CN102703438B - Molecular marker of brassica napus L. grain weight character and preparation method and application - Google Patents
Molecular marker of brassica napus L. grain weight character and preparation method and application Download PDFInfo
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Abstract
The invention belongs to the field of rape molecular breeding, and relates to a specific molecular marker of brassica napus L. grain weight and a preparation method and an application. Hybridization is performed by using brassica napus L. A254 as a female parent and using brassica napus L. A177 as a male parent to construct a dihaploid colony (DH); data of the genotype and 1000-grain weight of the DH colony are analyzed to obtain QTLs loci of the grain weight character. MINI3 and TTG2 genes of A254 and A177 are cloned by a homologous sequencing method; specific molecular marker MINI3a and TTG2a of the MINI3 and TTG2 genes are designed according to sequence different loci; the molecular marker MINI3a and TTG2a are positioned on two grain weight QTLs loci of a A5 linkage group; related verification and application demonstrate that the molecular marker prepared by the invention is a new genetic marker. The invention provides a new genetic marker for rape grain weight molecular breeding.
Description
Technical field
The present invention is that application number is dividing an application of 201010169042.X patent application.
The invention belongs to rape molecular breeding and biological technical field, be specifically related to discovery, the cloning and identification of swede type rape grain re-correlation gene, and the development and application of the specific molecular marker of genes involved.
Background technology
Swede type rape (Brassica napus L. is hereinafter to be referred as rape) is one of most important oil crops in the world.The seed of rape is not only the storage organ of oil and protein, is also simultaneously the organ of plant life cycle continuity.Seed size or weight are very important economic characters.At first grain is heavily one of three large factors that consist of yield per plant (individual plant effective angle fruit number, Seed number per pod, grain are heavy), so is also determining output (Clarke and Simpson, 1978; Butruille et al., 1999; Shi et al., 2009); Secondly, seed size also has relation (Morgan et al., 1998 with oleaginousness and protein content; Lionneton et al., 2004); Again, large seed has better adaptability usually in germination process.Therefore, understand fully the hereditary basis that seed size or weight form, very important to the improvement of yield of rape and quality.In addition, from the angle of evolving, understand fully that the variation of seed size also has very important meaning.
Although the seed size of rape is extremely important, at present its Genetic Control is still lacked deep understanding.Compare heritability higher (Liu et al., 1987 that grain is heavy with other Correlated Yield Characters; Qi et al., 2004; Shi et al., 2009).Along with the development of molecular marking technique, also located the heavy quantitative trait locus (Quantitative Trait Loci, QTL) of some rape grains at present.Quijada et al. (2006) utilizes pair tests in 2 years of four colonies to locate three and weighs relevant QTLs (being positioned at N7, N17 and N19) with grain, but there is no identical QTL between different groups; Udall et al. (2006) detect respectively between three different groups such as Hua Double Haploid (DH) colony, SYN DH colony and test cross colony respectively 6,4 with the heavy relevant QTLs of 5 grains, only have a QTL who is positioned on N14 stable detection to arrive between different groups and varying environment; Recently, Shi et al. (2009) utilizes two colonies of rape the heavy QTLs of 159 grains to be detected altogether under 10 varying environments, and these QTLs are distributed on other all karyomit(e)s except Cl.
Utilize model plant Arabidopis thaliana (Arabidopsis thaliana), utilize the means such as Analysis of Mutants in more than ten years in the past, the molecular regulation mechanism of seed size is studied.Alonson-Blanco et al., (1999) have located 11 QTLss relevant with seed size, have disclosed for the first time the hereditary complicacy of this proterties between differing materials.Recently, to the analysis of mass mutation body, further illustrated the molecule mechanism of many decision seed sizes.For example TTG2 (Transparent Testa Glabrous 2) gene mutation body, affect the accumulation of the Protoapigenone in kind of skin, usually can reduce grain heavy (Debeaujon et al., 2000,2003).And the sudden change of the transcription factors such as AP2 (APETELA2) or ARF2 (Auxin Response Factor 2) can make seed become large (Jofuku et al., 2005; Ohto et al., 2005; Schruff et al., 2005).Luo et al. (2005) has identified two seedlet mutant IKU2 (HAIKU2) and MINI3 (MINISEED3), and proposes first the genetically controlled possibility of seed size pathways metabolism.In view of rape and the very close kinship of Arabidopis thaliana, expection can utilize the information of Arabidopis thaliana, obtains the homologous gene of relevant controlling seed size from the rape genome.
