CN104342484B - A kind of molecular marker relevant to thousand grain weight of wheat and application thereof - Google Patents
A kind of molecular marker relevant to thousand grain weight of wheat and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of molecular marker relevant to thousand grain weight of wheat and application thereof.The present invention is by the Genetic Variation Analysis of 6 SFT genes in Semen Tritici aestivi natural variation colony, it is found to have two SNP, corresponding respectively to sequence 1 from 5 ' ends the 1371st and the 2450th, there are three kinds of haplotypes in the two SNP: haplotype first (G, G), haplotype second (G, A), haplotype third (A, G).Proved by association analysis, these three haplotype isozygoty in type, mass of 1000 kernel size is: the Semen Tritici aestivi that haplotype third isozygotys > Semen Tritici aestivi that isozygotys of haplotype first > Semen Tritici aestivi that isozygotys of haplotype second.Present invention also offers the CAPS labelling of detection said two SNP.It is demonstrated experimentally that by detection said two SNP, the Semen Tritici aestivi that mass of 1000 kernel is of a relatively high can be found.The present invention is that the molecular marker assisted selection breeding of Semen Tritici aestivi provides a new method, significant in cultivating High-Yield Wheat Cultivar or research.
Description
Technical field
The present invention relates to a kind of molecular marker relevant to thousand grain weight of wheat and application thereof.
Background technology
China is Wheat Production and the first big country of consumption in the world, and Wheat Production is closely related with national food security.
Arid always affects the main abiotic stress factor of Wheat Production.Levan is not only a kind of important shelf stability solvable
Property carbohydrate, the important carbon source of kernel grouting, is also main osmotic adjustment, and in plant, the accumulation of levan can
Improve its drought resisting, tolerance to cold.Therefore, study and utilize triticin synzyme (Sucrose:fructan6-
Fructosyltransferase, 6-SFT) gene is significant for the genetic improvement of high yield, drought resisting.Semen Tritici aestivi 6-SFT
Being one of key enzyme in levan building-up process, have two kinds of functions, one is directly with sucrose as substrate, on catalysing sucrose molecule
Fructosyl transfer on the C6 position of another sucrose molecule fructosyl, synthesize 6-ketose, two is with few levan as the end
Thing, generates branching type levan.Along with the fast development of molecular biotechnology, the function of important gene is the most revealed, is making
Thing genetic improvement efficiently utilize these genes become the important goal of Gene mining.Molecular Marker Assisted Selection Technology is for carrying
High objective trait efficiency of selection, Crop Improvement provides a new effective way.Functional molecular based on gene order exploitation
The direct marker gene of labelling, it is possible to high efficiency selected target gene itself, the highest attention of the person that therefore suffers from correlational study.But so far
Till the present, not yet develop the molecular marker of Semen Tritici aestivi 6-SFT gene, and the relation of evaluation of markers and yield traits.
CAPS technology is also called PCR-RFLP, be utilize oneself to know that the DNA sequence Resource Design in site goes out is a set of specific
PCR primer (19 27bp), then with a certain DNA fragmentation on this site of these primer amplifications, then with a kind of narrow spectrum
Restriction enzyme cleavage gained amplified production, gel electrophoresis separation endonuclease bamhi, dyes and carries out rflp analysis.GAPS labelling
Disclose is the information of the restricted length variation of special PGR fragment.CAPS is a class codominant marker, and its advantage is to keep away
Exempt from the step for that in rflp analysis, film transfers, the degree of accuracy of rflp analysis can have been kept again.Further, since a lot of restriction enzymes
Enzyme all can be with DNA amplification enzyme action, so detecting that polymorphism chance is bigger.
Summary of the invention
It is an object of the invention to provide a kind of molecular marker relevant to thousand grain weight of wheat and application thereof.
