[go: up one dir, main page]

WO2018125749A1 - Méthodes de production de lots de graines de type hybride - Google Patents

Méthodes de production de lots de graines de type hybride Download PDF

Info

Publication number
WO2018125749A1
WO2018125749A1 PCT/US2017/067907 US2017067907W WO2018125749A1 WO 2018125749 A1 WO2018125749 A1 WO 2018125749A1 US 2017067907 W US2017067907 W US 2017067907W WO 2018125749 A1 WO2018125749 A1 WO 2018125749A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
generations
inbred
plants
hybrid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2017/067907
Other languages
English (en)
Inventor
Sally Ann MACKENZIE
Michael Fromm
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Epicrop Technologies Inc
University of Nebraska Lincoln
University of Nebraska System
Original Assignee
Epicrop Technologies Inc
University of Nebraska Lincoln
University of Nebraska System
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Epicrop Technologies Inc, University of Nebraska Lincoln, University of Nebraska System filed Critical Epicrop Technologies Inc
Publication of WO2018125749A1 publication Critical patent/WO2018125749A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection

Definitions

  • Self-pollinated crops such as rice, wheat, soybeans, cotton, oats, rye, and barley generally suffer from a lack of investment by private industry due to a lack of recurring annual sales. Growers can buy the seeds, grow the crop, and then save some of the seeds from the crop for planting in the subsequent year(s). This is in contrast to hybrid crops where the better yields of hybrid seed relative to subsequent generations provides an economic incentive for the grower to buy seeds every year.
  • a biological system that provides the advantages of hybrids in a self-pollinated crop is needed to encourage investment in improving crop yields in these crops.
  • Plant genomes contain relatively large amounts of 5-methylcytosine (5meC; Kumar et al. 2013 J Genet 92(3): 629-666). Other than silencing transposable elements and repeated sequences, the biological roles of 5meC and its transgenerational stability are still emerging. Intercrossing a low methylation mutant plant with a normally methylated plant resulted in heritable changes in DNA methylation in the plant genome that affected some plant phenotypic traits in Arabidopsis (Cortijo et al. 2014 Science. 2014 Mar 7;343(6175): 1 145-8; Kooke et al. Plant Cell. 2015 Feb;27(2):337-48).
  • methylation include those described (Stroud et al., Cell 2013 152(1-2)352-64) that can alter DNA methylation patterns in progeny.
  • the present disclosure provides methods of producing hybrid-like yields in self- pollinated crops wherein the yields are highest in a targeted number of generations, wherein later generations of plants have lower relative yields.
  • the methods identify parent plants that can produce these hybrid-like yields that are highest (e.g., attain a peak yield) in a targeted number of generations with subsequent generations exhibiting lower yields.
  • Non-limiting embodiments methods of identifying an inbred plant that produces hybrid-like seeds with peak yields in a targeted number of generations comprising: a) producing candidate inbred plants with new epigenetic modifications; b) screening said candidate inbred plants of step (a) for inbred plants that display higher yields than a reference plant line in a targeted number of generations of hybrid-like plants, wherein said hybrid-like plant lines are produced by crossing or grafting a rootstock of a candidate inbred plant to a second parental plant and then self-pollinating the progeny for a targeted number of generations; and c) screening and selecting hybrid-like plant lines of step (b) with higher yields than a reference plant line in a targeted number of generations, for hybrid-like plant lines for which subsequent self-pollination for at least one generation produces plant populations with lower yields than the yield in the targeted number of generations, thereby identifying an inbred plant that produces hybrid-like seeds with peak yields in a targeted number of generations, are provided herein.
  • Non-limiting embodiments of methods of identifying an inbred plant that produces hybrid-like seeds with peak yields in a targeted number of generations comprising: a) producing candidate inbred plants with new epigenetic modifications by Mshl suppression- restoration to produce said candidate inbred plants; b) screening said candidate inbred plants of step (a) for inbred plants that display higher yields than a reference plant line in a targeted number of generations of hybrid-like plants, wherein said hybrid-like plant lines are produced by crossing or grafting a rootstock of a candidate inbred plant to a second parental plant and then self-pollinating the progeny for a targeted number of generations; and, c) screening and selecting hybrid-like plant lines of step (b) with higher yields than a reference plant line in a targeted number of generations, for hybrid-like plant lines for which subsequent self- pollination for at least one generation produces plant populations with lower yields than the yield in the targeted number of generations, thereby identifying an inbred plant that produces hybrid-like seeds
  • Non-limiting embodiments of methods of producing hybrid-like seeds with peak yields in a targeted number of generations comprising: a) producing candidate inbred plants with new epigenetic modifications; b) screening said candidate inbred plants of step (a) for inbred plants that display higher yields than a reference plant line in a targeted number of generations of hybrid-like plants, wherein said hybrid-like plant lines are produced by crossing or grafting a rootstock of a candidate inbred plant to a second parental plant and then self-pollinating the progeny for a targeted number of generations; c) screening and selecting hybrid-like plant lines of step (b) with higher yields than a reference plant line in a targeted number of generations, for hybrid-like plant lines for which subsequent self-pollination for at least one generation produces plant populations with lower yields than the yield in the targeted number of generations, thereby identifying an inbred plant that produces hybrid-like seeds with peak yields in a targeted number of generations; and, d) producing hybrid-like seeds with peak yield
