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CN109111511A - The breeding method of super long granule rice - Google Patents

The breeding method of super long granule rice Download PDF

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CN109111511A
CN109111511A CN201810206586.5A CN201810206586A CN109111511A CN 109111511 A CN109111511 A CN 109111511A CN 201810206586 A CN201810206586 A CN 201810206586A CN 109111511 A CN109111511 A CN 109111511A
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何予卿
夏朵
周浩
张庆路
高冠军
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Huazhong Agricultural University
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Abstract

The invention belongs to Rice molecular breeding technical fields.More particularly to a kind of breeding method of super long granule rice.The method that the present invention utilizes map based cloning, a new rice grain length gene GL3.3 (amino acid sequence of gene coding is as shown in 1-1275bp in SEQ ID NO:1) has been cloned in separation, which polymerize the rice material for being capable of forming overlength grain length with known grain length gene GS3 (amino acid sequence of gene coding is as shown in 1-696bp in SEQ ID NO:3).The advantage allele of the two can be effectively selected and polymerize in breeding using the KASP molecular labeling primer of GS3 and GL3.3, the family with the super long granule of gs3gl3.3 genotype combination is selected, the breeding efficiency of rice quality can be significantly improved.

Description

The breeding method of super long granule rice
Technical field
The invention belongs to Rice molecular breeding technical fields.More particularly to a kind of breeding method of super long granule rice.This hair It is bright to have cloned a new rice grain length gene GL3.3, the gene and known grain length gene using the method separation of map based cloning GS3 polymerize the rice material for being capable of forming overlength grain length.It can be effective using the KASP molecular labeling primer of GS3 and GL3.3 The advantage allele of the two is selected and polymerize in breeding, selects the family with the super long granule of gs3gl3.3 genotype combination System, can significantly improve the breeding efficiency of rice quality.
Background technique
Rice is the staple food crop in China, and the exterior quality of rice is the important finger for determining rice commodity value Mark.For a long time, China's rice breeding man and Seed company are always using yield as the ultimate aim of breeding, and rice quality Improvement then compare lag (Kong Xianqin etc., 2013).The rice quality of China's major part Approved variety is more general, Wu Faman The national demand to rice best in quality of foot, price is lower after being processed into commodity rice, has deepened " the strong rice of rice is weak " to a certain extent The phenomenon that, cause China's rice import amount rising (Kong Xianqin etc., 2013；Yu Hongyan and Liu Shiyi, 2016).Because of japonica rice product Matter is generally better than long-grained nonglutinous rice, recent years, and conventional japonica rice kind is mainly planted regional cultivated area in China's rice and expanded year by year. China's rice variety best in quality is considerably less, therefore the genetic improvement of the exterior quality of long-grained nonglutinous rice especially indica Hybrid Rice is compeled in eyebrow Eyelash.
The exterior quality of rice mainly determines that the long-grained nonglutinous rice that particle shape is slender, chalkness ratio is low is by consumer by particle shape and chalk are white Favor (Bao, 2014).The exterior quality of rice is improved in traditional breeding method, it is usually that the high strain of yield and quality is excellent Strain is hybridized, then constantly with the high strain selfing of yield to obtain all excellent strain of yield and quality.But Since grain length, chalk be white etc., exterior qualities belong to quantitative character, by multiple gene regulations, need to carry out in biggish group Selection, and character observation also compares and takes time and effort, and practical improved, process difficulty is larger.By the method for molecular breeding, utilize The molecular labeling for having cloned the white gene of particle shape, chalk condenses together advantageous allele, only need to identify genotype, be not necessarily to It identifies phenotype, saves manpower and material resources, be the effective ways for improveing Appearance Quality of Paddy Rice.
Molecular breeding has many advantages, but this method first has to reliable and stable genetic resources and genotype group It closes.So far, researcher utilizes different groups and method, clones some rice figures and the white major gene resistance of chalk.Product The result of study of kind of sequencing and association analysis all shows GW5 and GS3 respectively and is that rice grain is wide and grain length is main and the base of main effect Because (Takanokai etc., 2009；Duan etc., 2017；Zhou etc., 2017).And other particle shape major gene resistances belong to mostly it is dilute Have a variation, such as the wide gene GW2 of grain, grain length gene GL3, seed size gene GS2 (Song, 2007；Qi etc., 2012；Hu etc., 2015).There are also some important rather than main effect particle shape genes, such as seed size gene GS5, length-width ratio gene GW8 and length-width ratio Gene GW7 etc. (Li et al., 2011；Wang etc., 2012；Wang etc., 2015).And the white gene of chalk only has Chalk5 to be cloned at present (Li et al., 2014).These genes provide valuable genetic resources for the improvement of Appearance Quality of Paddy Rice.Although the particle shape base of clone Because many, but the combination available for improvement exterior quality is seldom.Wang etc. (2012) utilizes GS3 and GW8 is idle The allelotype gs3 and gw8 of energy are aggregated in short round shaped grain material HJZ74, which is successfully modified to slender particle shape.Wang Deng (2014) later by directly researching material typhoon excellent 55 and typhoon excellent 208 with slender particle shape, find them because just There is the genotype combination of gs3 and GW7 and shows as slender particle shape.
Therefore, it is more reasonable effective method that the combination of efficient gene type is directly excavated from current material.Applicant Hybridized using the super long granule material of naturally occurring with short grain material, then parses the reason of causing overlength particle shape, it is last total The combination of efficient gene type is born to be used to improve rice grain length.It is rice by developing the molecular labeling of effective gene type combination The particle shape improvement of rice provides effective technical support.
Summary of the invention
The object of the present invention is to provide a kind of methods for cultivating super long granule rice, pass through two grain length genes of testing inspection Functional label, can be horizontal with regard to the grain length of Accurate Prediction rice plant in seedling stage, to accelerate the selection of super long granule rice family Progress.