Have no in rape at present the clone of the heavy related gene of grain and the report of analysis.In view of the importance of grain principal characteristic shape, to discovery, the cloning and identification of the heavy related gene of grain in rape, and the exploitation of gene specific molecular marker is to promoting that yield of rape and quality breeding are very necessary.
Summary of the invention
The purpose of this invention is to provide the molecule marker of swede type rape grain re-correlation gene M INI3 and TTG2 and above-mentioned 2 gene specifics, and use it for the seed selection of rape grain principal characteristic shape.These molecule markers and method can be the swede type rape grain heavily breeding new means are provided, thereby accelerate the improvement process of swede type rape grain principal characteristic shape, improve accuracy and the efficiency of selection of swede type rape breeding.
The present invention is achieved by the following scheme.
a) swede type rape A-grade in the first class 254 (large grain pure lines, the seed of this material has been delivered Chinese Typical Representative culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on November 20th, 2009, its preserving number is CCTCC NO:P200909, the publication number of formerly patent application is: CN101962640A, open day: on 02 02nd, 2011, former number of patent application is 201010169042.X) with swede type rape A-grade in the first class 177 (granule pure lines, the seed of this material has been delivered Chinese Typical Representative culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on November 20th, 2009, its preserving number is CCTCC NO:P200908, the publication number of formerly patent application is: CN101962640A, open day: on 02 02nd, 2011, former number of patent application was 201010169042.X) hybridization, obtain F1,
B) bud by hybrid F1 obtains Doubled haploid line (DH system) colony of separation by microspores culture (lingering remnants of past customs group etc., 1997);
C) each strain that is colony to DH is carried out molecular marker analysis, and the genotype of each strain is described; Concrete grammar: separate each genomic dna that is of DH colony, adopt the SSR primer to carry out pcr amplification, amplified production is electrophoretic separation on the polyacrylamide gel of 6% (containing 5.7 gram acrylamides and 0.3 gram methene-bisacrylamide in 100ml polyacrylamide sol solution), after silver dyes, develops, obtain each strain genotype;
D) based on Mendelian and Morgan genetic linkage and law of segregation, build the swede type rape genetic linkage map with the Molecular Marker Information that obtains.The structure of genetic linkage map adopts MAPMAKER 3.0 (Lincoln et al., 1992) software to carry out;
E) measure the thousand seed weight numerical value of each mature seed that is of DH colony;
F) thousand seed weight of each strain of DH colony and the molecule marker in the swede type rape genetic linkage map are carried out chain and qtl analysis, QTL detects and adopts QTL Cartographer V2.0 (Wang et al., 2007) the composite interval mapping method (CIM) in software is carried out, take 2.0 as the LOD threshold value, there is a QTL site greater than 2.0 explanations.Obtain and the heavy relevant QTL site of grain: site such as QTLs such as 9 of TSW1, TSW2, TSW5a, TSW5b, TSW5c, TSW7a, TSW7b, TSW10 and TSW14 etc., wherein TSW7a and TSW7b are main effect QTL s sites, can explain the 27.64-37.90% that all heavily make a variation altogether.