  • Non-limiting embodiments of methods of producing hybrid-like seeds with peak yields in a targeted number of generations comprising: a) producing candidate inbred plants with new epigenetic modifications by Mshl suppression-restoration to produce said candidate inbred plants; b) screening said candidate inbred plants of step (a) for inbred plants that display higher yields than a reference plant line in a targeted number of generations of hybrid-like plants, wherein said hybrid-like plant lines are produced by crossing or grafting a rootstock of a candidate inbred plant to a second parental plant and then self-pollinating the progeny for a targeted number of generations; and, c) screening and selecting hybrid-like plant lines of step (b) with higher yields than a reference plant line in a targeted number of generations, for a hybrid-like plant line for which subsequent self-pollination for at least one generation produces plant populations with lower yields than the yield in the targeted number of generations, thereby identifying an inbred plant that produces hybrid-like seeds with peak yield
  • the targeted number of generations is selected from the group consisting of 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6, 5 to 6, and 6 generations. In certain embodiments of any of the aforementioned methods, the targeted number of generations is selected from the group consisting of 3, 4, 5, and 6. In certain embodiments of any of the aforementioned methods, a single plant harvester is used to measure yields in single plants in one or more generations of step (b).
  • the candidate inbred plants of step (a) are produced from an inbred parent which was suppressed for Mshl function or were derived from an ancestral plant suppressed for Mshl function.
  • the candidate inbred plants are produced from two genetically diverse parental plants at least one of which was suppressed for Mshl function or were derived from an ancestral plant suppressed for Mshl function.
  • seeds of said candidate inbred plants of step (a) are stored at about -18 to -20 C.
  • the hybrid-like plant lines of step (b) are produced by crossing or grafting a rootstock of a candidate inbred plant to a second parental plant, wherein said second parental plant is isogenic to said candidate inbred plant prior to any epigenetic modification steps.
  • the hybrid-like plant lines of step (b) are produced by crossing or grafting a rootstock of a candidate inbred plant to a second parental plant, wherein said second parental plant is isogenic to said candidate inbred plant and wherein said second parental plant is derived from an ancestral plant suppressed for Mshl .
  • the hybrid-like seeds are rice, wheat, soybeans, cotton, oats, rye, or barley seeds.
  • a soybean Mshl gene is subjected to suppression-restoration.
  • the soybean Mshl that is subjected to suppression-restoration encodes the polynucleotide of SEQ ID NO:6, SEQ ID NO: 8, or a variant of SEQ ID NO: 6 or SEQ ID NO: 8 having at least 80%, 90%, 95%, 98%, or 99% sequence identity thereto or having one, two, three , four, five or more nucleotide substitutions, deletions, and/or insertions.
  • the soybean Mshl gene that is subjected to suppression-restoration comprises a polynucleotide that encodes the polypeptide of SEQ ID NO: 7, SEQ ID NO: 9, or a variant of SEQ ID NO: 7 or SEQ ID NO: 9 having at least 80%, 90%, 95%, 98%, or 99% sequence identity thereto or having one, two, three , four, five or more amino acid residue substitutions, deletions, and/or insertions.
  • the candidate inbred plants are produced in step (a) by suppressing expression of Mshl in a parent plant and recovering a candidate inbred plant wherein Mshl expression has been restored.
  • the candidate inbred plants are produced in step (a) by introducing a loss-of- function mutation into an endogenous Mshl gene of a parent plant, obtaining a first progeny plant of the parent plant wherein Mshl gene expression is suppressed by the mutation, and recovering from the first progeny plant the candidate inbred plant, wherein Mshl gene expression is restored in the candidate inbred plant.
  • the phrase "producing candidate inbred plants with new epigenetic modifications by suppressing and then restoring Mshl” refers to a multi -generation process of suppressing Mshl in a plant for one or more plant generations and then restoring Mshl function in a later generation to create epigenetic modifications suitable for producing 'hybrid-like plants' .
  • reference plant line refers to a plant line lacking new epigenetic modifications but otherwise genetically similar or identical to a candidate plant.
  • a reference plant line facilitates comparison to lines with epigenetic contributions to yields and phenotypes.
  • new epigenetic modifications refers to epigenetic modifications introduced into a plant or plant line by a specific procedure as opposed to epigenetic modifications that occur by traditional breeding procedures.
  • exemplary non- limiting examples of specific procedures for introducing new epigenetic modifications are altering DNA methyltransferase levels, mutants in DNA methylation pathways, or Mshl suppression-restoration systems.
  • Mshl suppression-restoration refers to a multi- generation process of suppressing Mshl in a plant for one or more plant generations and then restoring Mshl function in a later generation to create epigenetic modifications in progeny plants and lines.