The present invention identifies the gene of two grain lengths, and provides functional molecular labeling or labeled primer.By Stigma appearing gene purposefully accurately can be imported and be polymerize by these Molecular Marker Assisted Selection Technologies, thus high efficiency Quick breeding has the family of overlength particle shape, saves human and material resources, and keeps the stable yields and high yield of the paddy rice cross breeding production of hybrid seeds.
The invention is realized by the following technical scheme:
Applicant passes through the functional molecular marker primer Kasp-GS3 using gene GS3 (number of logging in Os03g0407400) By PCR amplification, then read tape by quantitative fluorescent PCR to detect the genotype of the gene, the molecular labeling primer DNA sequence dna is as follows:
Left end primer sequence 1 (Kasp-GS3F1): GAAGGTGACCAAGTTCATGCTACGCTGCCTCCAGATGCTGC,
Left end primer sequence 2 (Kasp-GS3F2): GAAGGTCGGAGTCAACGGATTACGCTGCCTCCAGATGCTGA,
Right end primer sequence (SF28R): GTAGTACAAAAAGAAACAGC；
Applicant further through another gene GL3.3 (number of logging in Os03g0841800) functional molecular marker primer Kasp-GL3.3 is expanded by PCR, is then read tape by quantitative fluorescent PCR to detect the genotype of the gene, the molecule mark Remember that the DNA sequence dna of primer is as follows:
Left end primer sequence 1 (Kasp-GL3.3F1): GAAGGTGACCAAGTTCATGCTTTTTGGGGTTGTATATCAGG,
Left end primer sequence 2 (Kasp-GL3.3F2): GAAGGTCGGAGTCAACGGATTTTTTGGGGTTGTATATCAGA,
Right end primer sequence (Kasp-GL3.3R): CACATAGAGAAATTTTTAC；
Applicant passes through verifying, it was demonstrated that above-mentioned two molecular labeling primer (that is, Kasp-GS3 and Kasp-GL3.3) can be with Genetic improvement applied to breeding overlength particle shape rice family.
Applicant provide a kind of molecule labelling method for screening rice grain length major gene resistance GS3, step includes: to utilize The molecular labeling primer (Kasp-GS3) of grain length major gene resistance GS3 expands oryza sativa genomic dna to be checked, and utilizes fluorescent quantitation PCR detects resulting amplified production, and banding pattern cluster is in X-axis, and genotype of reading tape is Allele X (5016), it was demonstrated that rice grain length The presence of the synergy allele gs3 of major gene resistance GS3.
At the same time, applicant provide the molecule labelling method of another screening rice grain length gene GL3.3, steps It include: to expand oryza sativa genomic dna to be checked using the molecular labeling (that is, Kasp-GL3.3) of grain length gene GW5, and utilize glimmering Fluorescent Quantitative PCR detects resulting amplified production, and banding pattern cluster is in X-axis, and genotype of reading tape is Allele X (5016), it was demonstrated that water The presence of the synergy allele gl3.3 of the long grain major gene resistance GL3.3 of rice.
Applicant provide a kind of application using molecular labeling primer in the assisted Selection of breeding super long granule rice, institutes The applying step stated includes:
(1) the excellent short grain of character will be accredited as and middle long grain backbone material is combined with high overlength grain gene type respectively The cultivated rice " Nan Yang accounts for " of gs3gl3.3 hybridizes, and respectively obtains F1A and F1B；
(2) with F1A obtained in step (1) and short 5 generation of grain backbone material continuous backcross, in per generation, utilizes molecular labeling primer Kasp-GS3 and molecular labeling primer Kasp-GL3.3 identifies true hybrid, until obtaining near isogenic lines BC5F1A；With step (1) in F1B obtained in and middle long 5 generation of grain backbone material continuous backcross, per generation, are identified using molecular labeling primer Kasp-GL3.3 True hybrid, until obtaining near isogenic lines BC5F1B；
(3) BC5F1A is selfed to obtain BC5F2A, utilizes molecular labeling primer Kasp-GS3 and molecular labeling primer Kasp- It is the single plant that homozygous genotype combines gs3gl3.3 that GL3.3, which identifies genotype, the short grain backbone material after as improveing；It will BC5F1B is selfed to obtain BC5F2B, identifies genotype for homozygous genotype combination using molecular labeling primer Kasp-GL3.3 The single plant of gl3.3,
Wherein:
The nucleotide sequence of the molecular labeling primer Kasp-GS3 such as sequence table SEQ ID NO:5 to SEQ ID NO: Shown in shown in 7；
The nucleotide sequence of the molecular labeling primer Kasp-GL3.3 such as sequence table SEQ ID NO:8 to SEQ ID Shown in NO:10.
The present invention has the advantages that
(1) present invention finds a new grain length gene GL3.3, which can be improved the grain length of rice.And GL3.3 and grain length gene GS3 has been cloned there are epistatic interaction, the polymerization of the two is capable of forming the rice grain shape of overlength.Forefathers Although cloning some grain length genes, the assembly effect of these genes is not studied.And general two genes Assembly effect can be cumulative less than two gene individual effects, and the assembly effect of GL3.3 and GS3 is greater than two gene lists Only effect adds up.
(2) the grain length major gene resistance that the present invention identifies have passed through genetic transformation verifying.Using the transgenic line of GL3.3, Applicant, which demonstrates the gene, can effectively improve the grain length of rice.GL3.3 by increase glume longitudinal cell length come Improve width of the grain length of rice without influencing cell.Therefore GL3.3 is a kind of special type, with the particle shape found before Gene is different from, and can more effectively be used for the improvement of grain length.