G) in Arabidopis thaliana, MINI3 and TTG2 have been proved to be to control seed size and the heavy important gene of grain.By searching NCBI RiboaptDB (http://www.ncbi.nlm.nih.gov/nucleotide/), found two respectively with the Chinese cabbage BAC clone of Arabidopis thaliana MINI3 and TTG2 gene order height homology: AC189531 and AC232555, according to gene order information, designed primer MINI3F/R and the TTG2F/R of two pairs of amplification gene total lengths.Amplify respectively the genomic fragment of these two genes from swede type rape A-grade in the first class 254 and swede type rape A-grade in the first class 177, clone, order-checking.After carefully verifying, difference based on two parental gene group nucleotide sequences, developed a CAPs mark (the Pst I enzyme is cut) MINI3a of MINI3 gene and a SNP mark TTG2a of TTG2 gene, these two marks are positioned at respectively on the A5 linkage group of above-mentioned DH colony.MINI3a just in time is positioned at the peak value place of the heavy QTL site TSW5b of grain that detects, can explain 7% of thousand seed weight variation.TTG2a weighs the QTL peak value 5cM of place of QTL site TSW5c and is in its fiducial interval from another that detects, and can explain 7% of thousand seed weight variation, and TSW5c is adjacent to TSW5b and shows similar additive effect.Therefore can think that the MINI3 gene is the candidate gene of QTL site TSW5b; The TTG2 gene is the candidate gene of QTL site TSW5c.MINI3a and TTG2a control the gene M INI3 of thousand seed weight and the gene specific molecule marker of TTG2;
H) utilize TSW7a, TSW7b, TTG2a and MINI3a that the genotype of DH system is analyzed, have simultaneously above-mentioned four indicia band lines all and swede type rape A-grade in the first class 254 is consistent is a material greatly; It is opposite that to exist simultaneously above-mentioned four indicia band lines all consistent with swede type rape A-grade in the first class 177 be the granule material.
In aforesaid method, the nucleotide sequence of molecule marker primer pair used is as follows:
Primer pair (1) is numbered MINI3F/R:
Forward primer 5 '-ATGAATGCTTTTGATGGAACCTAC-3 ',
Reverse primer 5 '-CTAAAGGTTGAGACCAAAGTTGAGA-3 '.
Primer pair (2) is numbered TTG2F/R:
Forward primer 5 '-ATGGATGTGAAAGAGAGTGAAAGAA-3 ',
Reverse primer 5 '-TTAAATGGCTTGATTAGAATGTTGTG-3 '.
Primer pair (3) is numbered MINI3a:
Forward primer 5 '-AGACCATAACAATCACCGAACC-3 ',
Reverse primer 5 '-ACACGATCAATCTCTGGTTCATT-3 '.
Primer pair (4) is numbered TTG2a:
Forward primer 5 '-CCGCGGGTGATTCATCTAAG-3 ',
Reverse primer 5 '-GGAAGCTAAAAAATAAAGAGTTAAA-3 '.
wherein, the nucleotide sequence of SEQ ID NO:1 and SEQ ID NO:2 in primer pair MINI3F/R extension increasing sequence table is the A genome nucleotide sequence of the MINI3 gene of swede type rape A-grade in the first class 177 and A-grade in the first class 254, the nucleotide sequence of SEQ ID NO:5 and SEQ ID NO:6 in primer pair TTG2F/R extension increasing sequence table is the A genome nucleotide sequence of the TTG2 gene of swede type rape A-grade in the first class 177 and swede type rape A-grade in the first class 254, MINI3a is the CAPs mark, it is the primer of difference site design of the genomic nucleotide sequence of MINI3 Gene A of the swede type rape A-grade in the first class 177 that amplifies according to MINI3F/R and swede type rape A-grade in the first class 254, the nucleotide sequence of SEQ ID NO:3 and SEQ ID NO:4 in primer pair MINI3a extension increasing sequence table, after carrying out pcr amplification with the MINI3a primer pair, utilizing Pst I enzyme to carry out enzyme cuts, if can be digested be judged to be the genotype consistent with swede type rape A-grade in the first class 177, if can not be digested be judged to be the genotype (see Fig. 2) consistent with swede type rape A-grade in the first class 254, namely be decided to be the genomic molecule marker of MINI3 Gene A, TTG2a is the SNP mark, it is the primer of difference site design of the genomic nucleotide sequence of TTG2 Gene A of the swede type rape A-grade in the first class 177 that amplifies according to TTG2F/R and swede type rape A-grade in the first class 254, utilize the nucleotide sequence of SEQ ID NO:7 in primer pair TTG2a extension increasing sequence table, can carry out pcr amplification has and is the genotype consistent with A-grade in the first class 254 with what line occurred, and what can not carry out pcr amplification is the genotype (Fig. 2) consistent with A-grade in the first class 177.Be the genomic molecule marker of TTG2 Gene A.
In above-mentioned preparation method, step is a) to step f) method and the preparation process of the formerly patent application (number of patent application is 201010120725.6, and the applying date is on March 10th, 2010) of applicant Hua Zhong Agriculture University identical.From step g) to h) method be additional peculiar step of the present invention (that is, difference technical characterictic).
