CN102690822B - Molecular marker closely linked with major quantitative trait locus (QTL) of thousand-grain weight and grain length of wheat and obtaining method and application of molecular marker - Google Patents
Molecular marker closely linked with major quantitative trait locus (QTL) of thousand-grain weight and grain length of wheat and obtaining method and application of molecular marker Download PDFInfo
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Abstract
The invention discloses a molecular marker closely linked with a major quantitative trait locus (QTL) of the thousand-grain weight and grain length of wheat and an obtaining method and application of the molecular marker. A labeling primer wmc386 and barc216 are adopted to carry out polymerase chain reaction (PCR) amplification on the DNA of a wheat line Shannong 0431; and after the electrophoretic separation of the amplification product on 8% polyacrylamide gel, the molecular weights of the obtained amplification product are 190 bp and 100 bp respectively, i.e., the molecular marker of the major QTL of the thousand-grain weight and the grain length of the wheat. The molecular marker of the major QTL of the thousand-grain weight and the grain length of the wheat can be used for detecting whether the QTL for increasing the thousand-grain weight and the grain length is contained in a wheat variety or a wheat line, in order to fast screen out the wheat variety or the wheat line containing the QTL for increasing the thousand-grain weight and the grain length for breading; therefore, the selective breeding process for high-yield wheat varieties is much faster.
Description
Technical field
The present invention relates to wheat molecular biotechnology and Breeding Application field, specifically a kind of molecule marker and acquisition methods and application with increasing thousand grain weight of wheat and grain length main effect QTL compact linkage.
Background technology
Wheat is one of most important food crop in the world, so the seed selection of High-Yield Wheat Cultivar is paid much attention to by breeding man always.Yield Traits of Wheat is complicated quantitative character, be subject to a plurality of quantitative character gene locus therefors (Quantitative trait locus, QTL) control, there is low, affected by environment large, the high characteristic of selection difficulty of heritability, so when selecting and breeding high yielding wheat kind, the problem large, cost is high, achievement is little is grown, expended to the normal lifetime of traditional breeding method.Molecular mark is that a kind of molecule marker be associated with specific trait that adopts carries out the effective ways of breeding as supplementary means, have the envrionment conditions of not being subject to limit, in each tissue of development of plants and the advantage that growthdevelopmental stage all can detect, efficiency of selection is high.Studies have shown that, the thousand seed weight that is mainly subject to seed size affect trait is the most stable direct constituent element of output (Giura and Saulescu, Euphytica. 89:77-80,1996); Snape etc. once reported seed size and output height correlation (Euphytica. 154:401-408,2007).Visible, the output of wheat and seed size and thousand seed weight have significant correlation.Therefore, research thousand seed weight and seed size gene, by molecular marking technique, improve wheat grain size and thousand seed weight and obtain High-yield Wheat new variety, in breeding work, is extremely important.Wheat mountain agriculture strain 0431 is the F of 1994 " international Winter-Spring Mai Zajiaoshaixuanpu " (U.S., Cornell University)
3combination " Grtpi85504 // MI76-77-S29/ALD " seed selection the strain of large grain, disease-resistant (stripe disease, leaf rust, Powdery Mildew, root rot) and a hot-dry wind tolerance, comprehensive agronomy proterties is good, is the excellent germplasm resource of research thousand seed weight and seed size gene.At present, not yet have and its thousand seed weight and the mutually chain molecule marker of grain length main effect quantitative gene site QTL report.
Summary of the invention
Object of the present invention is just to provide molecule marker and acquisition methods and application a kind of and thousand grain weight of wheat and grain length main effect QTL compact linkage, that by invention, obtain and molecule marker thousand seed weight and grain length main effect QTL compact linkage, detect in wheat breed or strain whether there is the QTL that increases thousand seed weight and grain length, to accelerate the seed selection process of High-yield Wheat Varieties.