(3) the present invention provides the functional molecular markers of grain length major gene resistance GL3.3.Functional Markers for Detection Grain length gene loci in rice plant, only in seedling stage can Rapid identification go out the homozygous genotype single plant of long grain, eliminate in time Short grain single plant, not only saves production cost, but also substantially increase efficiency of selection, greatly the breeding week of shortening rice varieties Phase.And forefathers' research is mostly all without providing the functional molecular marker of gene.
Detailed description of the invention
Sequence table SEQ ID NO:1 be the code area (CDS) of grain length gene GL3.3 of the invention nucleotide sequence and its Corresponding amino acid sequence.
Sequence table SEQ ID NO:2 is the protein sequence of grain length gene GL3.3 coding.
Sequence table SEQ ID NO:3 is the nucleotide sequence and its corresponding amino of the code area (CDS) of grain length gene GS3 Acid sequence.
Sequence table SEQ ID NO:4 is the protein sequence of grain length gene GS3 coding.
Sequence table SEQ ID NO:5 is (the i.e. left end primer sequence of molecular labeling primer Kasp-GS3 of left end primer sequence 1 1)。
Sequence table SEQ ID NO:6 is (the i.e. left end primer sequence of molecular labeling primer Kasp-GS3 of left end primer sequence 2 2)。
Sequence table SEQ ID NO:7 is right end primer sequence (the i.e. right end primer sequence of molecular labeling primer Kasp-GS3 Column).
Sequence table SEQ ID NO:8 is (the i.e. left end primer of molecular labeling primer Kasp-GL3.3 of left end primer sequence 1 1)。
Sequence table SEQ ID NO:9 is (the i.e. left end primer of molecular labeling primer Kasp-GL3.3 of left end primer sequence 2 2)。
Sequence table SEQ ID NO:10 is right end primer sequence (i.e. the right end primer of molecular labeling primer Kasp-GL3.3).
Fig. 1: being general technical route map of the invention.
Fig. 2: the map based cloning of grain length gene GL3.3 of the present invention.Description of symbols: the A figure in Fig. 2 is parents and close etc. The mode of appearance of the seed of gene line；B figure in Fig. 2 is the result that particle shape QTL is just positioned in recombinant inbred lines；C in Fig. 2 D figure in figure and Fig. 2 is GL3.3 finely positioning process；E figure in Fig. 2 is equipotential difference of the candidate gene between parents.
Fig. 3: the genetic transformation verification process of grain length gene GL3.3 of the invention.Description of symbols: the A in Fig. 3 scheme to E figure is rice varieties treasure Shan 97 (ZS97) and the phenotypic difference of two kinds of seed progenies with CRISPR system knockout material: its In, the A figure in Fig. 3 is the rice paddy seed of the mutant material of ZS97 and knockout GL3.3 gene；B figure in Fig. 2 be ZS97 with With the comparison in difference of the nucleotide sequence of two kinds of mutant materials；C figure in Fig. 3 is the phenotype of transgenic paddy rice seed grain length Difference；D figure in Fig. 3 is the wide phenotypic difference of transgenic seed grain；E figure in Fig. 3 is the mass of 1000 kernel of transgenic paddy rice seed Phenotypic difference；I figure of the F figure into Fig. 3 is detection of the GL3.3 gene complementation to rice varieties phenotypic difference in Fig. 3, in which: figure F figure in 3 is the difference of the positive of rice varieties " Nanyang accounts for " and the seed phenotypes of negative-type；G figure in Fig. 3 is " Nanyang Account for " the positive and negative-type seed grain length；H figure in Fig. 3 is the positive of " Nanyang accounts for " and the seed of negative-type Grain is wide；I figure in Fig. 3 is the phenotypic difference of the mass of 1000 kernel of the positive of " Nanyang accounts for " and the seed of negative-type.
Fig. 4: grain length gene GL3.3 of the invention expression pattern and functional verification result.Description of symbols: in Fig. 4 A figure be GL3.3 subcellular localization；B figure in Fig. 4 is the GUS dyeing of various tissues；C figure in Fig. 4 is near isogenic lines Expression quantity of the middle GL3.3 in each period；D figure in Fig. 4 is the glume of near isogenic lines；E is that near isogenic lines is vertical in Fig. 4 To cellscan electron microscope；F and G is the comparison of near isogenic lines longitudinal cell number and cell length respectively in Fig. 4.
Fig. 5: grain length gene GL3.3 of the invention Population Genetics analysis.Description of symbols: in Fig. 5 A figure and: Fig. 5 In B figure be that allelic variation of the GL3.3 in 4726 kinds of cultivated rices and haplotype divide respectively；In C figure and Fig. 5 in Fig. 5 D figure is phenotypic difference of the GL3.3 difference allelotype in different subgroups；The figure of E is for the core of GL3.3 difference allelotype in Fig. 5 Sour polymorphic differences；F figure and G figure in Fig. 5 are that the extensibility haplotype homozygosity of the different allelotypes of GL3.3 is analyzed.
Fig. 6: grain length gene GL3.3 of the invention epistatic analysis test.Description of symbols: the A figure in Fig. 6 is The grain length for four kinds of genotype combinations that GS3 and GL3.3 are formed in japonica rice is distributed；B figure in Fig. 6 is GS3 and GL3.3 is in weight Mutually performing an analysis in group inbred line population；C figure in Fig. 6 is the seed of four kinds of genotype combination familys in recombinant inbred lines；Figure M figure of the D figure into Fig. 6 in 6 is that the phenotype of each economical character of GL3.3 near isogenic lines compares.All P values are all in figure It is test based on two tail t.