  • peak yields refers to yields obtained from a given generation of plants that exceed yields obtained from a parental plant line and that exceed yields obtained from a subsequent generation of plants.
  • plant line refers to one or more generations of a plant lineage, including ancestor plants, current plants, and multiple generations of progeny plants.
  • a plant line can be an inbred line or a line early in the process of becoming an inbred or can be more genetically heterogeneous than an inbred.
  • a plant line can be derived from a single ancestor or two common ancestors.
  • inbred refers to a plant or plant line sufficiently homozygous to have progeny with similar phenotypes. Typically an inbred plant has been self-pollinated for at least three generations or has been generated from a double haploid method to have a high degree of genetic and/or epigenetic homozygosity.
  • seeds of the candidate inbred plant refers to seeds from the candidate inbred plant or plant line, said seeds from earlier, current, or subsequent generations of said candidate inbred plant or plant line.
  • the phrases “candidate inbred plant” (singular) or “candidate inbred plants” (plural) refer to an inbred plant or plants, and/or an inbred plant line and includes seeds from the candidate inbred plant or plant line, said seeds from earlier, current, or subsequent generations of said candidate inbred plant or plant line. It is understood that the process of screening and/or identifying a candidate inbred plant or candidate inbred plants includes storing the seeds of these plants for later recovery from storage and propagation of plants from these seeds.
  • epigenetic trait refers to a trait dependent in part on epigenetic modifications. For example, a yield increase that is dependent in part upon DNA methylation modifications.
  • generation refers to one or several complete life cycle (an initial seed is grown to a mature plant that produces seeds).
  • progeny refers to a first, second, third, or later generations of plants derived from a parent or ancestor plant.
  • Fl refers to seeds produced from a cross of two parents or plants grown from said seeds. If only self-pollination is performed for a plant line, then Fl can refer to the first generation of plant grown.
  • F2 refers to seeds produced from self-pollination of plants grown from Fl seeds or plants grown from F2 seeds.
  • F3 refers to seeds produced from self-pollination of plants grown from F2 seeds or plants grown from F3 seeds.
  • Fn refers to seeds produced from self-pollination of plants grown from F(n-l) seeds or plants grown from Fn seeds.
  • hybrid-like is used to refer to plants or seeds that are epigenetically modified and that exhibit, or give rise to plants that exhibit, an increase in yield in comparison to a parental plant that lacks the epigenetic modifications.
  • the yield increase is diminished or lost in subsequent generations of plants obtained from selfs of certain hybrid like plants or from selfs of a plant obtained from certain hybrid-like seed.
  • Various methods for providing plants with hybrid-like yields are provided herein.
  • the methods provided herein can be applied to most crops that contain new epigenetics modifications that increase yields in hybrid-like plants.
  • the Mshl suppression-restoration system is one such suitable system.
  • Other systems for introducing epigenetic modifications include, but are not limited to, using recombinant DNA methyltransferases.
  • the methods of producing the candidate inbred plants as well as the two parental plants in the initial cross can be varied. One variation is to produce candidate inbred plants comprising genetics from two genetically different plants and concurrently creating new epigenetic modifications in said candidate inbred plants.
  • the two parental plants used to initiate the production of hybrid-like plants in the initial cross can have one or both parents comprising new epigenetic
  • the methods use Mshl suppression-restoration techniques.
  • Mshl suppression-restoration is a process that can occur over several generations and can lead to unusual plant phenotypes (Xu YZ, Santamaria Rde L, Virdi KS, Arrieta-Montiel MP, Razvi F, Li S, Ren G, Yu B, Alexander D, Guo L, Feng X, Dweikat FM, Clemente TE, Mackenzie SA. Plant Physiol. 2012 Jun; 159(2):710-20; Virdi KS, Wamboldt Y,
  • Methods and various Mshl nucleic acid sequences for producing epigenetically- modified plants using Mshl suppression in an ancestral plant or plants and restoration include, but are not limited to, those described in U. S. Patent Application Publication No. 20120284814, U.S. Patent Application Publication No. 20150052630, U.S. Patent
  • Mshl suppression can be effected by methods including, but not limited to, transgene-mediated suppression (e.g., by RNAi), VIGS-based suppression, introduction of inhibitory polynucleotides (e.g., encoding or comprising RNAi-inducing polynucleotides), or by introduction of mutations in the endogenous Mshl gene (e.g., in the Mshl promoter, transcribed region, splice donor and/or acceptor sites, polyadenylation/terminator region, or any combination thereof).
  • transgene-mediated suppression e.g., by RNAi
  • VIGS-based suppression introduction of inhibitory polynucleotides (e.g., encoding or comprising RNAi-inducing polynucleotides)
  • introduction of mutations in the endogenous Mshl gene e.g., in the Mshl promoter, transcribed region, splice donor and/or acceptor sites,
  • Types of mutations that provide for such Mshl suppression can include, but are not limited to, substitutions, deletions, and/or insertions into the endogenous Mshl gene.
  • Useful mutations in the endogenous Mshl gene include, but are not limited to, frameshift, nonsense, and missense mutations that cause a loss-of function.
  • Mutations in endogenous Mshl genes can be obtained from a variety of sources and by a variety of techniques.