Positively effect of the present invention:
The present invention successfully obtains the gene specific molecule marker with thousand grain weight properties genes involved TTG2 and MINI3, use these marks separable, differentiate, clone thousand seed weight genes involved, thereby can overcome the shortcoming that relies on phenotype to select in traditional breeding method.Utilize molecule marker that the present invention prepares can carry out the molecular marker assisted selection of rape grain principal characteristic shape, can obviously reduce the breeding work amount, shortening the breeding cycle, accelerate the process of rapeseed breeding.
Description of drawings
Sequence table SEQ ID NO:1 and SEQ ID NO:2 are the nucleotide sequences of the MINI3 gene of separating clone of the present invention.
SEQ ID NO:3 and SEQ ID NO:4 are the nucleotide sequences of the molecule marker MINI3a for preparing of the present invention.
SEQ ID NO:5 and SEQ ID NO:6 are the nucleotide sequences of the TTG2 gene of separating clone of the present invention.
SEQ ID NO:7 is the nucleotide sequence of the molecule marker TTG2a for preparing of the present invention.
SEQ ID NO:8 and SEQ ID NO:9 are the nucleotide sequences of the primer pair MINI3F/R of amplification MINI3 gene.
SEQ ID NO:10 and SEQ ID NO:11 are the nucleotide sequences of the primer pair TTG2F/R of amplification TTG2 gene.
SEQ ID NO:12 and SEQ ID NO:13 are the nucleotide sequences of primer pair of the molecule marker MINI3a of amplification MINI3 gene.
SEQ ID NO:14 and SEQ ID NO:15 are the nucleotide sequences of primer pair of the molecule marker TTG2a of amplification TTG2 gene.
Fig. 1: be techniqueflow chart of the present invention.
Fig. 2: be to utilize primer pair MINI3a, the TTG2a amplification in the genomic dna of swede type rape A-grade in the first class 254 and swede type rape A-grade in the first class 177 and F1 thereof.In figure, the Pst I enzyme of the pcr amplification product of MINI3a primer pair is cut the picture that the pcr amplification product of product and TTG2a primer pair is electrophoretic separation on 2% sepharose (Agarose that contains 2g in the TAE buffer of every 100ml).
Fig. 3: be the nucleotide sequence comparison result that utilizes primer pair MINI3F/R to amplify in the genomic dna of swede type rape pure lines A-grade in the first class 177 and A-grade in the first class 254.In figure, underline position is the binding site of primer pair MINI3a, and the site shown in arrow is the coding mutation in the 1751st site of sequence, thereby has caused the sudden change of the restriction enzyme site of Pst I enzyme.Utilize the product of primer pair MINI3a pcr amplification in the genomic dna of swede type rape pure lines A-grade in the first class 177 and A-grade in the first class 254, the Pst I enzyme that can carry out of A-grade in the first class 177 is cut, and the Pst I enzyme that can not carry out of A-grade in the first class 254 is cut.
Fig. 4: be the nucleotide sequence comparison result that utilizes primer pair TTG2F/R to amplify in the genomic dna of swede type rape pure lines A-grade in the first class 177 and A-grade in the first class 254.In figure, underline position is the binding site of primer pair TTG2a, trilateral represents the insertion mutation of 6 Nucleotide of the insertion mutation of 6 Nucleotide of the 223rd of sequence and the 461st, thereby utilizes these two insertion mutation sites to design the primer pair of TTG2a.Utilize primer pair TTG2a to increase in the genomic dna of swede type rape pure lines A-grade in the first class 177 and A-grade in the first class 254, A-grade in the first class 254 can carry out pcr amplification,
And A-grade in the first class 177 can not carry out pcr amplification.
Fig. 5: the positioning result that is the chromosomal genetic linkage map of the DH A5 of colony and the heavy QTLs of grain.In figure
QTLs site for detecting is followed successively by on A5 karyomit(e) from top to bottom: TSW5a, TSW5b and TSW5c.Wherein
Be QTL peak value position;
Fiducial interval for QTL.
