CN104774858B - Control major gene resistance TaTGW 7A and its CAPS labeling methods of thousand grain weight of wheat - Google Patents
Control major gene resistance TaTGW 7A and its CAPS labeling methods of thousand grain weight of wheat Download PDFInfo
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Abstract
The invention discloses the major gene resistance TaTGW 7A for providing control thousand grain weight of wheat.The invention also discloses the CAPS labeling methods for the major gene resistance TaTGW 7A for providing control thousand grain weight of wheat.The beneficial effects of the present invention are, utilize simplified genomic sequencing technique, identify the candidate region with Grain Weight in Common Wheat tight association, and the candidate gene in the region is cloned, through artificial colony and natural population's joint verification, the gene is a kind of to the significant new major gene resistance of grain ghost image sound, and develops functional label (TaTGW 7A).Through bioinformatic analysis, the gene is the phosphoglycerol synthase (IGPS) of indoles 3, is the key enzyme of auxin tryptophan synthetic pathway and non-tryptophan synthetic pathway, directly affects the synthesis of auxin, so as to further influence the formation of grain weight.The present invention provides new genetic resources for Yield Potential Breeding of Wheat, while further illustrates the molecular mechanism of yield composition.
Description
Technical field
The present invention relates to major gene resistance TaTGW-7A and its CAPS labeling method of control thousand grain weight of wheat.
Background technology
Wheat (Triticum aestivumL.) is the cereal crops that total output occupies second in the world, improves wheat production
Amount all has very important strategic importance to national food security and increase agriculture production benefit.Mass of 1000 kernel is wheat yield
One of three elements are formed, there is higher genetic force (Giura and Saulescu, 1996), therefore, the breeding process of wheat is very
It is exactly to improve constantly grain weight in big degree.However, the molecular regulation mechanism of grain weight is still unintelligible.
Forefathers' report is summarized, mass of 1000 kernel belongs to quantitative character, by main effect plus minor-polygene co- controlling.At present, grain weight
Related QTL site report is more, be widely distributed on 21 chromosomes of wheat (Araki etc., 1999；Shah etc., 1999；Kato
Deng 2000；Groos etc., 2003；Huang etc., 2003；Breseghello etc., 2006；Huang etc., 2006；Sun etc., 2009；
Marta etc., 2013；Simmonds etc., 2014).Wherein main effect QTL site is located at 1A, 3A, 4B, 5A, 6A 7A, 7B, 7D
(Campbell etc., 2003；Huang etc., 2003；Vasilis etc., 2010).In wheat, some functional gene of regulation and control grain weight is
Through being cloned.Control grain wide in such as Su (2011) homologous clone rice and the major gene resistance TaGW2 of grain weight.Ma etc. (2012)
The cell membrane invertase gene TaCWI related to Grain Weight in Common Wheat is cloned.Summary understands that the Genetic Mechanisms of mass of 1000 kernel are more multiple
Miscellaneous, different materials, genetic background is different, the major gene resistance of control grain weight also difference.More grain heavy phase correlation genes are identified,
Not only contribute to accelerate the process of multiple gene polymerization breeding, while help to further elucidate the molecular mechanism of yield composition.
The content of the invention
The technical problem to be solved in the present invention is to provide the major gene resistance TaTGW-7A and its CAPS of control thousand grain weight of wheat
Labeling method.
The present invention is achieved through the following technical solutions.
The major gene resistance TaTGW-7A of thousand grain weight of wheat is controlled, kind is capital 411, such as sequence table of its sequence.
The major gene resistance TaTGW-7A of thousand grain weight of wheat is controlled, kind is the red awns spring 21, such as sequence table of its sequence.