The object of the invention is to be achieved through the following technical solutions:
The molecule marker of a kind of and thousand grain weight of wheat provided by the present invention and grain length main effect QTL compact linkage, this molecule marker is
wmc386with
barc216, molecule marker described in it
wmc386primer left end nucleotide sequence as described in SEQ ID NO:1, right-hand member nucleotide sequence is as described in SEQ ID NO:2; Described molecule marker
barc216primer left end nucleotide sequence as described in SEQ ID NO:3, right-hand member nucleotide sequence is as described in SEQ ID NO:4.
And an acquisition methods for the molecule marker of thousand grain weight of wheat and grain length main effect QTL compact linkage, it comprises the following steps: adopt molecule marker
wmc386primer
Left end nucleotide sequence ATCACTGAAACGAAATGAGCGG(is as described in SEQ ID NO:1)
Right-hand member nucleotide sequence TGGTTGGCGGTTTTTCTCTACA(is as described in SEQ ID NO:2)
And molecule marker
barc216primer
Left end nucleotide sequence TGACGACCCAATCCATAGACA (as described in SEQ ID NO:3),
Right-hand member nucleotide sequence GGTGATTATTCGTGAGTTCCCTGTG(is as described in SEQ ID NO:4)
The DNA of wheat line mountain agriculture 0431 is carried out respectively to pcr amplification, and its pcr amplification system is 20 μ l, H
2o 5.75 μ l, 10 * PCR damping fluid, 10.0 μ l, Taq enzyme 0.25 μ l, left and right end primer 2 .0 μ l, DNA 2.0 μ l; Amplification condition is 94 ℃ of denaturation 4 min; 94 ℃ of sex change 40 s, 55 ℃ of annealing 45 s, 72 ℃ are extended 50 s, 35 circulations; 72 ℃ are extended 10 min; 10 ℃ of preservations; Amplified production is respectively on 8% polyacrylamide gel after electrophoretic separation, and the molecular weight of the amplified production of acquisition is respectively 190 bp and 100 bp, obtains the molecule marker with thousand grain weight of wheat and grain length main effect QTL linkage.
The application of the molecule marker of of the present invention and thousand grain weight of wheat and grain length main effect QTL compact linkage in selecting and breeding high yielding wheat, be the method for detection of wheat breed or strain by the molecule marker with thousand grain weight of wheat and grain length main effect QTL compact linkage, it comprises the following steps the method:
A. use
wmc386with
barc216labeled primer the DNA of wheat breed or strain is carried out respectively to pcr amplification, its pcr amplification system is 20 μ l, comprises H
2o 5.75 μ l, 10 * PCR damping fluid, 10.0 μ l, Taq enzyme 0.25 μ l, left and right end primer 2 .0 μ l, DNA 2.0 μ l; Amplification condition is 94 ℃ of denaturation 4 min; 94 ℃ of sex change 40 s, 55 ℃ of annealing 45 s, 72 ℃ are extended 50 s, 35 circulations; 72 ℃ are extended 10 min; 10 ℃ of preservations;
B. by above-mentioned amplified production respectively on 8% polyacrylamide gel after electrophoretic separation, if the amplified production molecular size range of wmc386 is that the molecule marker of 190 bp and the amplified production molecular size range of barc216 are that the molecule marker of 100 bp occurs simultaneously, this wheat breed or product be have increase thousand seed weight and grain length gene kind or strain, otherwise this wheat breed or strain belong to kind or the strain without increase thousand seed weight and grain length gene.