The present invention provides a kind of and utilizes Semen Tritici aestivi pleomorphism site to identify or the method for auxiliary qualification wheat genotypes, described many
State property site is X and two mononucleotide polymorphism sites of Y, and two mononucleotide polymorphism sites of described X and Y correspond respectively to
In Wheat volatiles DNA, sequence 1 is from 5 ' ends the 1371st and the 2450th；
Two mononucleotide polymorphism sites of described X with Y are followed successively by following I), II) with III) situation time corresponding base
Because type is A, B and C:
I) G/G and G/G；
II) G/G and A/A；
III) A/A and G/G.
It is the situation on a homologous chromosome before described "/", is the feelings on another homologous chromosome after described "/"
Condition.
Described acquisition genotype A, the method for B and C include that in the genomic DNA to Semen Tritici aestivi to be measured, any one section includes described
2 mononucleotide polymorphism site X and Y carry out PCR amplification at interior DNA fragmentation, and this pcr amplification product is carried out enzyme action mirror
Fixed step.
The target sequence of described PCR amplification is that sequence 1 is from 5 ' end the 1st 2663bp.
The specific primer that described PCR amplification uses is to for the single stranded DNA shown in sequence 2 and sequence 3
Shown single stranded DNA.
Described enzyme action is identified and is comprised the steps: PCR primer respectively with MboII and BsgI enzyme action, it is thus achieved that digestion products M
With digestion products B；
If described digestion products M is the DNA fragmentation of 762bp, 681bp, 465bp, 375bp, 225bp and 155bp, then described
Semen Tritici aestivi to be measured is that G/G, i.e. G isozygoty in described X site；
If described digestion products M is the DNA fragmentation of 1137bp, 681bp, 465bp, 225bp and 155bp, the most described to be measured
Semen Tritici aestivi is that A/A, i.e. A isozygoty in described X site；
If described digestion products B is the DNA fragmentation of 1955bp, 510bp and 198bp, the most described Semen Tritici aestivi to be measured is in described Y position
Point isozygotys for G/G, i.e. G；
If the DNA fragmentation that described digestion products B is 1955bp and 708bp, then it is at described Y by described Semen Tritici aestivi candidate to be measured
Site is that A/A, i.e. A isozygoty.
Any of the above-described described method can be used for identifying or auxiliary qualification thousand grain weight of wheat character；
Being confirmed as the Semen Tritici aestivi to be measured that genotype is C, its mass of 1000 kernel is the to be measured little of A and B higher than being confirmed as genotype
Wheat；Being confirmed as the Semen Tritici aestivi to be measured that genotype is A, its mass of 1000 kernel is higher than being confirmed as the Semen Tritici aestivi to be measured that genotype is B.
The present invention also protects the PCR primer pair expanding following DNA fragmentation: any one section on Wheat volatiles DNA includes
Two mononucleotide polymorphism sites of described X and Y are at interior DNA fragmentation.
Described PCR primer is to for the single stranded DNA shown in sequence 2 and the single stranded DNA shown in sequence 3.
The present invention also provides for a kind of reagent or test kit identifying or assisting in and differentiating thousand grain weight of wheat character, including sequence table
Single stranded DNA shown in single stranded DNA shown in sequence 2, sequence 3 and restricted enzyme MboII and BsgI.
The present invention by the Genetic Variation Analysis of 6-SFT gene in Semen Tritici aestivi natural variation colony, being found to have two SNP,
Correspond respectively to sequence 1 from 5 ' ends the 1371st and the 2450th, the two SNP three kinds of haplotypes of existence: monomer
Type first (G, G), haplotype second (G, A), haplotype third (A, G).Proved by association analysis, the type of isozygotying of these three haplotype
In, mass of 1000 kernel size is: the Semen Tritici aestivi that haplotype third isozygotys > Semen Tritici aestivi that isozygotys of haplotype first > Semen Tritici aestivi that isozygotys of haplotype second.This
The bright CAPS labelling additionally providing detection said two SNP.It is demonstrated experimentally that by detection said two SNP, thousand can be found
The Semen Tritici aestivi that weight is of a relatively high.The present invention is that the molecular marker assisted selection breeding of Semen Tritici aestivi provides a new method, is cultivating height
Yield wheat kind or research in significant.