Fig. 7: grain length gene function Kasp mark identification method of the invention.Description of symbols: the A figure in Fig. 7 is Kasp-GS3 amplified production quantitative fluorescent PCR tape reading result；B figure in Fig. 7 is Kasp-GL3.3 amplified production fluorescent quantitation PCR tape reading result.Wherein: X-axis and Y-axis respectively represent the power of two kinds of fluorescence signals.
Fig. 8: the stream of the GL3.3 gene of the invention molecular marker-assisted selection method that grain length is improved in cultivated rice application Cheng Tu.
Specific embodiment
General technical route of the invention:
The present invention is carried out according to following general technical route:
1, gene finely positioning；
2, gene function verifying and effect analysis (see Fig. 1).
General technical step 1, the finely positioning of gene are implemented (specifically according to map based cloning method common on science of heredity Reference implementation example 1).
(1) selection of material.The apparent parents of selection target Traits change construct genetic group more likely orientation effect Big QTL.The group of Primary Location is generally the F of biparent cross generation2Group or recombinant inbred lines (RIL) group (Fang Xuan An ancient unit of weight etc., 2000；Yano and Sasaki, 1997).The RIL group is due to that can repeat mostly for multi-environment close to pure lines Phenotype is investigated, detection that can be effective and stable and verifying QTL.
(2) near isogenic lines is constructed.In order to realize the finely positioning to QTL, need by being returned between parent, Construct near isogenic lines (NIL) makes the effect of target QTL be rendered as single Mendelian factor to eliminate the influence of background Variation (Tuinstra et al., 1997).BC is arrived in general backcrossing3Generation or BC4In generation, background is purer, can selection target The single plant of section heterozygosis carries out selfing and obtains a large amount of BC3F2Or BC4F2Single plant is recombinated for screening, realizes that QTL's is fine fixed Position.
(3) single plant is recombinated using bilateral label screening when just positioning, then by the section encrypted indicia, using dyeing The method that body step is moved positions section to reduce.If can not confirm the genotype of certain groups of single plants of recombination according to contemporary phenotype, It needs to carry out progeny test to these single plants to confirm their genotype.Progeny test is oneself for planting some recombination single plant F2 group is handed over, whether the phenotype for then investigating group separates.Confirm that the recombination single plant genotype is miscellaneous if separation occurs Mould assembly；Confirm that the genotype of the recombination single plant is homozygous if separation does not occur.QTL can be navigated to using the above method Sufficiently small section usually only includes a candidate gene.If bioinformatics can be combined containing multiple candidate genes Method and expression analysis method excluded.In addition, finding equipotential change by the way that candidate gene in parents is sequenced Different is also the effective ways for confirming and verifying candidate gene.
General technical step 2, the i.e. functional verification of gene are implemented using the method for conventional genetic conversion (with specific reference to implementation Example 2).
The preparation of genetic transformation is divided into two steps while carrying out: the first step is the culture of recipient cell；Second step is mesh Gene preparation.
Genetic transformation during map based cloning is typically chosen receptor parent as conversion object.In rice, japonica rice Transformation efficiency is much higher than long-grained nonglutinous rice, so applicant is to select japonica rice material as conversion object in many cases.It is right first Acceptor material, that is, brown rice carries out tissue cultures according to a conventional method, carries out after inducing differentiation to obtain callus conventional after being commissioned to train It supports.Construction of expression vector and target fragment is cloned on expression vector at the same time.Next good callus is selected Preculture is carried out, the carrier that thus can use after purification carries out Agrobacterium-mediated Transformation or other conversions.Cell after conversion is first First positive carry out plant regeneration culture is selected by screening.Phenotypic evaluation and genotype identification are carried out to regeneration plant, Until obtaining the positive singly copied and negative plant that offspring isolates.By the plantation of random district's groups to positive negative-type and Phenotype investigates the effect for determining target gene.
Embodiment 1: the positioning of grain length major gene resistance GL3.3
1. the building of recombinant inbred lines
(NYZ) is accounted for using super long granule japonica rice variety Nanyang and short grain rice variety treasure Shan 97 (ZS97) carries out hybridization and obtains F1 (parents' seed morphology referring to fig. 2 in A figure), be then selfed again, altogether obtain 190 plants of F2Group.The F of this 190 single plants2 Group can carry out constantly selfing until F according to the method (Fang Xuanjun etc., 2000) of " biography "7Generation (Fig. 1).
2. genotype identification and genetic map construction
The identification method of genotype is conventional ssr analysis method.Specific steps, first choice are screening polymorphism SSR primers.This 574 pairs of SSR primers have been screened in invention altogether, and wherein the primer of RM series is 452 pairs, and the primer of MRG series is 118 pairs.RM series Primer accounts for that have the label of polymorphism between precious Shan 97 be pair that MRG series primer is 54 in precious Shan 97 in rice varieties Nanyang It is right.Since polymorphism mark is more sufficient, genetic linkage maps are mainly constructed by RM series primer, and MRG series primer is mainly used To fill up missing (GAP).After the genotype for obtaining 190 labels using ssr analysis, applicant utilizes MAPMAKER/Exp3.0 " Kosambi " mapping function of software draws genetic linkage map.The genetic map overall length 1362.1cM finally obtained, between label Average distance be 7.77cM (Wang Lingqiang, 2007).
3. the Primary Location of grain length QTL
By 190 parts of RILs materials in 2001 and plantation in continuous 2 years of in May, 2002 to October in Wuhan City, Hubei Province China Middle agriculture university experimental plot.Each family plants 2 rows, 10 plants of every row, and seeding row spacing is 16.5cm × 26.4cm.Each family is examined Examine 8 plants intermediate of grain length, grain length phenotypic number of 8 plants of the mean value as the family.The identification of QTL be based on genotype, phenotype and Genetic linkage map is step-length complete using 2cM using the composite interval mapping method of WinQTLCart2.5 software (Zeng, 1994) The relevant QTL of genome detection grain length.The threshold value of QTL detection is LOD > 2.5, that is, thinking that the section exists influences grain length QTL.The peak shape of QTL scanning is desired to make money or profit with R Software on Drawing (Ihaka and Gentleman, 1996).6 grain lengths are detected in total QTL, wherein qGS3 the and qGL3.3 effect for being located at third chromosome is the most significant (see the B figure in Fig. 2).