The screening method of the molecule marker of provided by the invention and thousand grain weight of wheat and grain length main effect QTL compact linkage, it comprises the following steps:
(i) the wheat line mountain agriculture 0431 of take is hybridized and is obtained F as male parent as maternal, wheat breed Shandong wheat 21
1, F
1selfing produces F
2, adopt single seed descent to obtain the F that contains 177 strains
7daiRIL colony;
(ii) use modified CTAB method (Vander Beek et al., 1992) extract the DNA of each strain of described RIL colony, adopt diversity microarray technology mark (DArTs mark), simple sequence repeat marker (SSR mark) and the simple sequence repeat marker based on expressed sequence tag (EST-SSR mark) to carry out gene type assay to described strain, obtain the genotype data of described RIL colony;
(iii) utilize MAPMAKER 3.0 mapping softwares the genotype data of the described RIL colony obtaining to be built to the molecular genetic linkage map of wheat, its LOD >=3.0;
(iv) described RIL colony is carried out to field planting, each strain of Bing Dui RIL colony is carried out respectively phenotype investigation, obtains the thousand seed weight of each strain and the characteristic of grain length;
(v) utilize MAPMAKER 3.0 and Windows QTL Cartographer 2.5 mapping softwares, adopt composite interval mapping method, colony's genotype data of each molecule marker is carried out to linkage analysis with thousand seed weight and the grain length of corresponding each strain, in LOD>=3.0 o'clock, on karyomit(e) 5B
wPt-7029-wPt-9467in interval, the peak value of thousand seed weight QTL and grain length QTL all exists
wmc386-
barc216interval;
Its
wmc386with
barc216the molecular weight of the amplified production that labeled primer obtains in the DNA of wheat line mountain agriculture 0431 is respectively 190 bp and 100 bp, is the molecule marker with thousand grain weight of wheat and grain length main effect QTL compact linkage.
The MAPMAKER 3.0 adopting in aforesaid method, Windows QTL Cartographer 2.5 softwares are the popular software that carries out at present genetic mapping and qtl analysis, belong to commercial goods, can buy by commercial channel.
The DNA of wheat line of the present invention mountain agriculture 0431 refers to the DNA obtaining after the blade separation and Extraction of wheat line mountain agriculture 0431 plant.
8% polyacrylamide gel of the present invention refers to the methene acrylamide that contains 7.8 g acrylamides and 0.2 g in 100 ml polyacrylamide solutions.
The closely linked molecule marker of thousand grain weight of wheat disclosed by the invention and grain length
wmc386with
barc216the thousand seed weight of wheat breed or strain and the main effect QTL of grain length have been fully demonstrated, by this molecule marker to wheat derived varieties or strain pcr amplification, the QTL that can be very quickly whether wheat breed or strain be had to thousand seed weight and a grain length judges, thereby can rapid screening go out to have wheat breed or the strain that increases thousand seed weight and grain length QTL, accelerate the seed selection process of High-Yield Wheat Cultivar.
By provided by the invention, be used for wheat breeding with the molecule marking method of thousand grain weight of wheat and grain length main effect QTL compact linkage, not only screen fast accurate, not affected by environment, select target is clear and definite, and saved production cost, greatly improved efficiency of selection and the quality of High-Yield Wheat Cultivar or strain.
Accompanying drawing explanation
Fig. 1 is that thousand seed weight and the mapping of grain length main effect QTL on karyomit(e) 5B of wheat line mountain agriculture 0431 is interval.
In Fig. 1: hollow rectangle represents karyomit(e), middle solid black circle represents centromere position, and right side is the title of molecule marker, and left side numeral is the genetic distance between mark, and unit is cM; In figure, have two kinds of molecule markers, " wPt " and " rPt " is DArTs mark, and barc, gwm and wmc are SSR mark, indicate being labeled as of black underscore
wmc386with
barc216; The rectangle that the oblique line of karyomit(e) lower left is filled represents that QTL mapping is interval, and wherein GL represents grain length proterties; TGW represents thousand grain weight properties.
Fig. 2 is that wheat line mountain agriculture 0431 is parent's F
718 derivative strains of DaiRIL colony are used
wmc386labeled primer carries out the electrophoretic band figure of pcr amplification.
Fig. 3 is that wheat line mountain agriculture 0431 is parent's F
718 derivative strains of DaiRIL colony are used
barc216labeled primer carries out the electrophoretic band figure of pcr amplification.
Embodiment
Embodiment is used for further describing the present invention below, but does not limit in any form the present invention.