  • mutations an endogenous Mshl gene can introduced by physical mutagenesis methods, including but not limited to, chemical (e.g., EMS) or radiation mutagenic treatments,
  • TILLING gene editing techniques (e.g., CRISPR-CAS9, meganucleases, TALENS) and the like.
  • Comput Struct Biotechnol J. 2017; 15: 146-160 can be adapted for use in introducing mutations in an endogenous Mshl gene.
  • clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease e.g., Cas9, Cpfl, S. aureus Cas9 variants, eSpCas9
  • a guide RNA e.g., a homologous replacement sequence containing one or more loss of function mutations in the Mshl gene and homologous sequences at both ends of the double stranded break can provide for homologous
  • loss of function mutations include, but are not limited to, insertions, deletions, and
  • Loss-of-function mutations in an Mshl gene also include, but are not limited to, pre-mature translational stop codon insertions, deletions of one or more functional domains that include, but are not limited to, a DNA binding (Domain I), an ATPase (Domain V) domain, and/or a carboxy-terminal GIY-YIG type endonuclease domain, and the like. Also provided herein are mutations analogous the Arabidopsis mshl mutation that are engineered into endogenous Mshl gene to obtain similar effects. Methods for substituting endogenous chromosomal sequences by homologous double stranded break repair have been reported in tobacco and maize (Wright et al., Plant J.
  • a homologous replacement mshl sequence i.e. which provides a loss of function mutation in an Mshl target gene sequence
  • a homologous replacement mshl sequence can also be introduced into a targeted nuclease cleavage site by non-homologous end joining or a combination of nonhomologous end joining and homologous recombination (reviewed in Puchta, J. Exp. Bot. 56, 1, 2005; Wright et al., Plant J. 44, 693, 2005).
  • at least one site specific double stranded break can be introduced into the endogenous Mshl gene by a meganuclease.
  • meganucleases can provide for meganucleases that cut within a recognition sequence that exactly matches or is closely related to specific endogenous Mshl gene sequence (WO/06097853A1, WO/06097784A1, WO/04067736 A2, U.S. 20070117128A1). It is thus anticipated that one can select or design a nuclease that will cut within a target Mshl gene sequence. In other embodiments, at least one site specific double stranded break can be introduced in the endogenous Mshl gene target sequence with a zinc finger nuclease.
  • Restoration of Mshl function can be provided by crossing the plants that are homozygous for recessive mutations that provide for such Mshl suppression with plants having wildtype Mshl gene(s) and harvesting progeny plants that are heterozygous for the mshl mutation.
  • Methods and various nucleic acid sequences for producing epigenetically-modified plants using recombinant DNA methyltransferases include, but are not limited to, those described in U.S. Patent Application Publication No. 20160032310 and U.S. Patent
  • Methods of producing hybrid-like plants include the use of freezing seeds as a method of stably storing epigenetically modified seeds (U.S. Patent Application Publication No. 20170223914, which is incorporated herein by reference in its entirety).
  • Methods of producing hybrid-like plants include single plant harvesters and row harvesters for selecting individual plants or plant families (see U.S. Patent Application Publication No. 20170188512, which is incorporated herein by reference in its entirety.
  • suitable plants for the present disclosure both as self-pollinated plants as well as plants often produced as hybrids include but are not limited to, those from: barley (Hordeum vulgare), corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (for example, pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower
  • Improvements in yield in plant lines that include, but are not limited to, peak yields obtained by the methods provided herein, can be identified by direct measurements of wet or dry biomass including, but not limited to, grain, lint, leaves, stems, or seed. Improvements in yield can also be assessed by measuring yield related traits that include, but are not limited to, 100 seed weight, a harvest index, and seed weight. In certain embodiments, such yield improvements are improvements in the yield of a plant line relative to one or more parental line(s), subsequent generations, reference lines, controls, or checks and can be readily determined by growing plant lines obtained by the methods provided herein in parallel with the parental plants, subsequent generations, reference lines, controls, or checks.
  • field trials to determine differences in yield whereby plots of test and control plants are replicated, randomized, and controlled for variation can be employed (Giesbrecht FG and Gumpertz ML. 2004. Planning, Construction, and Statistical Analysis of Comparative Experiments. Wiley. New York; Mead, R. 1997. Design of plant breeding trials. In Statistical Methods for Plant Variety Evaluation, eds. Kempton and Fox. Chapman and Hall. London.).
  • Methods for spacing of the test plants (i.e. plants obtained with the methods of this invention) with check plants (parental or other controls) to obtain yield data suitable for comparisons are provided in references that include, but are not limited to, any of Cullis, B. et al. J. Agric. Biol. Env. Stat.11 :381-393; and Besag, J. and Kempton, RA. 1986. Biometrics 42: 231-251.).
  • peak yields obtained from targeted generation(s) of plants can be identified by comparing yields obtained from the targeted generation(s) to yields obtained from a parental line, from a subsequent generation of plants, from parental lines and subsequent generations, or from any other reference, check, or control plant line.