The major gene resistance TaTGW-7A of thousand grain weight of wheat CAPS labeling methods, step include：
(1) Wheat volatiles DNA is extracted；
(2) digestion is carried out with restriction enzyme HaeIII to genomic DNA, to obtained endonuclease bamhi Klenow
Fragment (3 ' → 5 ' exo -) (NEB) and dATP carries out 3 ' ends at 37 DEG C plus A processing, connection Dual-index sequencings connect
Head, PCR amplifications, purifying, sample mixing, cut glue choose purpose fragment, pcr amplification primer thing：FAATGATACGGCGACCACCGA,
RCAAGCAGAAGACGGCATACG, it is sequenced after library quality inspection is qualified with IlluminaHiSeqTM2500；
(3) primer is designed：
TaTGW-7A full length gene primers Q29F：GCAAGCCCGTCAACATGTACTAC, Q29R：
AATTTCTGGACTTATGGGGAGCC, Q35F：TCTACCACGCAGCAAATCGCC；Q35R：
CTGGACTTATGGGGAGCCTCT；Q37F：GCCCGTCAACATGTACTACTC, Q37R：GGTAGAGCTTCTCATAGCTGC,
Splice primer upstream 29PF：AAGGTTTGGGGAGTAAAGTTTT, 29PR：AGTCAGAATTGTGCCTAAGTGC；Downstream 31PF：
GAGGGGTAGGATGAAGGAAGA, 31PR：ATTTCTGGACTTATGGGGAGC, mRNA sequence SRF：
ACCCATCCCTCCATCCGTCG, SRR：ATCCGCCCACCTGGTAAAGC, digestion primer MQF：
GCTACATACACGCACCAACCC, MQR：GACGAGAAGATAGGCGGACAG
Primer Q29, Q35, Q37 are used to expand TaTGW-7A full length genes, PCR primer direct Sequencing；Primer 2 9P and 31P
TaTGW-7A upstream region of gene and downstream, PCR primer cloning and sequencing, and spliced gene total length are expanded respectively；Primer SR is used to expand
MRNA, product cloning sequencing；Primer MQ is used for CAPS marker developments, with BsmAI (NEB) at 55 DEG C digestion 2h, product is 2%
Agarose gel electrophoresis on separate, after dyeing under ultraviolet light observation note band.
Further, the step of (1) Wheat volatiles DNA is extracted includes：
1) wheat single grain is ground, take 0.1g add 0.7mLSDS extract solutions (0.1M Tris-HCl (PH=8.5),
0.1M NaCl, 0.05M EDTA (PH=8.0), 2%SDS) 45min is cracked under 60 DEG C of constant temperature, repeatedly concussion therebetween；
2) under the conditions of 4 DEG C of 12000rpm, 10min is centrifuged；
3) supernatant is taken to add isometric phenol：Chloroform：Isoamyl alcohol (25:24:1) turn upside down rotation to two-phase not quickly
Layering；
4) under the conditions of 4 DEG C of 12000rpm, 10min is centrifuged；
5) supernatant is taken to add isometric phenol：Chloroform：Isoamyl alcohol (25:24:1) rotation is turned upside down for several times；
6) under the conditions of 4 DEG C of 12000rpm, 10min is centrifuged；
7) take supernatant to add isometric isopropanol and stand 30min in -20 DEG C of refrigerators；
8) under the conditions of 4 DEG C of 12000rpm, 10min is centrifuged；
9) washed twice with 70% ethanol；
10) under the conditions of 4 DEG C of 12000rpm, 10min is centrifuged；
11) natural air drying is precipitated, 4 DEG C are deposited, standby.
Further, (2) are sequenced from arabidopsis as control.
Further, the PCR reaction systems of (3) CAPS marks：1 μ L 2.5mM dNTP, 0.25 μ L 10uM primers, 1 μ L
10*La Tap Buffer, 0.5U TaKaRa LaTap, 100ng DNA profilings, with double distilled water polishing to 10uL.
Further, the PCR response procedures of (3) CAPS marks：95 DEG C of denaturation 5min；95 DEG C of denaturation 45s；55 DEG C of annealing
50s；72 DEG C of extensions 50s, 72 DEG C of extension 10min.35 circulations.
Further, agarose gel electrophoresis：1.5-2% Ago-Gels, buffer system is 1 × TAE solution, in 100V
Under constant pressure, electrophoresis 50min, ethidium bromide (EB) is dyed, and gel imaging system is scanned and preserved.Polyacrylamide gel electrophoresis：
6% denaturing polyacrylamide gel, buffer system are 1 × TBE solution, voltage 1200V, electric current 55A, power 55W, electrophoresis
90-110min。
The present invention cardinal principle be：
(1) simplify genomic sequencing technique (1) and bioinformatics is utilized based on bioinformatics progress scheme system design
Method, network analysis is carried out to the reference gene group (or known BAC sequences) of wheat, according to the G/C content of genome, repeats sequence
The information such as row situation and genetic traits, marker development scheme is designed, to ensure the density of its molecular markers development, uniformity, effect
Rate and the accuracy of association analysis.(2) SLAF-seq library constructions and sequencing selection wheat build SLAF- as research material
Seq libraries, the scheme designed according to previous information method, screen specific long fragment, are obtained using high throughput sequencing technologies
Obtain magnanimity sequence label.(3) SLAF-seq data analyses carry out basic handling to data, and sequencing data is assessed, and obtain
Depth is sequenced and quality meets the SLAF fragments of requirement, to represent wheat cdna group information.The present invention utilizes SLAF-seq technologies
Identify the hot spot region of Grain Weight in Common Wheat related gene.