Embodiment 1
With a molecule marker for thousand grain weight of wheat and grain length main effect QTL compact linkage,
Adopt labeled primer
wmc386,
Left end primer sequence ATCACTGAAACGAAATGAGCGG(is as described in SEQ ID NO:1)
Right-hand member primer sequence TGGTTGGCGGTTTTTCTCTACA(is as described in SEQ ID NO:2)
With labeled primer barc216,
Left end primer sequence TGACGACCCAATCCATAGACA (as described in SEQ ID NO:3)
Right-hand member primer sequence GGTGATTATTCGTGAGTTCCCTGTG(is as described in SEQ ID NO:4)
The DNA of wheat line mountain agriculture 0431 is carried out respectively to pcr amplification, and its pcr amplification system is 20 μ l, H
2o 5.75 μ l, 10 * PCR damping fluid, 10.0 μ l, Taq enzyme 0.25 μ l, left and right end primer 2 .0 μ l, DNA 2.0 μ l; Amplification condition is 94 ℃ of denaturation 4 min; 94 ℃ of sex change 40 s, 55 ℃ of annealing 45 s, 72 ℃ are extended 50 s, 35 circulations; 72 ℃ are extended 10 min; 10 ℃ of preservations; The PCR instrument model of using is: in BIO-RAD S1000 THERMAL CYCLER(the present invention, all pcr amplifications all adopt this model PCR instrument); Amplified production is respectively on 8% polyacrylamide gel after electrophoretic separation, and the molecular weight of the amplified production of acquisition is respectively 190 bp and 100 bp, is the molecule marker of thousand grain weight of wheat and grain length main effect QTL.
The screening method of the molecule marker of provided by the invention and thousand grain weight of wheat and grain length main effect QTL compact linkage, it comprises the following steps:
(i) the large grain wheat line mountain agriculture 0431 of take is hybridized and is obtained hybrid F as male parent for female parent, the Common Wheat Varieties Shandong wheat 21 of take
1, F
1selfing produces F
2, adopt single seed descent to obtain the F that contains 177 strains
7daiRIL colony;
(ii) use improved method of CTAB, cetyl trimethylammonium bromide method (the Vander Beek et al. improveing, 1992) extract the DNA of each strain of above-mentioned RIL colony, adopt diversity microarray technology mark (DArTs mark), simple sequence repeat marker (SSR mark) and the simple sequence repeat marker based on expressed sequence tag (EST-SSR mark) to carry out gene type assay to described strain, obtain the genotype data of described RIL colony;
Wherein SSR mark and EST-SSR labeled analysis are that the DNA of screening strain is carried out to pcr amplification, amplified production carries out electrophoretic separation analysis on described 8% polyacrylamide gel, according to molecular marker screening result, filter out and between parent, have polymorphic primer, between parent, there is polymorphic primer to analyze in selected strain, obtain the genotype data of described RIL colony; DArTs labeled analysis be by and the technology of chip hybridization distinguish the method for polymorphism between different genes group, digestion with restriction enzyme by each strain genomic dna through two kinds of different cutting frequencies, the joint of the restriction endonuclease recognition sequence that recycling cutting frequency is low is connected with enzyme section is disconnected, then by realizing the pcr amplification to this special segment with this enzyme cut-grafting corresponding primer.Amplicon is used for building clone library, and then is passed through fluorescent mark, after sex change, put on chip as probe.The target dna detecting also needs through the cutting of same restriction endonuclease and pcr amplification, then just can with pass through fluorescently-labeled target sequence probe and hybridize, obtain the genotype data of described RIL colony;