Embodiment
Embodiment 1: the location of the heavy QTLs of grain in swede type rape
(1) structure of the DH colony of 254/ A-grade in the first class 177 of A-grade in the first class of swede type rape grain reorientation colony and field test and thousand seed weight are analyzed
employing is with swede type rape A-grade in the first class 254 (large grain pure lines, the seed of this material has been delivered Chinese Typical Representative culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on November 20th, 2009, its preserving number is CCTCC NO:P200909) for maternal, with swede type rape A-grade in the first class 177 (granule pure lines, the seed of this material has been delivered Chinese Typical Representative culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on November 20th, 2009, its preserving number is CCTCCNO:P200908) hybridize for male parent and obtain F1, the bud of F1 is carried out microspores culture obtain the DH segregating population.Obtain altogether the DH system of 238 systems, choose at random the location that 190 systems are used for structure and the heavy QTL of grain of complete genomic genetic linkage map.
DH system and its parent, F1 kind are planted two continuous times in 2007-2008 year and 2008-2009 year, and field test is taked randomized complete-block design, three repetitions, each is kind of two row, every row 11-12 individual plant, about the average 24cm of spacing in the rows, line space 30cm.All material is planted in Hua Zhong Agriculture University's rape experimental plot, Wuhan, is the winter rape planting environment.Field management is by general breeding field management.
With ripe test materials, take off seed from the individual plant of free pollination from the field harvesting annual May, cleans out impurity and not full seed, more than placing at least for 4 weeks, and seasoning in air.Each individual plant is got 500 full seeds at random, repeat for three times, in individual plant, error is no more than 0.1g, putting back to mixing after surpassing gets again, then calculate to average and be converted to thousand seed weight (weight of 1000 seeds) numerical value, each is to get 10-15 individual plant for parent, F1 and DH, and calculating averages is its thousand seed weight value (related data sees Table 1).
(2) genetic linkage map of DH colony builds and the heavy qtl analysis of grain
Being chosen at 190 DH of SSR primer pair that have amplification polymorphism between two parents is according to the method for having (Plieske and Struss, 2001; Suwabe et al., 2002; Lowe et al., 2004; Chen et al., 2009) analyze.Separate the genomic dna that each DH is, adopt the SSR primer that polymorphism is arranged that above-mentioned screening obtains to carry out pcr amplification, amplified production is electrophoretic separation on the polyacrylamide gel of 6% (containing 5.7 gram acrylamides and 0.3 gram methene-bisacrylamide in the 100ml polyacrylamide solution), after silver dyes, develops, obtain the molecule marker polymorphism data of each strain genotype and colony, the colony's genotype data that obtains is built the swede type rape genetic linkage map.The structure of genetic linkage map adopts MAPMAKER 3.0 (Lincoln et al., 1992) software carries out, and it is 9.0 that the parameter that linkage group is divided is set to the LOD value, and ultimate range is 30eM, each linkage group determine to utilize the orders such as order, try and ripple.Common mark in mapping population in linkage group and riveting calibration note are from Parkin et al. (1995), Lowe et al. (2004), Piquemal et al. (2005), " Kosambi " parameter is adopted in the calculating of the genetic distance between the information in the articles such as Qiu et al. (2006) and Chen et al. (2009), two sites.
the thousand seed weight data of each strain of DH colony and the molecule marker in the swede type rape genetic linkage map are carried out chain and qtl analysis, QTL Cartographer V2.0 (Wang et al. is adopted in the detection of QTL, 2007) the composite interval mapping method (CIM) in software is carried out, before QTL detects, its parameter setting is: select " forward-backward stepwise regression " pattern, the window size of assay intervals is selected 10cM, parameter setting is pattern 6:Pin=0.05, Pout=0.05, during detection, the LOD value is defaulted as 2.0, the fiducial interval of QTL determine with the peak value that LOD-1 was comprised two ends at peak value place the position of correspondence on genetic linkage map.Fiducial interval has lap to think to have the QTL of similar position between different environment and colony.
In the test of 2 years, altogether (A1, A2, A5, A7, A10 and C4) detects the QTLs of 9 thousand seed weight on 6 karyomit(e)s, and these QTLs can explain respectively the phenotypic variation (table 2) of 3.66-20.76%.From the allelotrope of A-grade in the first class 254 to TSW5a, TSW5b, TSW5c, TSW10 and TSW14 play positive acting, and TSW1 and TSW2 are played negative role.