(2) PCR (PCR), reactions steps：1. the denaturation of template DNA：Template DNA is heated to 93 DEG C
After the certain time of left and right, make template DNA double-strand or the double-stranded DNA to be formed dissociation is expanded through PCR, make single-stranded, so as to it
Combined with primer, prepared for lower whorl reaction；2. the annealing (renaturation) of template DNA and primer：Template DNA is heated to be denatured into list
After chain, temperature is down to 55 DEG C or so, and the primer complementary series pairing single-stranded with template DNA combines；3. the extension of primer：DNA moulds
Plate -- primer conjugate is in the presence of Taq DNA polymerase, and using dNTP as reaction raw materials, target sequence is template, by base complementrity
Pairing and semi-conservative replication principle, a new semi-conservative replication chain complementary with template DNA chain is synthesized, it is left by 30 circulations
The right side, purpose fragment are largely expanded.The present invention uses the software Design primers of Primer Premier 5, to grain weight correlation candidate
Gene is cloned.
Beneficial effects of the present invention：
Using genomic sequencing technique is simplified, the candidate region with Grain Weight in Common Wheat tight association is identified, and cloned this
The candidate gene in region, through artificial colony and natural population's joint verification, the gene is a kind of significant to grain ghost image sound new
Major gene resistance, and develop functional label (TaTGW-7A).Through bioinformatic analysis, the gene is indoles -3- phosphoglycerol
Synthase (IGPS), it is the key enzyme of auxin tryptophan synthetic pathway and non-tryptophan synthetic pathway, directly affects auxin
Synthesis, so as to further influence the formation of grain weight.The present invention provides new genetic resources for Yield Potential Breeding of Wheat, while enters one
Step illustrates the molecular mechanism of yield composition.
Brief description of the drawings
Fig. 1 is capital 411 and observation of red awns spring 21 note band schematic diagram；(1：Capital 411；2：The red awns spring 21；a：Before digestion；b：
After digestion)
Fig. 2 is the schematic diagram of TaTGW-7A Genes location；(red line, green line, blue line represent 2011,2012,2014 respectively
3.0) year grain tuple evidence, LOD value take
Embodiment
The present invention is described in further detail below according to drawings and examples.
(1) Wheat volatiles DNA is extracted
Test material for DNA extractions mixes pond, RIL RIL for 21, two height in parent capital 411 and red awns spring
(the red awns spring 21 of capital 411/) F9Each family of colony, 256 parts of Germplasms.Specific method is：
1st, wheat single grain is ground, take 0.1g add 0.7mLSDS extract solutions (0.1M Tris-HCl (PH=8.5),
0.1M NaCl, 0.05M EDTA (PH=8.0), 2%SDS) 45min is cracked under 60 DEG C of constant temperature, repeatedly concussion therebetween.
2nd, under the conditions of 4 DEG C of 12000rpm, 10min is centrifuged.
3rd, supernatant is taken to add isometric phenol：Chloroform：Isoamyl alcohol (25:24:1) turn upside down rotation to two-phase not quickly
Layering.
4th, under the conditions of 4 DEG C of 12000rpm, 10min is centrifuged.
5th, supernatant is taken to add isometric phenol：Chloroform：Isoamyl alcohol (25:24:1) rotation is turned upside down for several times.
6th, under the conditions of 4 DEG C of 12000rpm, 10min is centrifuged.
7th, take supernatant to add isometric isopropanol and stand 30min in -20 DEG C of refrigerators.
8th, under the conditions of 4 DEG C of 12000rpm, 10min is centrifuged.
9th, washed twice with 70% ethanol.
10th, under the conditions of 4 DEG C of 12000rpm, 10min is centrifuged.
11st, natural air drying is precipitated, 4 DEG C are deposited, standby.
(2) gene order-checking and digestion conceptual design are simplified
1st, reference gene group determines：It is final to choose wheat A bases according to information such as the Genome Size of wheat and G/C contents
Because a group conduct reference gene group carries out digestion prediction.