The step that improved method of CTAB is extracted DNA in blade is: get the fresh blade of 0.15～0.2 g and put into 2 ml centrifuge tubes, put into rapidly liquid nitrogen at least 10 s, wear into fast fine powder; Add 1 ml to be preheated to the CTAB extracting solution of 65 ℃, 65 ℃ of incubation 1 h, every 20 min put upside down and mix centrifuge tube or slightly shake incubation; Being placed in 4 ℃ of refrigerators or room temperature is cooled to below 15 ℃; What add 4 ℃ of 1 ml temperature is the phenol of 1:1 and chloroform by volume, mixes up and down 30 min, then centrifugal 20 min of 1000 rpm; Get 700 μ l supernatant liquors, adding isopyknic volume ratio is chloroform-primary isoamyl alcohol of 24:1, mixes up and down 30 min, guarantees that sample and chloroform are fully mixed; Centrifugal 20 min of 10000 rpm; Get supernatant liquor 500 μ l, add isopyknic ice Virahol, the pipe 15 times of turning upside down gently, can see nucleic acid precipitation; 4 ℃ of standing 30 min left and right of refrigerator; Centrifugal 30 min of 10000 rpm, abandon supernatant liquor, twice of the washing with alcohol precipitation of 1 ml 70%; Abandon ethanol, dry up DNA, with 250 μ l 1 * TE dissolving DNAs; The RNase that adds 1.0 μ l 2 mg/ml, 37 ℃ of water-bath 30 min remove RNA, obtain required DNA;
(iii) utilize MAPMAKER 3.0 mapping softwares, the genotype data of the RIL colony of acquisition is built to the molecular genetic linkage map of the wheat with 740 marks, take LOD >=3.0 as standard; Wherein 740 marks are comprised of 617 DArTs, 118 SSR and 5 EST-SSR;
(iv) JiangRIL colony has carried out 2 years field planting and the phenotypic evaluation of totally six testing circumstances, examine or check respectively grain characters and the thousand seed weight of each strain under different year and varying environment condition, wherein 2 years six testing circumstances are Tai'an in 2010, Tai'an in 2011, Heze in 2011, academy of agricultural sciences, Tai'an in 2011, Yantai in 2011 and Zibo in 2011.
Wheat planting method wherein: in RIL colony, each strain is planted 5 row, row long 2 m, line space 25 cm, 70 seeds of every row plantation, normal growth and results; Thousand seed weight measuring method: the wheat seed of each strain after results is got to 500 and weigh, repeat 3 times, average and calculate thousand seed weight; Grain length measuring method: the wheat seed of each strain after results is got to 20, and the end to end straight line amount length that is put into, repeats 3 times, calculates seed grain length.
(v) utilize the front and back homing method of MAPMAKER 3.0 and Windows QTL Cartographer 2.5 mapping softwares, take LOD >=3.0 as standard, colony's genotype data of each molecule marker is carried out to linkage analysis mapping with thousand seed weight and the grain length of corresponding each strain;
(vi) by qtl analysis, can be obtained: the interval of finding respectively to repeat under 4 envrionment conditionss to repeat under thousand seed weight QTL and 5 envrionment conditionss grain length QTL peak value on karyomit(e) 5B is
wmc386-
barc216, as shown in Fig. 1, table 1.
The qtl analysis result of table 1 thousand seed weight and grain length
As shown in Table 1, the peak value of these 9 QTL all exists
wmc386near, wherein, the peak value of the thousand seed weight QTL of the grain length QTL under (2011) 3 environment in Tai'an (2010), academy of agricultural sciences, Tai'an (2011) and Yantai and Tai'an (2011), (2011) 3 environment in academy of agricultural sciences, Tai'an (2011) and Yantai all exists
wmc386place.The thousand seed weight QTL peak value of Zibo (2011) environment from
wmc386distance be 1.0 cM, the QTL peak value of Tai'an (2011) and Zibo (2011) grain length from
wmc386distance be respectively 2.0 cM and 3.0 cM.And the contribution rate of these 9 QTL is up to 32.5%, minimum is 9.7%, and the additive effect of all QTL be on the occasion of.
wmc386-barc216in interval, in 5 environment, grain length QTL detected, and the average contribution rate of QTL is 16.5%; In 4 environment, thousand seed weight QTL detected, average contribution rate is 16.9%.Explanation exists
wmc386-barc216that in interval, detect is the QTL with thousand grain weight of wheat and mutually chain stable, the main effect of grain length, its labeled primer
wmc386with
barc216the molecular weight of the amplified production obtaining in the DNA of wheat line mountain agriculture 0431 is respectively 190 bp and 100 bp, is the molecule marker with thousand grain weight of wheat and grain length main effect QTL linkage.