The finely positioning of 4.GL3.3
For finely positioning GL3.3, applicant constructs the BC of ZS97 background4For near isogenic lines (NIL).NIL-NYZ With the grain length (the A figure in Fig. 2 and the G figure in Fig. 6) for being significantly higher than NIL-ZS97.In 5950 plants of BC4F2In single plant, applicant 187 recombination single plants exchanged between marking D3-3 and RM442 are identified.Single plant is recombinated using these, applicant will GL3.3 is navigated between D3-8 and D3-9, about the section of 15kb (see E figure of the C figure in Fig. 2 into Fig. 2).This section Comprising two open reading frame, Os03g0841800 and Os03g0841900.Compare sequencing discovery by parents, the latter is double Non-functional makes a variation between parent.And Os03g0841800 has the variation of G a to A after third exon, which leads to Nanyang The transcription product for accounting for the gene in (NYZ) lacks the third and fourth exon (see the E figure in Fig. 2).Therefore, applicant is preliminary Confirm that Os03g0841800 is the candidate gene of GL3.3
The functional verification of embodiment 2:GL3.3 and effect analysis
1, the functional verification of GL3.3
Os03g0841800 encodes the open reading frame (ORF) of a 6281kb, which includes 12 exons and 11 Introne with reference to annotation in genome is GSK5 (Kawahara et al., 2013) in japonica rice " OryzasativaLcv.Nipponbare ".In order to further Whether confirmation Os03g0841800 is exactly GL3.3, and applicant utilizes CRISPR/Cas9 system (Shan et al., 2013) to exist GSK5 gene has been knocked out in short grain long-grained nonglutinous rice treasure Shan 97.Applicant obtains two kinds of mutation for causing the genetic transcription to terminate in advance Body (E figure of the A figure into Fig. 3 in Fig. 3).Two kinds of mutants all have significant compared to wild type ZS97 (non-transgenic) Grain length and the grain weight of raising (see figure of the C figure into E Fig. 3 in Fig. 3).On the other hand, applicant's complementary conversion in " NYZ " The allelotype gene (Hiei et al., 1994) of GL3.3 in " ZS97 ".Analysis the result shows that, transgene negative plant With longer particle shape more significant than positive plant and higher mass of 1000 kernel (see I figure of the F figure in Fig. 3 into Fig. 3).These results Show that GSK5 is exactly GL3.3 and the gene is the gene of a negative regulation rice grain length and mass of 1000 kernel.
The expression pattern and function of 2.GL3.3
In order to position the position that GL3.3 is expressed in cell, applicant constructs the driving of CaMV 35S promoter GL3.3- yellow fluorescence protein merges (YFP) expression vector (Hajdukiewicz et al., 1994).Rice transformation plasm Body (Xue et al., 2008) the result shows that GL3.3-YFP is located in nucleus, this shows that GL3.3 is expressed in nucleus (the A figure in Fig. 4).Applicant utilizes quantitative fluorescent PCR (RT-PCR) (Livak et al., 2001) and the routine dyeing side GUS Method analyzes the spatial and temporal expression characteristic of GL3.3 gene.GUS coloration result shows GL3.3 gene in the rice varieties tested Endosperm, seedling, great expression (the B figure in Fig. 4) in stem and floral organ.The RT-PCR of NIL-NYZ and NIL-ZS97, as a result Show expression in seedling of the mRNA in rice varieties, young fringe, endosperm, stem and the root of GL3.3 gene (the C figure in Fig. 4).Therefore Presumption GL3.3 gene is the gene of an interim constitutive expression in each tissue of rice.Since the glume of NIL-NYZ exists Prefecundation just than NIL-ZS97 long, so applicant parses grain husk using the glume outer surface of two NILs of scanning electron microscope analysis The concrete reason of shell difference in length (see G figure of the D figure in Fig. 4 into Fig. 4).It was found that the longitudinal cell number of NIL-NYZ is significant Longitudinal cell number less than NIL-ZS97；And the longitudinal cell length of NIL-NYZ will be noticeably greater than NIL-ZS97 longitudinal cell Length.Therefore, the reduction of NIL-NYZ glume longitudinal cell number~10% and the increase of cell length~20% result in The increase of whole glume length.
The allelotype of 3.GL3.3 and artificial selection
Since GL3.3 is the gene of a negative regulation grain length, Nanyang accounts for the gl3.3 that (NYZ) contains a kind of saltant type. In 4726 parts of cultivated rice materials, the ratio of this allelotype contained by NYZ is 1%, and this allelotype is only present in In japonica rice, therefore applicant is called gl3.3Jap(A in Fig. 5 schemes, the B figure in Fig. 5).In japonica rice, there is gl3.3JapThis The kind of kind allelotype has significant longer particle shape (the D figure in Fig. 5) compared to the kind of wild type.By further analyzing The natural variation of GL3.3, applicants have discovered that another functional variation: that is, on the 6th exon one by C to A's Conversion, leads to variation of the amino acid sequence from histidine to glutamine.Ratio of this allelotype in 4726 parts of cultivated rices It is 5%, and is primarily present in the rice of Australia, therefore applicant is referred to as gl3.3Aus(A in Fig. 5 schemes, the B in Fig. 5 Figure).In the rice of Australia, the kind with this allelotype has significant longer particle shape (in Fig. 5 compared to the kind of wild type C figure).The polymorphic nucleic acid of two kinds of saltant types and wild type, applicant are calculated using DnaSP software (Rozas etc., 2003) It was found that two kinds of saltant types all have significantly lower polymorphism (the E figure in Fig. 5), it may be during breeding by remarkable Work selection.It is homozygous that applicant further studies the extensibility haplotype of GL3.3 wild type and saltant type in Australia rice and japonica rice Property, it is found that the linkage disequilibrium of wild type fails quickly, and saltant type has the slow of decline, shows that saltant type receives Artificial selection (F in Fig. 5 schemes, the G figure in Fig. 5).
