part, seed or progeny thereof, to a hybridization assay using a set of at least two specific probes, wherein at least one of said specific probes specifically recognizes the wild type IND allele, said at least one probe selected from the group consisting of: - a first probe that specifically recognizes the 5 'or 3' flanking region of the mutant and wild type IND allele, and a second probe that specifically recognizes the 3 'or 5' flanking region of the mutant and wild type IND allele, respectively, - a first probe that specifically recognizes the 5 'or 3' flanking region of the mutant and wild type IND allele, and a second probe that specifically recognizes the mutation region of the wild type IND allele, - a first probe that specifically recognizes the 5 'or 3' flanking region of the mutant and wild-type IND allele, and a second probe that specifically recognizes the junction region between the 3'0 5 'flanking region and the mutation region of the mutant-type IND allele wild respectively - a probe that specifically recognizes the junction region between the 5 'or 3' flanking region and the mutation region of the mutant IND allele, and wherein at least one of said specific probes specifically recognizes the mutant IND allele, said at least one probe selected from the group consisting of: - the first probe that specifically recognizes the 5 'or 3' flanking region of the mutant and wild type IND allele, and the second probe that specifically recognizes the 3 'or 5' flanking region of the mutant and wild type IND allele, respectively, - the first probe that specifically recognizes the 5 'or 3' flanking region of the mutant and wild-type IND allele mutant, and a third probe that specifically recognizes the mutation region of the mutant IND allele, - the first probe that specifically recognizes the 5 'or 3' flanking region of the wild type and mutant IND allele, and a third probe that specifically recognizes the junction region between the 3 'or 5' flanking region and the mutation region of the mutant IND allele, - a probe that specifically recognizes the junction region between the 5 'or 3' flanking region and the mutation region of the mutant IND allele. 12. A kit for identifying a mutant IND allele according to any one of claims 1 to 3 in a biological sample, comprising a set of primers or probes, said set being selected from the group consisting of: - a set of primers or probes, in which one of said primers or probes specifically recognizes the 5 'or 3' flanking region of the mutant IND allele and the other of said probes specifically recognizes the mutation region of the mutant IND allele, - a set of primers or probes, in which one of said primers specifically recognizes the 5 'or 3' flanking region of the mutant IND allele and the other of said primers or probes specifically recognizes the junction region between the 3 'flanking region or 5 'and the mutation region of the mutant IND allele, respectively, - a probe that specifically recognizes the junction region between the 5' or 3 'flanking region and the mutation region of the mutant IND allele, and in which - said 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 929 or from 931 to 1622 or the complementary thereof, respectively; said mutation region has the nucleotide sequence of nucleotide 930 of SEQ ID NO: 5 or complementary thereto; and said binding region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 930 or from 930 to 1622 or the complementary thereof, respectively or - said 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 995 or from 997 to 1622 or the complementary thereof, respectively; said mutation region has the nucleotide sequence of nucleotide 996 of SEQ ID NO: 5 or complementary thereto; and said binding region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 996 or 996 to 1622 or the complementary thereof, respectively or - said 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 1035 or from 1037 to 1622 or the complementary thereof, respectively; said mutation region has the nucleotide sequence of nucleotide 1036 of SEQ ID NO: 5 or complementary thereto; and said binding region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 1036 or from 1036 to 1622 or the complementary thereof, respectively or - said 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 902 or 904 to 1593 or the complementary thereof, respectively; said mutation region has the nucleotide sequence of nucleotide 903 of SEQ ID NO: 7 or complementary thereto; and said binding region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 903 or from 903 to 1593 or the complementary thereof, respectively or - said 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 910 or from 912 to 1593 or the complementary thereof, respectively; said mutation region has the nucleotide sequence of nucleotide 911 of SEQ ID NO: 7 or complementary thereto; and said binding region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 911 or from 911 to 1593 or complementary thereof, respectively or - said 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 7 from the nucleotide 1 to 919 or 921 to 1593 or the complementary thereof, respectively; said mutation region has the nucleotide sequence of nucleotide 920 of SEQ ID NO: 7 or complementary thereto; and said binding region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 920 or from 920 to 1593 or the complementary thereof, respectively. 