Accompanying drawing explanation
Fig. 1 is to develop CAPS labelling digestion products electrophoresis detection result according to two SNP of the present invention.Wherein, swimming lane M is for dividing
Sub-amount standard, left figure clip size from top to bottom be followed successively by 1500bp, 1000bp, 900bp, 800bp, 700bp, 600bp,
500bp、400bp、300bp、200bp、100bp；Right figure clip size from top to bottom be followed successively by 4500bp, 3000bp,
2000bp、1200bp、800bp、500bp、200bp.Left figure is the band that the PCR primer of different Semen Tritici aestivis carries out enzyme action with Mbo II
Type；Right figure is the banding pattern that the PCR primer of different Semen Tritici aestivis carries out enzyme action with Bsg I；Swimming lane G in left figure contains six from up to
Lower size is followed successively by the band of 762bp, 681bp, 465bp, 375bp, 225bp and 155bp, and swimming lane A contains five from top to bottom
Size is followed successively by the band of 1137bp, 681bp, 465bp, 225bp and 155bp；Swimming lane G in right figure contains three from top to bottom
Size is followed successively by the band of 1955bp, 510bp and 198bp, swimming lane A contain two from top to bottom size be followed successively by 1955bp and
The band of 708bp.
Fig. 2 is CAPS labelling digestion products electrophoresis detection result in DH colony (drought selects No. 10 × Shandong wheat 14).Wherein, swimming lane
M is molecular weight standard, clip size from top to bottom be followed successively by 1500bp, 1000bp, 900bp, 800bp, 700bp, 600bp,
500bp、400bp、300bp、200bp、100bp；Swimming lane H is that drought selects No. 10；Swimming lane L is Shandong wheat 14；Swimming lane 1-150 is respectively DH
150 strains in colony.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Wheat lines used in following embodiment is all from National crop genebank of China (http://icscaas.com.cn/
Jiguoku/zhongzhiku.htm), material information is shown in Crops In China kind matter Information Network, network address: http: //
icgr.caas.net.cn。
2 SNP and PCR-RFLP polymorphic detections that embodiment 1 is relevant to thousand grain weight of wheat
1, special primer and the sequence analysis of the genomic DNA fragment containing 2 SNP of Semen Tritici aestivi are expanded
Gene on Wheat volatiles 6-SFT(Genbank is FJ228688) 2 SNP of middle discovery, correspond respectively to
Sequence 1 is from the 1371st of 5 ' ends the (named X site, there is the polymorphism of G and A), and sequence 1 is from 5 '
2450th (there is the polymorphism of G and A in named Y-site) of end；The two site X and Y is in Semen Tritici aestivi natural variation colony
Three kinds of haplotypes of middle existence:
Haplotype first: G, G；
Haplotype second: G, A；
Haplotype third: A, G；
According to the sequence difference of Semen Tritici aestivi different genes group, design specific primer PCR amplification comprises these 2 SNP site and exists
Interior DNA fragmentation:
F:5'-CTCTCTAGACATAATCAAAAGGGA-3'(sequence 2)；
R:5'-TTCTTTGATCCAATGTAGCTTCA-3'(sequence 3)；
Shown in the sequence 1 of the target sequence such as sequence table of described PCR amplification (2663bp), this sequence includes Semen Tritici aestivi 6-SFT base
Because organizing part the 3rd exon, the 3rd intron and part the 4th exon of DNA.Restriction analysis shows, the two site polymorphic
Property can be identified by Mbo II and Bsg I respectively.
2, PCR-RFLP polymorphic detection and the foundation of methods of genotyping
1) genomic DNA of Semen Tritici aestivi to be measured is extracted；
2) with the genomic DNA of step 1) as template, PCR amplification, the system (15 μ L) of PCR amplification are carried out with primers F and R
For: ddH2O8.0 μ L, 5 × PCR buffer3.0 μ L, primers F 2(5 μm ol L-1) and R2(5 μm ol L-1) each 0.6 μ L, dNTP
(2.5 μm ol L-1) 0.4 μ L, transfastpfu enzyme (5U) 0.3 μ L, template DNA (20ng μ L-1) 2.1 μ L；
PCR amplification condition is: 95 DEG C of 5min；95 DEG C of 1min, 55 DEG C of 45s, 72 DEG C of 3min30s, 35 circulations；72℃
10min, 15 DEG C of preservations.