2nd, digestion scheme determines：Digestion prediction is carried out to reference gene group using digestion forecasting software, selects most suitable digestion
Scheme.
3rd, experiment flow：According to selected most suitable digestion scheme, to detecting qualified each sample genomic DNA with restricted
Restriction endonuclease HaeIII (New England Biolabs, NEB), carry out digestion.Obtained endonuclease bamhi (SLAF labels) is used
Klenow Fragment (3 ' → 5 ' exo -) (NEB) and dATP carry out 3 ' ends plus A processing, connection Dual-index at 37 DEG C
Sequence measuring joints, PCR amplification (pcr amplification primer things：F AATGATACGGCGACCACCGARCAAGCAGAAGACGGCATACG), it is pure
Change (Agencourt AMPure XP beads (Beckman Coulter, High Wycombe, UK)), sample mixing, cut glue selection
Purpose fragment, it is sequenced after library quality inspection is qualified with IlluminaHiSeqTM2500.To assess the accuracy of digestion experiment,
It is sequenced from arabidopsis (Arabidopsis thaliana ecotype Columbia) as control (Control).
4th, information analysis flow：It is identified using the Dual-index initial data obtained to sequencing, obtains each sample
The reads of product.After filtering sequencing reads joint, the assessment of sequencing quality and data volume is carried out.Commented by Control data
Digesting efficiency is estimated, with the accuracy and validity of this judgment experiment process.Method by being clustered between reads, in parent and mix
SLAF labels are developed in pond, find the SLAF labels that polymorphism in parent be present.Obtained polymorphism SLAF labels are carried out
Association analysis, the SLAF labels with character tight association are obtained, and are located in reference gene group, determine candidate region,
Molecular labeling and candidate region of the acquisition with Grain Weight in Common Wheat tight association, and gene cloning and work(are carried out to the transcript in region
Can marker development.
(3) design of primers
1st, TaTGW-7A full length genes primer Q29F：GCAAGCCCGTCAACATGTACTAC Q29R：
AATTTCTGGACTTATGGGGAGCC Q35F：TCTACCACGCAGCAAATCGCC；Q35R：
CTGGACTTATGGGGAGCCTCT；Q37F：GCCCGTCAACATGTACTACTC Q37R：GGTAGAGCTTCTCATAGCTGC.
Splice primer (being used for cloning and sequencing) upstream 29PF：AAGGTTTGGGGAGTAAAGTTTT 29PR：
AGTCAGAATTGTGCCTAAGTGC；Downstream 31PF：GAGGGGTAGGATGAAGGAAGA31PR：
ATTTCTGGACTTATGGGGAGC.MRNA sequence SRF：ACCCATCCCTCCATCCGTCG SRR：
ATCCGCCCACCTGGTAAAGC.Digestion primer MQF：GCTACATACACGCACCAACCC MQR：
GACGAGAAGATAGGCGGACAG.SSR primers information comes from Gremene databases (http://wheat.pw.usda.gov/
GG2/index.shtml).Primer has the Software for Design of Primer Premier 5, is synthesized by Shanghai life work.
2nd, PCR reaction systems：1 μ L 2.5mM dNTP, 0.25 μ L 10uM primers, 1 μ L10*La Tap Buffer, 0.5U
TaKaRa LaTap, 100ng DNA profilings, with double distilled water polishing to 10uL.
PCR response procedures：95 DEG C of denaturation 5min；95 DEG C of denaturation 45s；55 DEG C of annealing 50s (annealing temperature is depending on primer)；
72 DEG C of extensions 50s, 72 DEG C of extension 10min.35 circulations.
3rd, PCR primer agarose gel electrophoresis：1.5-2% Ago-Gels, buffer system is 1 × TAE solution, in 100V
Under constant pressure, electrophoresis 50min, ethidium bromide (EB) is dyed, and gel imaging system is scanned and preserved.Polyacrylamide gel electrophoresis：
6% denaturing polyacrylamide gel, buffer system are 1 × TBE solution, voltage 1200V, electric current 55A, power 55W, electrophoresis
90-110min。
(4) TaTGW-7A full length genes are cloned
Primer Q29, Q35, Q37 are used to expand TaTGW-7A full length genes, PCR primer direct Sequencing；Primer 2 9P and 31P
TaTGW-7A upstream region of gene and downstream, PCR primer cloning and sequencing, and spliced gene total length are expanded respectively；Primer SR is used to expand
MRNA, product cloning sequencing；Primer MQ is used for CAPS marker developments.