13. A kit according to claim 12, wherein - said primer that specifically recognizes the 5 'or 3' flanking region of the mutant IND allele may consist of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the 5 'or 3' flanking region of the mutant or complementary IND allele thereof, respectively or - said primer that specifically recognizes the mutation region of the mutant IND allele consists of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the mutation sequence of the mutant IND allele or the complement thereof, or - said primer that specifically recognizes the junction region between the 5 'or 3' flanking region and the mutation region of the mutant IND allele consists of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from a sequence spanning the junction region between the 5 'or 3' flanking region and the mutant region of the mutant IND allele or complement thereof, wherein said 17 to 200 consecutive nucleotides do not derive exclusively from the mutation or flanking sequences, - said probe that specifically recognizes the 5 'or 3' flanking region of the mutant IND allele, consists of a nucleotide sequence of 13 to 1000 consecutive nucleotides selected from the 5 'or 3' flanking sequence of the mutant IND allele or the complement thereof , respectively, or a sequence that has at least 80% sequence identity with it, or - said probe that specifically recognizes the mutation region of the mutant IND allele, consists of a nucleotide sequence of 13 to 1000 consecutive nucleotides selected from the mutation sequence of the mutant IND allele or the complement thereof, or a sequence that has at least 80% sequence identity with it, or - said probe that specifically recognizes the junction region between the 5 'or 3' flanking region and the mutation region of the mutant IND allele, consists of a nucleotide sequence of 13 to 1000 consecutive nucleotides selected from a sequence that encompasses the region of junction between the 5 'or 3' flanking region and the mutant region of the mutant or wild-type IND allele or the complement thereof, respectively, in which said 13 to 1,000 consecutive nucleotides are not exclusively derived from the mutation or flanking sequences or a sequence having a sequence identity of at least 80% with it. 14. A kit for determining the zygosity status of a mutant IND allele according to any one of claims 1 to 3 in a plant, or a cell, part, seed or progeny thereof, comprising a set of primers or probes, wherein at least two of said primers or at least one of said probes specifically recognize the wild-type IND allele and wherein at least two of said primers or at least one of said probes specifically recognize the mutant IND allele, selected from the group consisting of: - a set of at least three primers or probes, in which a first primer or probe specifically recognizes the 5 'or 3' flanking region of the mutant and the wild type IND allele, a second primer or probe specifically recognizes the mutation region of the mutant IND allele, and a third primer or probe specifically recognizes the mutation region of the wild type IND allele, - a set of at least three primers or probes, in which a first primer or probe specifically recognizes the 5 'or 3' flanking region of the mutant and the wild-type IND allele, a second primer or probe specifically recognizes the binding region between the 3 'or 5' flanking region and the mutation region of the mutant IND allele, respectively, and a third primer or probe specifically recognizes the junction region between the 3 'or 5' flanking region and the mutation region of the IND allele wild-type respectively - a set of at least two probes, in which a first probe specifically recognizes the junction region between the 5 'or 3' flanking region and the mutation region of the mutant IND allele and a second probe specifically recognizes the junction region between the 5 'or 3' flanking region and the mutation region of the wild type IND allele, and in which - said 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 929 or from 931 to 1622 or the complementary thereof, respectively; said mutation region of the wild type IND allele has the nucleotide sequence of nucleotide 930 of SEQ ID NO: 5 or complementary thereto; said mutant region of the mutant IND allele has sequence a or complementary to it; said binding region of the wild-type IND allele may comprise the nucleotide sequence of SEQ ID NO: 5 of nucleotide 1 to 930 or 930 to 1622 or of the complementary thereof, respectively; and said junctional region of the mutant IND allele comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 929 followed by a or followed by the nucleotide sequence SEQ ID NO: 5 from nucleotide 931 to 1622 