Embodiment 2: the acquisition of the gene specific molecule marker of thousand grain weight properties in swede type rape
In Arabidopis thaliana, MINI3 and TTG2 gene have been proved to be to control seed size and the heavy important gene of grain.In order to probe into the possibility of the gene specific mark that utilizes the heavy related gene MINI3 of grain and TTG2 in swede type rape, designed the experiment that separates homologous sequence in swede type rape, by searching NCBI RiboaptDB (http://www.ncbi.nlm.nih.gov/nucleotide/), found two respectively with the Chinese cabbage BAC clone of Arabidopis thaliana MINI3 and TTG2 gene order height homology: AC189531 and AC232555, and these two BAC clones all are positioned on A5 karyomit(e).According to gene order information, designed primer MIN13F/R and the TTG2F/R of two pairs of amplification gene total lengths.Amplify the genomic fragment of these two genes from two parent A-grade in the first class 254 of DH colony and A-grade in the first class 177, clone, order-checking.After carefully verifying, developed SNP mark of TTG2 gene and a CAPs mark of MINI3 gene (called after TTG2a and MINI3a respectively) based on the difference of two parental gene group nucleotide sequences, these two marks have been positioned at respectively on the A5 linkage group of DH colony.MINI3a just in time has been positioned at the peak value place of the heavy QTL site TSW5b of grain that detects, can explain 7% of thousand seed weight variation.TTG2a can explain 7% of thousand seed weight variation from the QTL peak value 5cM of place of another the heavy QTL site TSW5c that detects and in its fiducial interval.TSW5c is adjacent to TSW5b and shows similar additive effect effect.Therefore can think that the MINI3 gene is the candidate gene of QTL site TSW5b; The TTG2 gene is the candidate gene of QTL site TSW5c.TTG2a and MINI3a control the gene TTG2 of thousand seed weight and the gene specific molecule marker of MINI3;
Embodiment 3: the validation verification of thousand grain weight properties gene specific molecule marker in swede type rape
(1) checking of thousand grain weight properties gene specific molecule marker in swede type rape
Compare with two sites on A7, TTG2a and MINI3a site are less to the contribution of phenotypic variation, in order to detect TTG2a and MINI3a site to the hereditary effect of phenotypic variation, with the genotype in these two sites to DH colony be divide into groups and calculate the mean value of its thousand seed weight, each thousands of the tuple values of two kinds of genotype in the TTG2a of DH colony and MINI3a two sites have obvious difference (table 3).
(2) the combined effect checking in the heavy QTLs of grain site in swede type rape
Detect the combined effect in the heavy QTLs of grain site in DH colony, DH colony be the variation (table 4) of dividing into groups and comparing its weight with the genotype of the QTLs on A5 and A7.Because three QTLs on A5 are chained together closely, seldom obtain recombination system in DH colony, three sites on A5 are reduced to a site and are used for genotypic classification.Genotypic combination (table 4) thereby three sites should have 8 in DH colony in.Can obtain a conclusion from the data of table 4: although the QTLs site effect on A5 is less, but its contribution to thousand seed weight can not be out in the cold, data by table 4 can find out, the thousand seed weight data of first group (comprising three all forward additive alleles) are all higher than the numerical value of other all groups.
Utilize TSW7a, TSW7b, TTG2a and MINI3a that the genotype of DH system is analyzed, have simultaneously that above-mentioned four indicia band lines are all consistent with A-grade in the first class 254 is a material greatly, with the thousand seed weight positive result of TTG2 and MINI3 gene; Opposite to exist simultaneously above-mentioned four indicia band lines all consistent with A-grade in the first class 177 be the granule material, with the thousand seed weight negative-effect of TTG2 and MINI3 gene.
By checking the genotype in all thousand seed weight QTLs of DH colony site, 75# system has the QTLs site of all positive results, and it all has maximum thousand seed weight numerical value (table 5) in the phenotypic number of 2 years.On the contrary, 87# is the QTLs site that has all boomerang effects, and it all had minimum thousand seed weight numerical value in 2 years.