4.GL3.3 and GS3 epistatic interaction form overlength particle shape
Although gl3.3AusAnd gl3.3JapThe natural variation of rice grain length is all influenced, but compared to GL3.3 in mapping population In additive effect (a=0.35mm), gl3.3AusEffect (a=0.24mm) less than normal and gl3.3JapEffect it is significantly bigger than normal (a=1.36mm) (see the C figure in Fig. 5, the D in Fig. 5 schemes).It has been more than known since effect of the GL3.3 in japonica rice is excessive Any particle shape major gene resistance, applicant speculate may GL3.3 in japonica rice and some grain length gene exist it is long-range chain It is uneven.By analyzing the genotype of particle shape major gene resistance GS3, it is found by the applicant that 92% has gl3.3JapThe japonica rice of allelotype Kind also has gs3 allelotype, show gs3 in japonica rice with gl3.3JapLong-range chain (see the A figure in Fig. 6).Forefathers' research Show gs3 in breeding process by artificial selection (Takanokai etc., 2009), therefore gl3.3JapIn overlength together with gs3 It is selected in grain japonica rice variety.The effect of GS3 is considered in fact, and effect (a=0.45mm) of the GL3.3 in japonica rice is still Greater than the effect (the A figure in Fig. 6) in mapping population, therefore applicant suspects that GL3.3 may there are interactions with GS3.Pass through weight The effect of GS3 and GL3.3 in new analysis recombinant inbred lines, it is found by the applicant that there are apparent epistasis for the two genes Interaction (B in Fig. 6 schemes, the C figure in Fig. 6).This interaction makes the japonica rice of GL3.3 effect under the non-functional background of GS3, That is gs3gl3.3 will form a kind of particle shape of overlength.Effect of the GL3.3 in " NYZ " complementary transgenic line is greater than " ZS97 " The effect for knocking out transgenic line also further illustrates this point (see Fig. 3).
Application of the embodiment 3:GL3.3 in the improvement of rice grain length
The GL3.3 that the present invention navigates to be rare and main effect a grain length gene, and can with GS3 interaction and play Stronger function forms super long granule phenotype, can effectively improve the grain length character of existing cultivation rice varieties.It can be in breeding process The middle molecular labeling using GL3.3, which carries out assisted Selection, can reduce cultivated area, simplifies breeding procedures and save manpower object Power, to improve breeding efficiency.Therefore will there is GL3.3 in rice germplasm material (such as kind " Nanyang accounts for " in this experiment) Nonfunctional allelotype (gl3.3AusAnd gl3.3Jap) material be used for the improvement of rice grain length character, have and very extensive answer Use prospect.
The technology path of the present embodiment can be found in method described in Fig. 7, and Fig. 7 is the utilization GL3.3 advantage of applicant's design The general technical process of allele improvement rice grain length.The techniqueflow include: first selection production on short grain or in Long grain backbone rice varieties material, i.e., abbreviation " backbone material " (these " backbone materials " be all it is available, be included in In production have merit and grain length needs the conventional rice variety and sterile line improved and restorer, identify above-mentioned backbone The method of material is all the method for existing report, and there is no exploration labour, the method for obtaining sterile line and restorer is also all The method of existing report and the common knowledge of this field), with the cultivated rice material for having preponderant genotype and combining gs3gl3.3 (such as " Nanyang accounts for ", but not limited to this) hybridized, short grain backbone material cenospecies F can be respectively obtained1Generation (or F1A) and Middle long grain backbone material F1Generation (or F1B).By F1AWith short 5 generation of grain backbone material continuous backcross (this is the known method in field) It can be obtained near isogenic lines, per generation selects the two sites using molecular labeling primer Kasp-GS3 and Kasp-GL3.3 True hybrid single plant (Fig. 7), until obtaining BC5F1A.It is corresponding, F1BIt is obtained with middle long 5 generation of grain backbone material continuous backcross close etc. In gene line, per generation, all utilize molecular labeling primer Kasp-GS3 and Kasp-GL3.3 to select the true hybrid single plant in the two sites, Until obtaining BC5F1B.The near isogenic lines BC of short grain backbone material and middle long grain backbone material5F1AAnd BC5F1BIt is selfed respectively, Obtain BC5F2AAnd BC5F2B.Molecular labeling primer Kasp-GS3 and Kasp-GL3.3 is recycled to identify BC5F2AIn have homozygosis Short grain backbone rice material after the improvement of the single plant of gs3gl3.3 genotype combination, as stigma exposing ratio.Recycle molecule mark Note primer Kasp-GS3 and Kasp-GL3.3 identify BC5F2BIn the single plant with homozygosis gl3.3 genotype combination, as column cap Middle long grain rice backbone material after exposing ratio improvement.Long grain backbone material in the short grain backbone material of rice and rice after improvement Material particle shape has become super long granule, therefore can be referred to as rice super long granule backbone material.