  • peak yields obtained from the targeted generation of plants will exceed yields obtained from any of the other aforementioned plants or plant lines by at least about 1%, 2%, 5%, 7%, 8%, 10%, 12%, 14%, or 15%.
  • peak yields obtained from the targeted generation of plants will exceed yields obtained from any of the other
  • peak yields obtained from the targeted generation of plants will exceed yields obtained from any of the other aforementioned plants or plant lines by at least about 25%, 50%, or 100%.
  • the methods for introducing heritable epigenetic or genetic variation in a plant or progeny thereof can comprise the step of grafting rootstock obtained from a plant or a parent plant thereof had been subjected to suppression of a MSH1 gene.
  • the heritable epigenetic variation provides a useful trait is selected from the group consisting of improved yield, delayed flowering, non-flowering, increased biotic stress resistance, increased abiotic stress resistance, enhanced lodging resistance, enhanced growth rate, enhanced biomass, enhanced tillering, enhanced branching, delayed flowering time, delayed senescence, increased flower number, improved architecture for high density planting, improved photosynthesis, increased root mass, increased cell number, improved seedling vigor, improved seedling size, increased rate of cell division, improved metabolic efficiency, and increased meristem size in comparison to the control plant.
  • the plant, progeny of the plant, or scion contain(s) one or more epigenetic changes in one or more nuclear chromosomes, wherein the epigenetic changes are absent from nuclear chromosomes of the control plant or are absent from nuclear chromosomes of a plant from which the scion was obtained.
  • the epigenetic change(s) are also present in the rootstock that had been subjected to MSH1 gene suppression.
  • the epigenetic changes in the plant, progeny of the plant, scion, or rootstock are associated with the improvement in the useful trait.
  • the epigenetic changes in the plant, progeny of the plant, scion, or rootstock induced by suppression of a MSH1 gene In certain embodiments, the plant, progeny of the plant, scion, or rootstock contain(s) one or more epigenetic changes in one or more nuclear chromosomes that are absent from nuclear chromosomes of rootstock obtained from a plant or are absent from nuclear chromosomes of a parent plant thereof had not been subjected to MSH1 gene suppression.
  • the plant, progeny of the plant, scion and/or the rootstock exhibit CG hypermethylation of a region encompassing a MSHl locus in comparison to a control plant that had not been subjected to the MSH1 gene suppression. In certain embodiments, the plant, progeny of the plant, scion and/or the rootstock exhibit pericentromeric CHG hyper-methylation in comparison to a control plant that had not been subjected to the MSHl gene suppression.
  • the plant, progeny of the plant, scion and/or the rootstock exhibit CG hypermethylation and/or CHG hypermethylation at one or more nuclear chromosomal loci in comparison to corresponding nuclear chromosomal loci of a control plant that had not been subjected to the MSHl gene suppression.
  • the plant is selected from the group consisting of a crop plant, a tree, a bush, and a vine.
  • the crop plant is selected from the group consisting of corn, soybean, cotton, canola, wheat, rice, tomato, tobacco, millet, potato, sugarbeet, cassava, alfalfa, barley, oats, sugarcane, sunflower, strawberry, and sorghum.
  • the tree is selected from the group consisting of an apple, apricot, grapefruit, orange, peach, pear, plum, lemon, coconut, poplar, eucalyptus, date palm, palm oil, pine, and an olive tree.
  • the bush is selected from the group consisting of a blueberry, raspberry, and blackberry bush.
  • the vine is a grape vine. Also provided are plants or progeny thereof obtained by any of the aforementioned methods. Also provided are plant parts obtained from the plant or progeny thereof that were made by any of the aforementioned methods.
  • grafted plants comprising a scion to which a rootstock had been grafted, wherein the rootstock is obtained from a plant or a parent plant thereof that had been subjected to MSH1 gene suppression, as well as progeny plants and clonal propagates obtained from the grafted plant.
  • Such rootstocks can be also used to introduce epigenetic and/or genetic variation into varietal or non-hybrid plants that result in useful traits as well as useful plants, plant parts including, but not limited to, seeds, plant cells, and processed plant products that exhibit, carry, or otherwise reflect benefits conferred by the useful traits.
  • such rootstocks can also be used to introduce epigenetic and/or genetic variation into plants that are also amenable to hybridization.
  • Rootstocks useful for introducing epigenetic and/or genetic variation into plants can be obtained from a variety of rootstock source plants that had been subjected to MSH1 gene suppression.
  • the rootstock source plant is a plant that had itself been subjected to MSH1 gene suppression.
  • the rootstock source plant is the progeny of a parental plant that had itself been subjected to MSH1 gene suppression.
  • Various methods of making rootstock source plants by MSH1 gene suppression are provided herein. Plants that can serve as rootstock source plants and methods of making such plants are also disclosed in US Patent Application Publication No. 20120284814, which is specifically incorporated herein by reference in its entirety. The use of plants with useful traits and methods of making such plants disclosed in para. [0072], [0085], and [0089] in US Patent Application Publication No. 20120284814 as rootstock sources is specifically provided, and each of those paragraphs is specifically incorporated herein by reference in their entireties.
  • a population of progeny plants obtained from the grafted plant are screened and individual progeny plants are selected for one or more useful traits.