3) by step 2) PCR primer that obtains is respectively with MboII and BsgI enzyme action, it is thus achieved that digestion products M and digestion products
B, carries out 1% sepharose electrophoresis detection, each clip size in record digestion products, and judges according to following method and record to be measured
Semen Tritici aestivi in described X and the situation of Y-site:
If described digestion products M is the DNA fragmentation of 762bp, 681bp, 465bp, 375bp, 225bp and 155bp, then described
Semen Tritici aestivi to be measured is that G isozygotys the Semen Tritici aestivi (the swimming lane G in the left figure of Fig. 1) of (being expressed as G/G) in described X site；
If described digestion products M is the DNA fragmentation of 1137bp, 681bp, 465bp, 225bp and 155bp, the most described to be measured
Semen Tritici aestivi is that A isozygotys the Semen Tritici aestivi (the swimming lane A in the left figure of Fig. 1) of (being expressed as A/A) in described X site；
If described digestion products B is the DNA fragmentation of 1955bp, 510bp and 198bp, the most described Semen Tritici aestivi to be measured is at described Y
Site is that G isozygotys the Semen Tritici aestivi (the swimming lane G in the right figure of Fig. 1) of (being expressed as G/G)；
If the DNA fragmentation that described digestion products B is 1955bp and 708bp, the most described Semen Tritici aestivi to be measured is at described Y-site to be
A isozygotys the Semen Tritici aestivi (the swimming lane A in the right figure of Fig. 1) of (being expressed as A/A)；
4) according to the result of step 3), it is following I III types that Semen Tritici aestivi is divided into the situation at described X and Y-site:
I: G/G and G/G(i.e. haplotype first is isozygotied)；
II: G/G and A/A(i.e. haplotype second is isozygotied)；
III: A/A and G/G(i.e. haplotype third isozygotys)；
It is the situation on a homologous chromosome before described "/", is the feelings on another homologous chromosome after described "/"
Condition.
3, two CAPS labellings are utilized natural population to carry out typing and is associated analyzing with thousand grain weight properties
In the natural population of 154 parts of hexaploid wheats composition each Semen Tritici aestivi as Semen Tritici aestivi to be measured according to the side of step 2
Method carries out typing, at random the amplified production size of part Semen Tritici aestivi is carried out sequence verification, and result is as shown in table 1.