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Sequence table
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Met Ala Tyr Ser Gly Leu Arg His Gly Gly Val Gly Ser Ser Ser Arg
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Pro Gly His Gly Phe Lys Gly Pro Ala Ser Ser Val Glu Cys Leu Gly
20 25 30
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Arg Glu Met Leu Glu Met Gln Leu Arg Asp Ser Lys Pro Asp Val Gly
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Asp Glu Lys Asn Thr Glu Arg Asp Val Val Asp Gly Ser Ser Ala Glu
50 55 60
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Ala Gly His Ile Ile Ala Thr Thr Ile Arg Gly Arg Asn Gly Leu Pro
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Phe Gly Val Val Tyr Gln Ala Lys Cys Arg Glu Thr Gly Glu Ile Val
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Ala Ile Lys Lys Val Leu Gln Asp Lys Arg Tyr Lys Asn Arg Glu Leu
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Gln Ile Met His Met Leu Asp His Pro Asn Ile Val Gly Leu Lys His
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Tyr Phe Phe Ser Thr Thr Glu Arg Asp Glu Leu Tyr Leu Asn Leu Val
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Leu Glu Tyr Val Pro Glu Thr Val Asn Arg Ile Ala Arg Gln Tyr Ser
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Arg Met Asn Gln Arg Val Pro Leu Ile Tyr Val Lys Leu Tyr Thr Tyr
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Gln Ile Cys Arg Ala Leu Ala Tyr Ile His Asn Cys Val Gly Ile Cys
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His Arg Asp Ile Lys Pro Gln Asn Val Leu Val Asn Pro His Thr His
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Glu Pro Asn Ile Ser Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala Pro Glu
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Leu Ile Phe Gly Ala Thr Glu Tyr Thr Thr Ala Ile Asp Leu Trp Ser
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Thr Gly Cys Val Met Ala Glu Leu Leu Leu Gly Gln Pro Leu Phe Pro
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Gly Glu Ser Gly Val Asp Gln Leu Val Glu Ile Ile Lys Val Leu Gly
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Thr Pro Thr Arg Glu Glu Ile Lys Cys Met Asn Pro Asn Tyr Thr Glu
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Arg Glu Met Leu Glu Met Gln Leu Arg Asp Ser Lys Pro Asp Val Gly
35 40 45
Asp Glu Lys Asn Thr Glu Arg Asp Val Val Asp Gly Ser Ser Ala Glu
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Ala Gly His Ile Ile Ala Thr Thr Ile Arg Gly Arg Asn Gly Leu Pro
65 70 75 80
Lys Gln Ser Val Thr Tyr Ile Ala Glu His Val Val Gly Thr Gly Ser
85 90 95
Phe Gly Val Val Tyr Gln Ala Lys Cys Arg Glu Thr Gly Glu Ile Val
100 105 110
Ala Ile Lys Lys Val Leu Gln Asp Lys Arg Tyr Lys Asn Arg Glu Leu
115 120 125
Gln Ile Met His Met Leu Asp His Pro Asn Ile Val Gly Leu Lys His
130 135 140
Tyr Phe Phe Ser Thr Thr Glu Arg Asp Glu Leu Tyr Leu Asn Leu Val
145 150 155 160
Leu Glu Tyr Val Pro Glu Thr Val Asn Arg Ile Ala Arg Gln Tyr Ser
165 170 175
Arg Met Asn Gln Arg Val Pro Leu Ile Tyr Val Lys Leu Tyr Thr Tyr
180 185 190
Gln Ile Cys Arg Ala Leu Ala Tyr Ile His Asn Cys Val Gly Ile Cys
195 200 205
His Arg Asp Ile Lys Pro Gln Asn Val Leu Val Asn Pro His Thr His
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Gln Leu Lys Ile Cys Asp Phe Gly Ser Ala Lys Val Leu Val Lys Gly
225 230 235 240
Glu Pro Asn Ile Ser Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala Pro Glu
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Leu Ile Phe Gly Ala Thr Glu Tyr Thr Thr Ala Ile Asp Leu Trp Ser
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Thr Gly Cys Val Met Ala Glu Leu Leu Leu Gly Gln Pro Leu Phe Pro
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Gly Glu Ser Gly Val Asp Gln Leu Val Glu Ile Ile Lys Val Leu Gly
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Phe Lys Phe Pro Gln Ile Lys Ala His Pro Trp His Lys Val Phe Gln
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Lys Arg Leu Pro Pro Glu Ala Val Asp Leu Val Ser Arg Phe Leu Gln
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Tyr Ser Pro Asn Leu Arg Cys Thr Ala Met Glu Ala Cys Met His Pro
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Phe Phe Asp Glu Leu Arg Asp Pro Asn Thr Arg Leu Pro Asn Gly Arg
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Pro Leu Pro Pro Leu Phe Asn Phe Arg Thr Gln Glu Leu Asn Gly Ile
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Asp Pro Cys Gly Arg His Arg Leu Gln Leu Ala Val Asp Ala Leu His
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His Ser His His Phe Leu Lys Lys Phe Arg Cys Leu Cys Arg Ala Ser
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Ala Cys Cys Leu Ser Tyr Leu Ser Trp Ile Cys Cys Cys Ser Ser Ala
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Ala Gly Gly Cys Ser Ser Ser Ser Ser Ser Ser Phe Asn Leu Lys Arg