or complementary to the same, respectively or - said 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 995 or from 997 to 1622 or the complementary thereof, respectively; said mutation region of the wild type IND allele has the nucleotide sequence of nucleotide 996 of SEQ ID NO: 5 or complementary thereto; said mutant region of the mutant IND allele has sequence a or complementary to it; said binding region of the wild-type IND allele may comprise the nucleotide sequence of the SEQ ID NO: 5 of nucleotide 1 to 996 or 996 to 1622 or complementary thereto, respectively; and said junctional region of the mutant IND allele comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 995 followed by a or followed by the nucleotide sequence SEQ ID NO: 5 from nucleotide 997 to 1622 0 of the complementary of same, respectively or - said 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 1035 or from 1037 to 1622 or the complementary thereof, respectively; said mutation region of the wild type IND allele has the nucleotide sequence of nucleotide 1036 of SEQ ID NO: 5 or complementary thereto; said mutation region of the mutant IND allele has the t or complementary sequence thereof; said binding region of the wild-type IND allele may comprise the nucleotide sequence of SEQ ID NO: 5 of nucleotide 1 to 1036 or 1036 to 1622 or of the complementary thereof, respectively; and said junctional region of the mutant IND allele comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 1035 followed by tot followed by the nucleotide sequence SEQ ID NO: 5 from nucleotide 1037 to 1622 or complementary to the same, respectively or - said 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 902 or 904 to 1593 or the complementary thereof, respectively; said wild type IND allele mutation region has the nucleotide sequence of nucleotide 903 of SEQ ID NO: 7 or complementary thereto; said mutant region of the mutant IND allele may have the t or complementary sequence thereof; and said binding region of the wild-type IND allele comprises the nucleotide sequence of SEQ ID NO: 7 of nucleotide 1 to 903 or 903 to 1593 or complementary thereof, respectively; and said junctional region of the mutant IND allele comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 902 followed by tot followed by the nucleotide sequence SEQ ID NO: 7 from nucleotide 904 to 1593 or complementary to the same, respectively or - said 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 910 or from 912 to 1593 or the complementary thereof, respectively; said wild-type IND allele mutation region has the nucleotide sequence of nucleotide 911 of SEQ ID NO: 7 or complementary thereto; said mutant region of the mutant IND allele has sequence a or complementary to it; and said binding region of the wild-type IND allele may comprise the nucleotide sequence of SEQ ID NO: 7 of nucleotide 1 to 911 or 911 to 1593 or complementary thereof, respectively; and said junctional region of the mutant IND allele comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 910 followed by a or followed by the nucleotide sequence SEQ ID NO: 7 from nucleotide 912 to 1593 0 of the complementary of same, respectively or - said 5 'or 3' flanking region comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 919 or from 921 to 1593 or the complementary thereof, respectively; said mutation region of the wild type IND allele has the nucleotide sequence of nucleotide 920 of SEQ ID NO: 7 or complementary thereto; said mutant region of the mutant IND allele may have the t or complementary sequence thereof; and said binding region of the wild type IND allele may comprise the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 920 or from 920 to 1593 or complementary thereof, respectively; and said junctional region of the mutant IND allele comprises the nucleotide sequence of SEQ ID NO: 7 from nucleotide 1 to 919 followed by tot followed by the nucleotide sequence SEQ ID NO: 7 from nucleotide 921 to 1593 or complementary to the same, respectively. 15. A method for increasing the yield of the Brassica plant comprising at least two IND genes, which comprises introducing by mutagenesis two homozygous mutant IND alleles as described in any one of claims 1 to 3 into its genome. 16. Use of a mutant IND allele according to any one of claims 1 to 3 to increase seed yield or resistance to pod shelling in a Brassica plant. 17. A procedure to alter the dehiscence properties of the fruit of a Brassica plant that comprises at least two iNd genes, comprising the steps of: - generating and / or selecting a Brassica plant comprising at least two IND genes, in which at least two alleles of the at least two IND genes are partial defective ind alleles as described in any one of claims 1 to 3, - select a plant with altered fruit dehiscence properties, in particular, a plant in which the threshing of seeds is reduced or delayed until after harvest, while the pods maintain at the same time an agronomically relevant threshing capacity, wherein said partial defective alleles are generated by mutagenesis.