X and the situation of Y-site in table 1, Semen Tritici aestivi natural population
| Sequence number | Wheat breed title | Type |
| 1 | 04-030 | Ⅰ |
| 2 | 04-037 | Ⅲ |
| 3 | 04-044 | Ⅰ |
| 4 | 04-067 | Ⅰ |
| 5 | 04-111 | Ⅰ |
| 6 | 04-112 | Ⅰ |
| 7 | 04-135 | Ⅲ |
| 8 | 04-208 | Ⅰ |
| 9 | 124-1 in peace 85 | Ⅲ |
| 10 | Peace 86 in 17 | Ⅱ |
| 11 | Beijing 10 | Ⅰ |
| 12 | Beijing 14 | Ⅱ |
| 13 | Beijing 837 | —— |
| 14 | Beijing 8686 | Ⅲ |
| 15 | Single R8043 | Ⅲ |
| 16 | Single R8093 | Ⅱ |
| 17 | Single R8108 | Ⅲ |
| 18 | Single R8194 | Ⅲ |
| 19 | Single R9062 | Ⅰ |
| 20 | Rich anti-13 | Ⅲ |
| 21 | Feng You 5 | Ⅲ |
| 22 | Capital 411 | Ⅲ |
| 23 | Capital spring 70-5321 | Ⅱ |
| 24 | East, Jingdone district 82 307 | Ⅱ |
| 25 | East, Jingdone district 83 65 | Ⅲ |
| 26 | Capital core 8922 | Ⅰ |
| 27 | Capital agriculture 79-15 | Ⅲ |
| 28 | Capital agriculture 80 reflects 107 | Ⅲ |
| 29 | Capital agriculture 84-6786 | Ⅲ |
| 30 | Capital agriculture 84-6789 | Ⅲ |
| 31 | Capital product 10 | Ⅰ |
| 32 | Capital product 11 | Ⅰ |
| 33 | Capital product 30 | Ⅰ |
| 34 | Capital product 3 | Ⅲ |
| 35 | Capital double 16 | Ⅲ |
| 36 | Double No. 2 of capital | Ⅲ |
| 37 | Capital selects 20 | Ⅰ |
| 38 | Capital selects 25 | Ⅰ |
| 39 | 85 mirror 28 are prolonged in capital | Ⅲ |
| 40 | Section loses 26 | —— |
| 41 | Red good No. 4 | Ⅱ |
| 42 | Agricultural university 146 | Ⅲ |
| 43 | Agricultural university 183 | Ⅰ |
| 44 | Agricultural university 20074 | Ⅰ |
| 45 | Agricultural university 81146 | Ⅲ |
| 46 | The former winter 834 | Ⅱ |
| 47 | The former winter 8445 | Ⅲ |
| 48 | The former winter 847 | Ⅱ |
| 49 | The former winter 856 | Ⅲ |
| 50 | Fortune drought 2028 | Ⅲ |
| 51 | Early fringe 21 | Ⅰ |
| 52 | Early fringe 65 | Ⅰ |
| 53 | Early fringe 66 | Ⅰ |
| 54 | In 7902 | Ⅲ |
| 55 | In 84 I-40551 | Ⅲ |
| 56 | In 8502 | Ⅲ |
| 57 | Hill 8 | Ⅰ |
| 58 | In 86 I-50455 | Ⅲ |
| 59 | In big 86-mirror 2 | —— |
| 60 | In big 91-product 9 | Ⅲ |
| 61 | In big 92-mirror 49 | Ⅲ |
| 62 | In big 92-product 8 | Ⅱ |
| 63 | Zhongyou9507 | Ⅲ |
| 64 | Middle work 60064 | Ⅲ |
| 65 | Middle work 60115 | Ⅰ |
| 66 | Middle work 634 | Ⅲ |
| 67 | Bai Qimai | Ⅱ |
| 68 | Xifeng 20 | Ⅲ |
| 69 | Cangzhou Semen Tritici aestivi | Ⅰ |
| 70 | Euphorbia royleana Boiss. | Ⅲ |
| 71 | Ji 92-5203 | Ⅱ |
| 72 | Ji wheat 26 | Ⅲ |
| 73 | Ji wheat 30 | Ⅱ |
| 74 | Ji wheat 32 | Ⅰ |