  • populations of progeny plants can be obtained by methods including, but not limited to, selfing or outcrossing the grafted plant comprising the rootstock to obtain seed that give rise to the population.
  • populations of progeny plants can also be obtained by methods including, but not limited to, growing a population of plants that are derived from independent clonal propagates obtained from the grafted plant comprising the rootstock.
  • Such selected individual progeny plants that exhibit the useful trait can then be sexually or asexually propagated to yield populations of plants that exhibit the useful trait or seed lots that exhibit or harbor the useful trait. Such sexual propagation can be accomplished by selfing or outcrossing the selected individual progeny plants that exhibit the useful trait.
  • the rootstock source plant is the progeny of a parental plant that had been subjected to MSHl suppression
  • the rootstock source plant itself can be a plant that was selected for one or more useful traits. Grafting rootstock from a plant that had been selected for a useful trait to a scion that does not exhibit the trait can impart the trait to the resultant grafted plant or to progeny thereof. Resultant grafted plants or progeny thereof that exhibit the useful trait can then be sexually or asexually propagated to yield populations of plants that exhibit the useful trait or seed lots that exhibit or harbor the useful trait.
  • MSHl gene suppression in the rootstock can be continuous and ongoing or can be transient.
  • Non-limiting and exemplary methods for effecting continuous and ongoing MSHl gene suppression in the rootstock include suppressing expression of a MSHl gene with mutations in the endogenous MSHl gene or with a transgene that yields a product that suppresses expression of the MSHl endogenous gene.
  • the MSHl gene suppression in the rootstock can be transient or have occurred in a parental plant from which the rootstock was obtained but not in the rootstock that was used in the graft.
  • Non-limiting and exemplary methods for effecting transient suppressing of MSHl function in the rootstock include suppressing expression of a MSHl target gene with a transgene that provides for inducible or repressible expression of a product that suppresses expression of the endogenous gene, with a transgene that can be excised, or with a heterozygous transgene insert that is removed from the rootstock by segregation. Any of the methods described herein for restoring MSHl function after MSHl suppression can be used to generate rootstock used in certain embodiments.
  • Grafting can be effected by any method that provides for establishment of a vascular connection between the rootstock and the scion.
  • Methods of grafting that can be used to effect the connection between the scion and the rootstock include, but are not limited to, apical graftage, side graftage, bark graftage, and root graftage.
  • Such methods for effecting grafts of scions to rootstock are disclosed in "Plant Propagation: Principles and Practices; Chapter 12: Techniques of Grafting” Ed. Hartman, Kester, Davies, and Geneve, 7 th Edition.
  • Rootstocks subjected to MSH1 gene suppression or obtained from a parental plant that had been subjected to MSH1 gene suppression can exhibit modifications of one or more nuclear chromosomes.
  • such rootstocks can exhibit characteristic DNA methylation and/or gene transcription patterns that occur in plants subjected to suppression of an MSH1 target gene.
  • Such characteristic DNA methylation and/or gene transcription patterns that occur in plants or seeds subjected to suppression of an MSH1 target gene can include, but are not limited to, those patterns disclosed in Example 5.
  • rootstock of first generation progeny of a plant subjected to suppression of a MSHl gene will exhibit CG differentially methylated regions (DMR) of various discrete chromosomal regions that include, but are not limited to, regions that encompass the MSHl locus.
  • DMR differentially methylated regions
  • a CG hypermethylated region that encompasses the MSHl locus will be about 5 to about 8 MBp (mega base pairs) in length.
  • rootstock of first generation progeny of a plant subjected to suppression of a MSHl gene will also exhibit changes in plant defense and stress response gene expression.
  • a rootstock, a scion grafted thereto, and/or a plant cell, a seed, a progeny plant, plant populations, seed populations, and/or processed products obtained therefrom that has been subject to suppression of a MSHl gene will exhibit pericentromeric CHG
  • CHG hypermethylation across chromosomes that have been previously observed (U.S. Patent 6,444,469).
  • Such CG and CHG hypermethylation can be assessed by comparing the methylation status of a sample from rootstocks, scions of plants grafted to root stocks, plants or seed that had been subjected to suppression of a MSHl gene, or a sample from progeny plants or seed derived therefrom, to a sample from control plants or seed that had not been subjected to suppression of a MSHl gene.
  • control plants include, but are not limited to, plants, grafted plants, scions thereof and rootstocks thereof that had not been subjected to MSHl gene suppression.
  • such aforementioned changes in the methylation patterns exhibited by scions that are grafted to the rootstocks, or exhibited by a plant cell, a seed, a progeny plant, plant populations, seed populations, and/or processed products obtained from the grafted plant be used to monitor the effectiveness of the graft in transmitting desirable epigenetic changes or to identify a plant cell, a seed, a progeny plant, plant populations, seed populations, and/or processed products obtained from the grafted plant.