Above presentation of results can utilize the information of these marks to be used for the molecular marker assisted selection of grain principal characteristic shape, and heavy genotype selection is also very accurately to grain to use these marks.
The thousand seed weight data of parent, F1 and the segregating population of table 1:DH colony
Remarks:
1)P1=is maternal, the p2=male parent; Capitalization after numerical value and lowercase refer under t test between the parent significance of difference under 0.01 level and 0.05 level respectively.
2)h
B 2: be broad-sense heritability.
Table 2: the heavy QTLs site information of the grain that detects in DH colony
Remarks:
1)The name of QTL is to add the numeral of its place linkage group according to the initial capitalization of proterties name; If when detecting more than 1 QTL, add in order alphabetical a or b in its back on a linkage group;
2)Interval: from two nearest side marks of peak value; Peak value: the figure spectral position (cM) at LOD value peak value place; Mark: from the nearest mark of peak value:
3)A: additive effect; Positive result refers to can increase from the allelotrope of female parent the value of thousand seed weight;
4)The phenotypic variation ratio that QTL can explain.
In table 3:DH colony, four grains of 2 years weigh genotype and the thousand seed weight tag-related in site
Remarks:
1)AA and BB refer to identical with A-grade in the first class 177 with A-grade in the first class 254 respectively genotype.The number that is of each genoid type during numerical value in bracket.χ
2The=3.84th, in the situation that 0.05 horizontal degree of freedom is 1 value;
2)Capitalization and lowercase refer to the significance of difference under 0.01 and 0.05 level respectively.
The allelotrope of table 4:DH colony is marked at the combined effect in the heavy QTLs of the grain site on A5 and A7 linkage group
Remarks:
1)AA and BB refer to identical with A-grade in the first class 177 with A-grade in the first class 254 respectively genotype.Upper closely linked three the QTLs sites of A5 are seen as a genotype site and classify;
2)N: the sample size that this genotype kind is included;
3)Lowercase refers to the significance of difference of the Duncan test under 0.05 level.
Table 5: the heavily performance of grain of two DH systems of all positive results in all that detect heavy QTLs site and negative-effect polymerization
Remarks: AA and BB refer to identical with A-grade in the first class 177 with A-grade in the first class 254 respectively genotype.
Table 6: numbering and the nucleotide sequence thereof of the molecule marker primer pair of the present invention's design
Remarks: CAPs: enzyme is cut the extension increasing sequence polymorphism; SNP: single nucleotide polymorphism.
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Claims (5)
1. the molecule marker TTG2a of 254 principal characteristic shapes of a cabbage type rape variety A-grade in the first class, its nucleotide sequence is as shown in SEQ ID NO:7.
2. the increase primer pair of molecule marker TTG2a of 254 principal characteristic shapes of cabbage type rape variety A-grade in the first class as claimed in claim 1, its nucleotide sequence is as shown in SEQ IDNO:14 and SEQ IDNO:15.
3. the preparation method of the specific molecular marker of a swede type rape grain principal characteristic shape, its step comprises:
A) be that maternal and swede type rape A-grade in the first class 177 are paternal hybrid with swede type rape A-grade in the first class 254, obtain F1;
B) plant the F1 that obtains, obtain double haploid (DH) colony of separation by microspores culture from the bud of described F1 plant;
C) each strain in the DH colony that obtains is carried out molecular marker analysis, the genomic dna of each strain of separation DH colony, adopt the SSR primer to carry out pcr amplification, amplified production concentration is 6% polyacrylamide gel electrophoresis separation, after silver dyes, develops, obtain the genotype of each strain;
D) build the swede type rape genetic linkage map with obtaining genotype;
E) measure the thousand seed weight numerical value of mature seed of each strain of DH colony;
F) molecule marker in thousand seed weight numerical value and swede type rape genetic linkage map is carried out chain and qtl analysis, the QTL site of controlled grain principal characteristic shape; QTL detects the composite interval mapping method that adopts in QTL Cartographer V2.0 software to carry out;
It is characterized in that, step is as follows:
1) search the NCBI RiboaptDB, find a Chinese cabbage BAC with the genes involved TTG2 gene order height homology of Arabidopis thaliana control seed size to clone AC232555, sequence information according to AC232555, the primer pair TTG2F/R of design pair for amplification TTG2 full length gene, its nucleotide sequence is as shown in SEQ ID NO:10 and 11; Amplify the genomic fragment of TTG2 gene from swede type rape A-grade in the first class 177 and A-grade in the first class 254, Cloning and sequencing;
2) according to the difference of swede type rape A-grade in the first class 177 and A-grade in the first class's 254 genome nucleotide sequences, design the SNP mark TTG2a of TTG2 gene, the nucleotide sequence of this molecule marker primer pair that increases is as shown in SEQ ID NO:14 and SEQ ID NO:15; Utilize described molecule marker TTG2a, repeating step c) method, obtain the genotype of each strain; Repeating step f) linkage analysis method is positioned at step b with described molecule marker TTG2a) the A5 linkage group of DH colony on;
3) carry out pcr amplification according to the primer pair shown in SEQ ID NO:14 and SEQ ID NO:15, obtain distinguishing the specific molecular marker TTG2a of the TTG2 gene of swede type rape large seed and small-sized seed, the nucleotide sequence of described molecule marker TTG2a is as shown in SEQ ID NO:7; Wherein can carry out the large grain of being judged to be of pcr amplification material, that can not carry out pcr amplification is judged to be the granule material.