| 75 | Ji wheat 41 | Ⅲ |
| 76 | Ji wheat No. 6 | Ⅲ |
| 77 | Ji wheat No. 1 | Ⅰ |
| 78 | High excellent 504 | Ⅱ |
| 79 | Handan 4589 | Ⅲ |
| 80 | Weighing apparatus wheat No. 2 | Ⅲ |
| 81 | Stone spy 14 | Ⅲ |
| 82 | The four red hourglass heads of rib | Ⅰ |
| 83 | Hundred agricultures 3217 | Ⅱ |
| 84 | White rough wheat | Ⅱ |
| 85 | Wenmai 6 | Ⅲ |
| 86 | Lay down and open up No. one | Ⅱ |
| 87 | Henan wheat No. 13 | Ⅱ |
| 88 | Henan wheat 18 | Ⅱ |
| 89 | Henan wheat No. 2 | Ⅲ |
| 90 | Henan wheat No. 8 | Ⅱ |
| 91 | Luohan No.2 | Ⅱ |
| 92 | Luoyang 8628 | Ⅱ |
| 93 | Luoyang 9048 | Ⅲ |
| 94 | Purple stalk Bai Mangxian | Ⅱ |
| 95 | Changle 5 | Ⅰ |
| 96 | Jinan 13 | Ⅱ |
| 97 | Shandong wheat 14 | Ⅲ |
| 98 | Shandong wheat 15 | Ⅰ |
| 99 | Shandong wheat 19 | Ⅲ |
| 100 | Cigarette 881414 | Ⅲ |
| 101 | Locust wheat | Ⅲ |
| 102 | Red Buddhist monk | Ⅰ |
| 103 | Face drought 6105 | Ⅲ |
| 104 | Face drought 917 | Ⅰ |
| 105 | Face drought 935 | Ⅲ |
| 106 | Face anti-5108 | Ⅱ |
| 107 | Pingyang 348 | Ⅲ |
| 108 | Changzhi 6878 | Ⅰ |
| 109 | Little lay down 54 | Ⅱ |
| 110 | Drought selects 11 | Ⅰ |
| 111 | Drought selects 12 | Ⅰ |
| 112 | Drought selects No. 3 | Ⅰ |
| 113 | Shanxi 2148-7 | Ⅰ |
| 114 | Shanxi wheat 17 | Ⅱ |
| 115 | Shanxi wheat 33 | Ⅱ |
| 116 | Shanxi wheat 39 | Ⅰ |
| 117 | Shanxi wheat 44 | Ⅲ |
| 118 | Shanxi wheat 47 | Ⅲ |
| 119 | Shanxi wheat 50 | Ⅲ |
| 120 | Shanxi wheat 54 | Ⅲ |
| 121 | Shanxi wheat 57 | Ⅲ |
| 122 | Drought selects No. 10 | Ⅰ |
| 123 | Shanxi wheat 68 | Ⅲ |
| 124 | Shanxi wheat 72 | Ⅲ |
| 125 | Golden light | Ⅱ |
| 126 | Changwu 131 | Ⅲ |
| 127 | Changwu 89 (1) 3-4 | Ⅲ |
| 128 | Dali 1 | Ⅲ |
| 129 | Shan 225-9 | Ⅱ |
| 130 | Shan 229 | Ⅲ |
| 131 | Shan drought 8675 | Ⅲ |
| 132 | Shan closes No. 6 | Ⅰ |
| 133 | Shan agriculture 7859 | Ⅱ |
| 134 | Shaanyou 225 | Ⅱ |
| 135 | Shan money 1869 | Ⅲ |
| 136 | Weihe wheat No. 4 | Ⅰ |
| 137 | Little Qi wheat | Ⅱ |
| 138 | PANDAS | Ⅰ |
| 139 | SALGEMMA | Ⅲ |
| 140 | Spring 029th-3 | Ⅱ |
| 141 | Spring 039th-4 | Ⅱ |
| 142 | Spring 049th-5-1 | Ⅱ |
| 143 | Spring 079th-7 | Ⅲ |
| 144 | Spring 179th-18 | Ⅱ |
| 145 | Spring 219th-24 | Ⅲ |
| 146 | Spring 229th-25 | Ⅲ |
| 147 | Spring 239th-26 | Ⅲ |
| 148 | Spring 259th-28 | Ⅲ |
| 149 | Spring 349th-38 | Ⅲ |
| 150 | Spring 359th-39 | Ⅲ |
| 151 | Spring 379th-41 | Ⅲ |
| 152 | Spring 409th-44 | Ⅲ |
| 153 | Spring 429th-46 | Ⅱ |
| 154 | Spring 459th-50-1 | Ⅱ |
Note: " " in table indicates without PCR primer.