(5) gene structure and sequence analysis
TaTGW-7A genes include from translation initiation codon to terminator total length 5892bp comprising 9 extrons and 8
Son, the long 2241bp of gene reading frame, encode 743 amino acid.Pass through bigger seed capital 411 and the sequence in fine grain red awns spring 21
Row difference, it is found that 4 SNP sites be present makes a variation.It is positioned at T → C on First Exon at first, causes have the ammonia of granule kind third
Acid mutation is big grain kind valine；Positioned at T → A in First Intron at second；3rd positioned at T → A in the 3rd introne；
4th C → T in the 5th introne；TaTGW-7A contains TIM-barrel fold structures, thus it is speculated that sweet for indoles -3-
Oleophosphoric acid synthase (IGPS).IGPS is the key enzyme of auxin tryptophan synthetic pathway and non-tryptophan synthetic pathway, direct shadow
The synthesis of auxin is rung, it is likely to the formation for further influenceing grain weight.
(6) marker development, Genes location and the correlation with grain weight
According to the base mutation of First Exon, design digestion primers F MQ：GCTACATACACGCACCAACCC RMQ：
GACGAGAAGATAGGCGGACAG, exploitation CAPS marks.PCR response procedures：95 DEG C of denaturation 5min；95 DEG C of denaturation 45s；62℃
Anneal 50s；72 DEG C of extension 50s, 35 circulations, 72 DEG C of extension 10min.BsmAI (NEB) digestion 2h at 55 DEG C are finally used, are produced
Thing separates on 2% agarose gel electrophoresis, and band (Fig. 1) is remembered in observation under ultraviolet light after dyeing.
The present invention utilizes two kind of groups (natural population that RIL and 256 parts of Germplasms are formed) simultaneously
Carry out TaTGW-7A and grain weight correlation analysis.
As a result show, under 7 years environment (2006-2008,2010-2012,2014), TaTGW-7A is in RIL group
19.9-26.2% phenotypic variation can be explained in body.Under 3 years (2011,2012,2014) environment, TaTGW-7A is in nature
Display and the correlation of grain weight reach the pole level of signifiance in colony, can explain 10.4-12.6% phenotypic variation.Illustrate TaTGW-
7A is really more significant to grain ghost image sound, for the main effect QTL of regulation and control grain weight.The present invention continues with above-mentioned recombinant inbred lines,
Choose the SSR primers being evenly distributed on 7A and carry out Genes location analysis, the results showed that TaTGW-7A and Xbarc222 close linkages
(Fig. 2), and the gene is just positioned at the section of original oriented main effect QTL, thus further infers that TaTGW-7A conducts
The candidate gene in QTL regions influences the phenotype of grain weight.
The above embodiments merely illustrate the technical concept and features of the present invention, and its object is to allow be familiar with this art
Personage can understand present invention and be carried out, and it is not intended to limit the scope of the present invention.It is all according to the present invention
The equivalent change or modification that Spirit Essence is made, it should all cover within the scope of the present invention.