4. the application of molecule marker claimed in claim 1 in 254 principal characteristic shape marker assisted selection of swede type rape A-grade in the first class.
5. the application of primer pair claimed in claim 2 in 254 principal characteristic shape marker assisted selection of swede type rape A-grade in the first class.
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| KR101576794B1 (en) * | 2013-01-29 | 2015-12-11 | 삼성에스디에스 주식회사 | System and method for aligning of genome sequence considering read length |
| RS61283B1 (en) * | 2014-12-18 | 2021-02-26 | Dow Agrosciences Llc | Fine mapping and validation of qtl underlying fiber content and seed coat color traits and identification of snp markers for marker assisted selection of these traits derived from yellow seed coat (ysc) canola line yn01-429 and its lineage |
| CN105063033B (en) * | 2015-08-15 | 2018-06-15 | 华中农业大学 | A kind of molecular labeling of Brassica Napus knee ospc gene and its application in anti-clubroot breeding |
| WO2017088144A1 (en) * | 2015-11-26 | 2017-06-01 | 北京市农林科学院 | Snp combination for analyzing diversity of chinese cabbage germplasm resource and for molecular breeding, and use thereof |
| CN108754011B (en) * | 2018-06-21 | 2021-05-14 | 贵州省油菜研究所 | Major QTL (quantitative trait locus) site for thousand grain weight trait of brassica napus, SNP (Single nucleotide polymorphism) molecular marker and application |
| CN111172315B (en) * | 2020-02-25 | 2023-04-14 | 贵州省油菜研究所 | A01 chromosome major QTL site with main inflorescence grain weight character of brassica napus, SNP molecular marker and application |
| CN114058734B (en) * | 2022-01-17 | 2022-04-19 | 华智生物技术有限公司 | SNP molecular marker combination for detecting rape varieties and application thereof |
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| CN101248753A (en) * | 2008-03-27 | 2008-08-27 | 华中农业大学 | Method for assist-breeding low erucic acid, low sulfuric glucoside cabbage type rape self-incompatible line with microspore cultivation and SSR making |
| CN101336611A (en) * | 2008-07-04 | 2009-01-07 | 江苏丘陵地区镇江农业科学研究所 | Breeding method of good quality, double-low, disease-resistant, high yield, lodging-resistant rape |
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| CN101248753A (en) * | 2008-03-27 | 2008-08-27 | 华中农业大学 | Method for assist-breeding low erucic acid, low sulfuric glucoside cabbage type rape self-incompatible line with microspore cultivation and SSR making |
| CN101336611A (en) * | 2008-07-04 | 2009-01-07 | 江苏丘陵地区镇江农业科学研究所 | Breeding method of good quality, double-low, disease-resistant, high yield, lodging-resistant rape |
Non-Patent Citations (1)
| Title |
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| Isabelle Debeaujon et al..Proanthocyanidin-accumulating cells in Arabidopsis testa：regulation of differentiation and role in seed development.《The Plant Cell》.2003,第15卷(第11期),2514-2531. * |
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