2008 and 2009, at Institute of Crop Science, Chinese Academy of Agricultural Science experimental farm (Changping County, Beijing) field planting
The Semen Tritici aestivi of above-mentioned natural population, normal water and fertilizer management, investigate the mass of 1000 kernel of each wheat breed, with Tassel2.1 software to thousand
The situation of weight and X and Y-site is associated analyzing, and in order to prevent spurious correlation, by the group structure information of K=4, (Zhang Jianan etc. use
The 83 pairs of SSR marker by Structure2.1(Prichard et al, 2000) group to 154 shown in table 1 part wheat lines
Body structure is analyzed, and finds there is obvious flex point during K=4, therefore 154 parts of materials of table 1 is divided into 4 groups) substitute into
Tassel2.1 is associated analyzing, and selecting P < 0.05 is significance level, and result is as shown in table 2.
The situation of X and Y-site and the association analysis result of mass of 1000 kernel in table 2 Semen Tritici aestivi natural population
aMass of 1000 kernel mean+SD represents；*Represent P < 0.05.
The association analysis result of table 2 shows, the three of the natural population formation of 154 shown in table 1 part hexaploid wheat composition
The mass of 1000 kernel difference of type all reaches significant level (P < 0.05).Wherein, the thousand grain weight of wheat of type III is the highest, and I takes second place, and II
Mass of 1000 kernel minimum.In 2 years the mass of 1000 kernel of the wheat lines of type III respectively relatively II Semen Tritici aestivi height 2.58g and 3.13g.To certainly
So the research of colony shows, type III is to improve the favorable genes type of thousand grain weight of wheat.
4, utilize two SNP that DH colony carries out typing and mass of 1000 kernel detection and association analysis
DH(doubled haploid) colony: drought is selected in No. 10 (serial numbers 122 in table 1, type is I) and Shandong wheat 14(table 1
Serial number 97, type is III) be carry out in hybridization 1 generation that parent obtains Anther Culture (method is according to document: Jing Ruilian etc. use
Anther Culture creates Semen Tritici aestivi doubled haploid mapping population. biotechnology .1999, and 9 (3): 4-9), obtain by 150 strain groups
The DH colony become；
In above-mentioned DH colony, each strain is as Semen Tritici aestivi to be measured, carries out typing according to the method for step 2, result such as figure
Shown in 2；At 2001,2005,2006,2,009 2,011 6 years Beijing area field planting above-mentioned DH colonies and parent, normal water
Fertile management, investigates the mass of 1000 kernel of each Semen Tritici aestivi；Carrying out t inspection according to genotyping result and mass of 1000 kernel result thereof, result shows, DH colony
In two kinds of situations, appreciable impact thousand grain weight of wheat, illustrate type III be improve mass of 1000 kernel favorable genes type (table 3).
The association analysis of the situation of X and Y-site and mass of 1000 kernel in table 3 Wheat DH colony
*P<0.05；**P<0.01；***P<0.001。
Claims (7)
1. utilize the method that Semen Tritici aestivi pleomorphism site is identified or auxiliary identifies thousand grain weight of wheat character, described pleomorphism site
For X and two mononucleotide polymorphism sites of Y, two mononucleotide polymorphism sites of described X and Y correspond respectively to wheat cdna
In group DNA, sequence 1 is from 5 ' ends the 1371st and the 2450th；
Two mononucleotide polymorphism sites of described X with Y are followed successively by following I), II) with III) situation time corresponding genotype
For A, B and C:
I) G/G and G/G；
II) G/G and A/A；
III) A/A and G/G；
Being confirmed as the Semen Tritici aestivi to be measured that genotype is C, its mass of 1000 kernel is higher than being confirmed as the Semen Tritici aestivi to be measured that genotype is A and B；Quilt
Being defined as the Semen Tritici aestivi to be measured that genotype is A, its mass of 1000 kernel is higher than being confirmed as the Semen Tritici aestivi to be measured that genotype is B；
Described method includes that in the genomic DNA to Semen Tritici aestivi to be measured, any one section includes two single nucleotide polymorphism of described X and Y
Site carries out PCR amplification at interior DNA fragmentation, and this pcr amplification product carries out the step of enzyme action qualification；
Identify that the clip size of obtained digestion products determines that the genotype of described Semen Tritici aestivi to be measured is A, B or C according to enzyme action.