Claims (6)
1. controlling the major gene resistance TaTGW-7A of thousand grain weight of wheat CAPS labeling methods, it is characterised in that kind is capital 411,
Its sequence such as SEQ ID NO:1, its CDS area is SEQ ID NO:2；
The major gene resistance TaTGW-7A of thousand grain weight of wheat CAPS labeling methods, step include：
(1) Wheat volatiles DNA is extracted；
(2) digestion is carried out with restriction enzyme HaeIII to genomic DNA, to obtained endonuclease bamhi Klenow
Fragment and dATP carries out 3 ' ends plus A processing at 37 DEG C, connects Dual-index sequence measuring joints, PCR amplifications, purify, be mixed
Sample, cut glue selection purpose fragment, pcr amplification primer thing：
F AATGATACGGCGACCACCGA
R CAAGCAGAAGACGGCATACG
It is sequenced after library quality inspection is qualified with IlluminaHiSeqTM2500；
(3) primer is designed：
TaTGW-7A full length gene primers：
Q29：Q29F：GCAAGCCCGTCAACATGTACTAC
Q29R：AATTTCTGGACTTATGGGGAGCC
Q35：Q35F：TCTACCACGCAGCAAATCGCC
Q35R：CTGGACTTATGGGGAGCCTCT
Q37：Q37F：GCCCGTCAACATGTACTACTC
Q37R：GGTAGAGCTTCTCATAGCTGC
Splice primer：
29P：29PF：AAGGTTTGGGGAGTAAAGTTTT
29PR：AGTCAGAATTGTGCCTAAGTGC
31P：31PF：GAGGGGTAGGATGAAGGAAGA
31PR：ATTTCTGGACTTATGGGGAGC
MRNA sequence：
SR；SRF：ACCCATCCCTCCATCCGTCG
SRR：ATCCGCCCACCTGGTAAAGC
Digestion primer：
MQ：MQF：GCTACATACACGCACCAACCC
MQR：GACGAGAAGATAGGCGGACAG；
Primer Q29, Q35, Q37 are used to expand TaTGW-7A full length genes, PCR primer direct Sequencing；Primer 2 9P and 31P distinguish
Expand TaTGW-7A upstream region of gene and downstream, PCR primer cloning and sequencing, and spliced gene total length；Primer SR is used to expand mRNA,
Product cloning is sequenced；Primer MQ is used for CAPS marker developments, and with BsmAI, digestion 2h, product coagulate in 2% agarose at 55 DEG C
Separated on gel electrophoresis, band is remembered in observation under ultraviolet light after dyeing.
2. the major gene resistance TaTGW-7A of thousand grain weight of wheat according to claim 1 CAPS labeling methods, its feature exist
In, in step (1) Wheat volatiles DNA extract the step of include：
1) wheat single grain is ground, takes 0.1g to add 0.7mLSDS extract solutions and 45min is cracked under 60 DEG C of constant temperature, therebetween repeatedly
Concussion；
2) under the conditions of 4 DEG C of 12000rpm, 10min is centrifuged；
3) supernatant is taken to add isometric phenol：Chloroform：Isoamyl alcohol (25:24:1) rotation of turning upside down does not divide quickly to two-phase
Layer, isometric phenol：Chloroform：Isoamyl alcohol ratio is 25:24:1；
4) under the conditions of 4 DEG C of 12000rpm, 10min is centrifuged；
5) supernatant is taken to add isometric phenol：Chloroform：Isoamyl alcohol turns upside down rotation for several times, isometric phenol：Chloroform：
Isoamyl alcohol ratio is 25:24:1；
6) under the conditions of 4 DEG C of 12000rpm, 10min is centrifuged；
7) take supernatant to add isometric isopropanol and stand 30min in -20 DEG C of refrigerators；
8) under the conditions of 4 DEG C of 12000rpm, 10min is centrifuged；
9) washed twice with 70% ethanol；
10) under the conditions of 4 DEG C of 12000rpm, 10min is centrifuged；
11) natural air drying is precipitated, 4 DEG C are deposited, standby.
3. the major gene resistance TaTGW-7A of thousand grain weight of wheat according to claim 1 CAPS labeling methods, its feature exist
In step (2) is middle to be sequenced from arabidopsis as control.
4. the major gene resistance TaTGW-7A of thousand grain weight of wheat according to claim 1 CAPS labeling methods, its feature exist
In the PCR reaction systems that CAPS is marked in step (3)：1 μ L 2.5mM dNTP, 0.25 μ L 10uM primers, 1 μ L 10*La
Tap Buffer, 0.5U TaKaRa LaTap, 100ng DNA profilings, with double distilled water polishing to 10uL.
5. the major gene resistance TaTGW-7A of thousand grain weight of wheat according to claim 1 CAPS labeling methods, its feature exist
In the PCR response procedures that CAPS is marked in step (3)：95 DEG C of denaturation 5min；95 DEG C of denaturation 45s；55 DEG C of annealing 50s；72 DEG C are prolonged
Stretch 50s, 72 DEG C of extension 10min, 35 circulations.
6. the major gene resistance TaTGW-7A of thousand grain weight of wheat according to claim 1 CAPS labeling methods, its feature exist
In agarose gel electrophoresis：1.5-2% Ago-Gels, buffer system is 1 × TAE solution, under 100V constant pressures, electrophoresis
50min, ethidium bromide staining, gel imaging system are scanned and preserved, polyacrylamide gel electrophoresis：6% modacrylic
Acrylamide gel, buffer system are 1 × TBE solution, voltage 1200V, electric current 55A, power 55W, electrophoresis 90-110min.
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