  • the second plant can also be a grafted plant comprising a scion grafted to rootstock that had been subjected to MSH1 gene suppression, a progeny plants obtained from a grafted plant comprising a scion grafted to rootstock that had been subjected to MSH1 gene suppression, any other ungrafted plant that had been subjected to MSH1 gene suppression, or any other ungrafted plant obtained from one or more parental plants that had been subjected to MSH1 gene suppression.
  • Such second plants can be plants that were selected for a useful trait and that were progeny of any plant or grafted plant that had subjected to MSH1 gene suppression.
  • Control plants used as comparators to identify progeny of the cross that exhibit an improvement in the useful trait include, but are not limited to: progeny of a cross between a plant which lacks a graft to the rootstock and a plant that is isogenic to the second plant, progeny of a self of a plant that lacks a graft to the rootstock, progeny of a self of the second plant; progeny of a cross between a plant that is isogenic to the plant source of the scion of the grafted plant and a plant that is isogenic to the second plant; and, progeny of a cross between a plant that is isogenic to the plant source of the scion of the grafted plant and that is isogenic to the plant source of a scion of the second plant when the second plant is a grafted plant. Also provided are methods where at least the scion of the first plant is from a different heterotic group than the second plant or where at least the scion of the first plant is from the same heterotic group
  • the selfed plant is a grafted plant where the rootstock source plant is the progeny of a parental plant that had been subjected to MSH1 gene suppression and the rootstock source plant itself was selected for and exhibits one or more useful traits.
  • Control plants used as comparators to identify progeny of the self that exhibit an improvement in the useful trait include, but are not limited to: progeny of a self of a plant which lacks a graft to the rootstock, progeny of a self of a plant that has a graft to rootstock that had not been subjected to MSH1 gene suppression, and progeny of a self of a plant that is isogenic to the plant source of the scion of the grafted plant.
  • Example 1 Enhanced yields in Sorghum occurs in the F3 and F4 hybrid-like
  • Sorghum MSHl-dr plants used in these experiments were derived as described (Xu YZl, Arri eta-Monti el MP, Virdi KS, de Paula WB, Widhalm JR, Basset GJ, Davila JI, Elthon TE, Elowsky CG, Sato SJ, Clemente TE, Mackenzie SA. Plant Cell. 2011 Sep;23(9):3428- 41).
  • RNAi transgene constructions segments encoding 157 amino acids from the MSH1 C terminus were amplified from total cDNA of sorghum using primers zm-msf8 (SEQ ID NO: 1) and zm-msr8 (SEQ ID NO: 2). PCR products were cloned in forward and reverse orientation, separated by an intron sequence in a base vector:
  • pUCRNAi- intron which harbors the second intron of the Arabidopsis small nuclear riboprotein (At4g02840), was rationally provided by H. Cerutti (University of Kansas, Lincoln, NE).
  • the vector pPTN290 a derivative of binary plasmid pPZP212, was used to construct the MSH1 -RNAi cassettes under the control of the maize (Zea mays)
  • Agrobacterium tumefaciens strain NTL4 was used for inoculating embryos from sorghum Tx430 lines.
  • Six T 3 individuals displaying the MSHl-dr phenotype but null for the MSHl-RNAi transgene were used as females in crosses to wild type inbred Tx430 to derive F 1 seed. Another three T 3 individuals were used as males in the reciprocal crosses to Tx430.
  • the number of F 1 plants derived from each cross ranged from 5 to 19 individuals.
  • Parents and F 1 progeny were grown under greenhouse conditions on a 14 hr/10 hr day-night cycle with 28°C/22°C day-night temperatures. Self-pollinated seed of F 1 plants was harvested individually to generate corresponding F 2 families.
  • the 2012 multi -location experiment included Lincoln, NE (40° 51 'N, 96° 35W) and Mead, NE (41° 9'N, 96° 24 W) sites, which received 178 mm and 158 mm of precipitation over the growing season, respectively.
  • lines were grown in two-row plots arranged in a randomized complete block design with two replicates.
  • grain yield was estimated by taking threshed panicles from a meter-length area of each row and converting to grams/m 2 .
  • Soybean variety Thorne was transformed with an RNAi construct containing a 556 bp cDNA segment (SEQ ID NO: 3) encoding amino acid 945 to 1131 of Soybean MShl, which represents the region after domain V to the end of GIY-YIG homing endonuclease domain, was PCR amplified from cDNA using primers Soy-MSF4 (SEQ ID NO: 4) and Soy-3Rbam (SEQ ID NO: 5). The PCR amplified fragment was cloned in forward and reverse orientation flanking the second intron of the Arabidopsis small nuclear riboprotein (At4g02840) in the pUCRNAi vector provided by Dr. H.
  • the CaMV 35S promoter and transcription terminator regulate expression of the construction and the neomycin phosphotransferase II (npt II) reporter gene, and the insert is flanked by right border (RB) and left border (LB) integration sequences.
  • RB right border
  • LB left border
  • Agrobacterium tumefaciens stain C58Cl/pMP90 was used for soybean transformation of the variety Thorne.
  • T8 and T9 refer to epigenetically modified dr-inbred parental lines crossed to Thorne to produce Fl seeds. Fl plants and later generations were then self-pollinated for 3 generations to produce F4 seeds for the field trial. Symbols: WT or Wt is the Thorne parent line; Bulk means no selection in previous F3 generation while Top 50% means the top 50% of F3 plants were selected to produce F4 seeds; Individual lineages end with R-# or P-#. * p-value ⁇ 0.05; *** p-value ⁇ 0.001.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Botany (AREA)
  • Developmental Biology & Embryology (AREA)
  • Environmental Sciences (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