Method the most according to claim 1, it is characterised in that: the target sequence of described PCR amplification is that sequence 1 is from 5 '
End the 1st 2663bp.
Method the most according to claim 2, it is characterised in that: the specific primer that described PCR amplification uses is to for sequence
Single stranded DNA shown in table sequence 2 and the single stranded DNA shown in sequence 3.
Method the most according to claim 3, it is characterised in that: described enzyme action is identified and is comprised the steps: to divide PCR primer
Yong MboII and BsgI enzyme action, it is thus achieved that digestion products M and digestion products B；
If described digestion products M is the DNA fragmentation of 762bp, 681bp, 465bp, 375bp, 225bp and 155bp, the most described to be measured
Semen Tritici aestivi is G/G in described X site；
If described digestion products M is the DNA fragmentation of 1137bp, 681bp, 465bp, 225bp and 155bp, the most described Semen Tritici aestivi to be measured
It is A/A in described X site；
If described digestion products B is the DNA fragmentation of 1955bp, 510bp and 198bp, the most described Semen Tritici aestivi to be measured at described Y-site is
G/G；
If the DNA fragmentation that described digestion products B is 1955bp and 708bp, then it is at described Y-site by described Semen Tritici aestivi candidate to be measured
For A/A.
5. Semen Tritici aestivi pleomorphism site is in the application identified or in auxiliary qualification thousand grain weight of wheat character；
Described pleomorphism site is X and two mononucleotide polymorphism sites of Y, two mononucleotide polymorphism sites of described X and Y
Correspond respectively in Wheat volatiles DNA sequence 1 from 5 ' ends the 1371st and the 2450th；
Two mononucleotide polymorphism sites of described X with Y are followed successively by following I), II) with III) situation time corresponding genotype
For A, B and C:
I) G/G and G/G；
II) G/G and A/A；
III) A/A and G/G；
Being confirmed as the Semen Tritici aestivi to be measured that genotype is C, its mass of 1000 kernel is higher than being confirmed as the Semen Tritici aestivi to be measured that genotype is A and B；Quilt
Being defined as the Semen Tritici aestivi to be measured that genotype is A, its mass of 1000 kernel is higher than being confirmed as the Semen Tritici aestivi to be measured that genotype is B.
6. one kind identifies or assisting in the primer pair differentiating thousand grain weight of wheat character, it is characterised in that: described primer is to for sequence table
Single stranded DNA shown in sequence 2 and the single stranded DNA shown in sequence 3.
7. one kind identifies or assisting in reagent or the test kit differentiating thousand grain weight of wheat character, it is characterised in that: described reagent or examination
Agent box includes the single stranded DNA shown in the single stranded DNA shown in sequence 2, sequence 3 and restricted enzyme MboII
And BsgI.
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| CN106318936B (en) * | 2015-06-17 | 2018-12-21 | 中国农业大学 | Wheat stablizes molecular labeling and its application of grain weight main effect QTL |
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| CN107794308B (en) * | 2016-08-29 | 2020-05-12 | 中国农业科学院作物科学研究所 | Specific SNP for identifying wheat grain traits and application thereof |
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| CN111187852B (en) * | 2020-01-16 | 2021-09-21 | 中国科学院遗传与发育生物学研究所 | SNP (single nucleotide polymorphism) site related to wheat yield and application thereof |
| CN111593136B (en) * | 2020-05-18 | 2022-03-11 | 安徽农业大学 | Wheat thousand grain weight related SNP marker and application thereof |
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| CN114317807B (en) * | 2022-01-10 | 2023-06-30 | 河北师范大学 | SNP locus related to thousand grain weight characters of wheat and application thereof |
| CN115927718B (en) * | 2022-08-29 | 2023-08-15 | 河北省农林科学院粮油作物研究所 | KASP molecular marker for identifying grain weight and grain width of wheat and application |
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