Embodiment 2
The F that molecule marker provided by the invention and thousand grain weight of wheat and grain length main effect QTL compact linkage is parent in wheat line mountain agriculture 0431
718 derivative strains in DaiRIL colony and the application in Lu Mai 21
With large grain, disease-resistant, hot-dry wind tolerance strain mountain agriculture 0431 work female parent, with granule, many fringes kind Shandong wheat 21, make male parent, by single seed descent (SSD method), built F
7recombinant inbred lines (RIL colony), and carried out utilize GaiRIL colony carry out seed and Correlated Yield Characters QTL location and with the screening of main effect QTL compact linkage molecule mark.Its concrete steps are as follows:
(1) 18 derivative strains in the RIL colony of wheat breed mountain agriculture 0431 are carried out to field planting, get the DNA that the plant leaf of each strain adopts the CTAB method of described improvement to carry out separation and Extraction acquisition;
Derived varieties or the strain of described wheat line mountain agriculture 0431 refer to: take wheat line mountain agriculture 0431 as parent, by adopting conventional hybridization and multiplying to F
2generation above, tissue culture or corn and wheat hybridizing induction monoploid, then the wheat breed or the strain that by colchicine, double to obtain double haploid or adopt genetic transforming method acquisition;
(2) adopt labeled primer
wmc386with
barc216the DNA of acquisition is carried out to pcr amplification, and its pcr amplification system 20 μ l, comprise H
2o 5.75 μ l, 10 * PCR damping fluid, 10.0 μ l, Taq enzyme 0.25 μ l, left and right end primer 2 .0 μ l, DNA 2.0 μ l; PCR processing condition are: 94 ℃ of denaturation 4 min; 94 ℃ of sex change 40 s, 55 ℃ of annealing 45 s, 72 ℃ are extended 50 s, 35 circulations; 72 ℃ are extended 10 min; 10 ℃ of preservations; Pcr amplification product is electrophoretic separation on the non-denaturing polyacrylamide gel of acrylamide and methene the proportion of acylamide 39:1, then detects with argentation;
(3) detected result is analyzed, the amplified production molecular size range of its wmc386 is that the molecule marker of 190 bp and the amplified production molecular size range of barc216 are that the molecule marker of 100 bp occurs simultaneously, amplified production all product consistent with the amplified production of mountain agriculture 0431 be to there is the derivative strain that increases grain length, thousand seed weight QTL.
Concrete outcome is analyzed as follows: utilize
wmc386with
barc216labeled primer the DNA of mountain agriculture 0431, a 1-18 derivative strain and Shandong wheat 21 is carried out to pcr amplification, its result respectively as shown in Figures 2 and 3:
In Fig. 2 and Fig. 3, swimming lane 1-18 represents respectively the electrophoretic separation band of 18 derivative strain amplified productions of Shan Nong 0431, CK is the electrophoretic separation band (Shandong wheat 21 is granule, many fringes kind) of Shandong wheat 21 amplified productions, and what SN represented is the electrophoretic separation band of mountain agriculture 0431 amplified production.
From Fig. 2, Fig. 3, can find out, in 18 derivative strains, what its banding pattern was identical with mountain agriculture 0431 banding pattern is to carry the derivative strain that increases grain length, thousand seed weight QTL, derives strain 2,13,15 and 18, and these derivative strains can be used for the strain of next step SOYBEAN IN HIGH-YIELD BREEDING; And the electrophoretic separation banding pattern of the amplified production of Shandong wheat 21 is different from mountain agriculture 0431 banding pattern, Shandong wheat 21 is not have the kind that increases thousand seed weight and grain length QTL.
Prove thus: utilize
wmc386with
barc216carry out molecular marker assisted selection, can have kind or the strain that increases thousand seed weight and grain length QTL by Effective selection, this mark can improve efficiency of selection and the quality of High-Yield Wheat Cultivar or strain greatly for wheat assistant breeding.