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Pro Ser Cys Cys Cys Asn Cys Asn Cys Asn Cys Cys Ser Ser Ser Ser
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Ser Ser Cys Gly Ala Ala Leu Thr Lys Ser Pro Cys Arg Cys Arg Arg
145 150 155 160
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Arg Ser Cys Cys Cys Arg Arg Cys Cys Cys Gly Gly Val Gly Val Arg
165 170 175
gcg tgc gcg agc tgc agc tgc tcc ccg ccg tgc gcg tgc tgc gcg ccg 576
Ala Cys Ala Ser Cys Ser Cys Ser Pro Pro Cys Ala Cys Cys Ala Pro
180 185 190
ccg tgc gcg gga tgc tcg tgc cgc tgc acc tgc ccg tgc ccg tgc ccc 624
Pro Cys Ala Gly Cys Ser Cys Arg Cys Thr Cys Pro Cys Pro Cys Pro
195 200 205
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Gly Gly Cys Ser Cys Ala Cys Pro Ala Cys Arg Cys Cys Cys Gly Val
210 215 220
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Pro Arg Cys Cys Pro Pro Cys Leu
225 230
<210> 4
<211> 232
<212> PRT
<213>rice (Oryza sativa)
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Met Ala Met Ala Ala Ala Pro Arg Pro Lys Ser Pro Pro Ala Pro Pro
1 5 10 15
Asp Pro Cys Gly Arg His Arg Leu Gln Leu Ala Val Asp Ala Leu His
20 25 30
Arg Glu Ile Gly Phe Leu Glu Gly Glu Ile Asn Ser Ile Glu Gly Ile
35 40 45
His Ala Ala Ser Arg Cys Cys Arg Glu Val Asp Glu Phe Ile Gly Arg
50 55 60
Thr Pro Asp Pro Phe Ile Thr Ile Ser Ser Glu Lys Arg Ser His Asp
65 70 75 80
His Ser His His Phe Leu Lys Lys Phe Arg Cys Leu Cys Arg Ala Ser
85 90 95
Ala Cys Cys Leu Ser Tyr Leu Ser Trp Ile Cys Cys Cys Ser Ser Ala
100 105 110
Ala Gly Gly Cys Ser Ser Ser Ser Ser Ser Ser Phe Asn Leu Lys Arg
115 120 125
Pro Ser Cys Cys Cys Asn Cys Asn Cys Asn Cys Cys Ser Ser Ser Ser
130 135 140
Ser Ser Cys Gly Ala Ala Leu Thr Lys Ser Pro Cys Arg Cys Arg Arg
145 150 155 160
Arg Ser Cys Cys Cys Arg Arg Cys Cys Cys Gly Gly Val Gly Val Arg
165 170 175
Ala Cys Ala Ser Cys Ser Cys Ser Pro Pro Cys Ala Cys Cys Ala Pro
180 185 190
Pro Cys Ala Gly Cys Ser Cys Arg Cys Thr Cys Pro Cys Pro Cys Pro
195 200 205
Gly Gly Cys Ser Cys Ala Cys Pro Ala Cys Arg Cys Cys Cys Gly Val
210 215 220
Pro Arg Cys Cys Pro Pro Cys Leu
225 230
<210> 5
<211> 41
<212> DNA
<213>rice (Oryza sativa)
<220>
<221> primer_bind
<222> (1)..(41)
<400> 5
gaaggtgacc aagttcatgc tacgctgcct ccagatgctg c 41
<210> 6
<211> 41
<212> DNA
<213>rice (Oryza sativa)
<220>
<221> primer_bind
<222> (1)..(41)
<400> 6
gaaggtcgga gtcaacggat tacgctgcct ccagatgctg a 41
<210> 7
<211> 20
<212> DNA
<213>rice (Oryza sativa)
<220>
<221> primer_bind
<222> (1)..(20)
<400> 7
gtagtacaaa aagaaacagc 20
<210> 8
<211> 41
<212> DNA
<213>rice (Oryza sativa)
<220>
<221> primer_bind
<222> (1)..(41)
<400> 8
gaaggtgacc aagttcatgc tttttggggt tgtatatcag g 41
<210> 9
<211> 41
<212> DNA
<213>rice (Oryza sativa)
<220>
<221> primer_bind
<222> (1)..(41)
<400> 9
gaaggtcgga gtcaacggat tttttggggt tgtatatcag a 41
<210> 10
<211> 19
<212> DNA
<213>rice (Oryza sativa)
<220>
<221> primer_bind
<222> (1)..(19)
<400> 10
cacatagaga aatttttac 19

Claims (4)

1. a kind of grain length major gene resistance GL3.3 is cultivating the application in super long granule rice, which is characterized in that the GL3.3 base Because the amino acid sequence of coding is as shown in 1-1275bp in SEQ ID NO:1.
2. the super long granule genotype combination gs3gl3.3 of grain length major gene resistance GS3 and GL3.3 are cultivating answering in super long granule rice With, which is characterized in that the amino acid sequence of the GL3.3 gene coding is described as shown in 1-1275bp in SEQ ID NO:1 GS3 gene coding amino acid sequence as shown in 1-696bp in SEQ ID NO:3.
3. a kind of molecular labeling primer Kasp-GS3 is cultivating the application in super long granule rice, which is characterized in that the molecule The nucleotide sequence of labeled primer Kasp-GS3 is as shown in SEQ ID NO:5 to SEQ ID NO:7.
4. a kind of molecular labeling primer Kasp-GL3.3 is cultivating the application in super long granule rice, which is characterized in that point The nucleotide sequence of sub- labeled primer Kasp-GL3.3 is as shown in SEQ ID NO:8 to SEQ ID NO:10.
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CN111979233A (en) * 2019-05-22 2020-11-24 江苏省农业科学院 Method for increasing rice grain type and application thereof
CN111849999A (en) * 2019-10-24 2020-10-30 扬州大学 Rice GS3 mutant gene, its molecular marker and application
CN111849999B (en) * 2019-10-24 2022-11-29 扬州大学 Rice GS3 mutant gene, its molecular marker and application
CN112485216A (en) * 2020-11-20 2021-03-12 华中农业大学 Multi-source information fusion Thailand jasmine rice adulteration identification method
CN112485216B (en) * 2020-11-20 2022-02-01 华中农业大学 Multi-source information fusion Thailand jasmine rice adulteration identification method

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