L'invention concerne une méthode de production de rendements de type hybride dans des cultures normalement auto-pollinisées, les rendements étant les plus élevés dans un nombre ciblé de générations, et les générations ultérieures de plantes ayant des rendements relatifs inférieurs.
PCT/US2017/067907 2016-12-30 2017-12-21 Méthodes de production de lots de graines de type hybride Ceased WO2018125749A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201662440845P 2016-12-30 2016-12-30
US62/440,845 2016-12-30

Publications (1)

Publication Number Publication Date
WO2018125749A1 true WO2018125749A1 (fr) 2018-07-05

Family

ID=62709994

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2017/067907 Ceased WO2018125749A1 (fr) 2016-12-30 2017-12-21 Méthodes de production de lots de graines de type hybride

Country Status (1)

Country Link
WO (1) WO2018125749A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150113679A1 (en) * 2013-09-25 2015-04-23 Board Of Regents Of The University Of Nebraska Methods and Compositions for Obtaining Useful Plant Traits
US20150189842A1 (en) * 2013-11-07 2015-07-09 Board Of Regents Of The University Of Nebraska Methods and Compositions for Obtaining Useful Plant Traits
US20160192608A1 (en) * 2014-01-23 2016-07-07 The Board Of Regents Of The University Of Nebraska Epigenetically Enhanced Double Haploids
US20170223914A1 (en) * 2016-02-04 2017-08-10 Michael E Fromm Similar performance from seeds with epigenetic traits

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150113679A1 (en) * 2013-09-25 2015-04-23 Board Of Regents Of The University Of Nebraska Methods and Compositions for Obtaining Useful Plant Traits
US20150189842A1 (en) * 2013-11-07 2015-07-09 Board Of Regents Of The University Of Nebraska Methods and Compositions for Obtaining Useful Plant Traits
US20160192608A1 (en) * 2014-01-23 2016-07-07 The Board Of Regents Of The University Of Nebraska Epigenetically Enhanced Double Haploids
US20170223914A1 (en) * 2016-02-04 2017-08-10 Michael E Fromm Similar performance from seeds with epigenetic traits

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DE LA ROSA SANTAMARIA, R ET AL.: "MSH1-lnduced Non-Genetic Variation Provides a Source of Phenotypic Diversity in Sorghum bicolor", PLOS ONE, vol. 9, no. 10, 27 October 2014 (2014-10-27), pages 1 - 8, XP055516749 *
VIRDI, KS ET AL.: "MSH1 Is a Plant Organellar DNA Binding and Thylakoid Protein under Precise Spatial Regulation to Alter Development", MOLECULAR PLANT, vol. 9, no. 2, February 2016 (2016-02-01), pages 245 - 260, XP055516754 *

Similar Documents

Publication Publication Date Title
US9750205B2 (en) Quartet breeding
CA2937429A1 (fr) Plantes modifiees
US20220090118A1 (en) Powdery mildew resistant cannabis plants
AU2020285344B2 (en) Gene for parthenogenesis
CN116987727A (zh) 玉米基因krn2及其用途
JP2013507977A (ja) 増加した硬度を有するトマト果実
US20220162632A1 (en) Delayed harvest of short stature corn plants
CN109068605A (zh) 单性结实植物及其产生方法
US20250163445A1 (en) Domestication of a legume plant
CN114672492A (zh) 一种调控水稻株型的基因及其应用
WO2025052377A1 (fr) Plants de niébé présentant des caractéristiques améliorées et procédés pour les produire
JP2013128484A (ja) 近逆育種方法
IL288219B2 (en) Cannabis plants with improved yields
US20230193305A1 (en) Methods for increasing powdery mildew resistance in cannabis
WO2020025631A1 (fr) Résistance au cysdv chez des membres de la famille des cucurbitacées
US20210032645A1 (en) Targeted recombination between homologous chromosomes and uses thereof
EP4129050A1 (fr) Procédé de production d'un plant stérile mâle sensible à la température
AU2022206708A1 (en) Methods For Increasing Powdery Mildew Resistance In Cannabis
WO2018125749A1 (fr) Méthodes de production de lots de graines de type hybride
CN114786474A (zh) 灼烧病毒抗性基因
Mohr et al. CRISPR-Cas9 Gene Editing of the Sal1 Gene Family in Wheat. Plants 2022, 11, 2259
US20230203513A1 (en) Cucumber plant habit
US20220186243A1 (en) Cannabis plants with improved yield
WO2025131041A1 (fr) Protéine et gène mutants liés à la stérilité femelle de riz, et marqueur moléculaire et son utilisation
WO2024095258A1 (fr) Qtl de tomate épistatiques pour augmenter le rendement

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17886175

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17886175

Country of ref document: EP

Kind code of ref document: A1