SEQUENCE LISTING
<110> Shandong Agricultural University
The molecule marker of <120> and thousand grain weight of wheat and grain length main effect QTL compact linkage
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213> wmc386
<400> 1
atcactgaaa cgaaatgagc gg 22
<210> 2
<211> 22
<212> DNA
<213> wmc386
<400> 2
tggttggcgg tttttctcta ca 22
<210> 3
<211> 21
<212> DNA
<213> barc216
<400> 3
tgacgaccca atccatagac a 21
<210> 4
<211> 25
<212> DNA
<213> barc216
<400> 4
ggtgattatt cgtgagttcc ctgtg 25
Claims (4)
1. with the molecule marker of thousand grain weight of wheat and grain length main effect QTL compact linkage, it is characterized in that this molecule marker is for adopting
wmc386with
barc216labeled primer the gene PCR amplification of wheat line mountain agriculture 0431 is obtained respectively to the amplified production that molecular weight is 190 bp and 100 bp; Described in it
wmc386upstream primer sequence as described in SEQ ID NO:1, downstream primer sequence is as described in SEQ ID NO:2; Described
barc216upstream primer sequence as described in SEQ ID NO:3, downstream primer sequence is as described in SEQ ID NO:4.
2. with the acquisition methods of the molecule marker of thousand grain weight of wheat and grain length main effect QTL compact linkage, it is characterized in that it comprises the following steps:
With
wmc386with
barc216labeled primer carries out respectively pcr amplification to the DNA of wheat line mountain agriculture 0431, and its pcr amplification system is: H
2o 5.75 μ l, 10 * PCR damping fluid, 10.0 μ l, Taq enzyme 0.25 μ l, upstream and downstream primer 2 .0 μ l, DNA 2.0 μ l; Amplification condition is: 94 ℃ of denaturation 4 min; 94 ℃ of sex change 40 s, 55 ℃ of annealing 45 s, 72 ℃ are extended 50 s, 35 circulations; 72 ℃ are extended 10 min; 10 ℃ of preservations; Amplified production is respectively on 8% polyacrylamide gel after electrophoretic separation, and the molecular weight that obtains corresponding amplified production is respectively 190 bp and 100 bp, is the molecule marker of thousand grain weight of wheat and grain length main effect QTL; Wherein
wmc386mark upstream primer sequence as described in SEQ ID NO:1, downstream primer sequence is as described in SEQ ID NO:2;
barc216mark upstream primer sequence as described in SEQ ID NO:3, downstream primer sequence is as described in SEQ ID NO:4.
3. the preparation method of the molecule marker of according to claim 2 and thousand grain weight of wheat and grain length main effect QTL compact linkage, is characterized in that the DNA of described wheat line mountain agriculture 0431 refers to the DNA obtaining after the blade separation and Extraction of wheat line mountain agriculture 0431 plant.
4. the preparation method of the molecule marker of according to claim 2 and thousand grain weight of wheat and grain length main effect QTL compact linkage, is characterized in that described 8% polyacrylamide gel refers to the methene acrylamide that contains 7.8 g acrylamides and 0.2 g in 100 ml polyacrylamide solutions.
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| CN104342484B (en) * | 2013-07-23 | 2016-08-10 | 中国农业科学院作物科学研究所 | A kind of molecular marker relevant to thousand grain weight of wheat and application thereof |
| CN104087580B (en) * | 2014-07-10 | 2017-03-01 | 山东农业大学 | Semen Tritici aestivi 4B chromosome grain weight QGW4B CAPS molecular marker and its application |
| CN105200143B (en) * | 2015-10-22 | 2018-02-13 | 中国农业大学 | Grain Weight in Common Wheat main effect QTL QTgw 4A.1 close linkage mark and its application |
| CN105713990B (en) * | 2016-04-29 | 2019-04-05 | 中国农业科学院作物科学研究所 | Wheat molecular marker and its application in identification wheat yield correlated traits |
| CN107794307B (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 |
| CN110982933B (en) * | 2020-03-02 | 2020-06-30 | 鲁东大学 | Molecular marker closely linked with major QTL (quantitative trait locus) of wheat grain length and application thereof |
| CN111518933B (en) * | 2020-05-18 | 2022-03-11 | 安徽农业大学 | Wheat grain length related SNP marker and application thereof |
| CN115976263B (en) * | 2022-12-19 | 2024-01-12 | 中国农业大学 | KASP molecular marker of wheat thousand grain weight major QTL and application thereof |
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