CN101775439A - Auxiliary method for screening wheat of different 1000-grain weights and special marker thereof - Google Patents
Auxiliary method for screening wheat of different 1000-grain weights and special marker thereof Download PDFInfo
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Abstract
The invention discloses an auxiliary method for screening wheat of different 1000-grain weights and a special marker thereof. The method detects the 2708th deoxyribonucleotide from the end of 5' of the ribotide as showed in Sequence 2 in a genome DNA of the wheat to be detected is A or G to determine that the genotype of the wheat to be detected is AA or GG; the AA genotype is the homozygote of the 2708th deoxyribonucleotide from the end of 5' of the ribotide as shown in Sequence 2 in the genome DNA of the wheat to be detected which is A; the GG genotype is the homozygote of the 2708th deoxyribonucleotide from the end of 5' of the ribotide as shown in Sequence 2 in the genome DNA of the wheat to be detected which is G; the AA genotype is an excellent allelic variation of the wheat, the 1000-grain weight and/or the grain width of the AA genotype wheat is higher than the GG genotype wheat. The marker has good repeatability and low cost and has good application prospect in the auxiliary selection of the molecular marker for improving the 1000-grain weight (particle width) of the wheat and in molecular design breeding.
Description
Technical field
The present invention relates to different thousand seed weight method of wheat of a kind of assisting sifting and special marker thereof.
Background technology
Wheat is one of main food crop of China, and the height of its output directly influences people's standard of living and national food safety.Along with improving constantly of Increase of population, cultivated area minimizing and grain-production cost, SOYBEAN IN HIGH-YIELD BREEDING will be the eternal theme of China's wheat breeding.Yet China's improving yield of wheat breeding at present is in ramp up.Utilize advanced Protocols in Molecular Biology excavation, the utilization functional gene directly related with wheat yield, for genetic resources is stocked and provided to wheat molecular mark and transgenic breeding, to improve China's wheat breeding level, quicken breeding process, significantly to improve wheat yield significant.
Wheat yield mainly is made of mu spike number, grain number per spike and thousand seed weight, and grain number per spike becomes certain negative correlation with the mu spike number in the output three elements, and promptly grain number per spike is less relatively at most for the mu spike number, and vice versa, and thousand seed weight is then relatively independent.Therefore, the raising of thousand grain weight of wheat will be from now on one of main path of promoting of China's yield of wheat (Hu Yanji, Zhao Tanfang. the research that the grain recast is used in the improving yield of wheat breeding. Acta Agronomica Sinica, 1995,21:671-678; Xu Weigang, Hu Lin, Wu Zhaosu, Gai Junyi. the research of Guanzhong area wheat breed output and output structure genetic improvement. Acta Agronomica Sinica, 2000,26:352-358).Thousand seed weight mainly by grain length, grain is wide and grain factor such as thick constitutes, it is a typical quantitative character, be subjected to controlled by multiple genes (Ammiraju J S S, Dholakia B B, SantraD K, et al.Identification of inter simple sequence repeat (ISSR) markersassociated with seed size in wheat.Theor Appl Genet, 2001,102:726-732; Groos C, Robert N, Bervas E, Charmet G.Genetic analysis of grainprotein-content, grain yield and thousand-kernel weight in bread wheat.Theor Appl Genet, 2003,106:1032-1040; Huang X Q,
H, Ganal M W,
M S.Advanced backcross QTL analysis for the identification of quantitativetrait loci alleles from wild relatives of wheat (Triticum aestivum L.) .TheorAppl Genet, 2003,106:1379-1389; Kumar N, Kulwal P, Gaur A, Tyagi A, KhuranaJ, Khurana P, Balyan H, Gupta P.QTL analysis for grain weight in commonwheat.Euphytica, 2006,151:135-144).Three integrants of thousand seed weight and the thousand seed weight relation that all is proportionate, and do not have negative correlation between them.Therefore, can reach the purpose that improves thousand grain weight of wheat by the arbitrary integrant that improves thousand seed weight.Result of study shows, in the constituent element of thousand grain weight of wheat, grain wide relational degree and the highest (the Sun X Y of contribution rate to thousand seed weight, Wu K, Zhao Y.QTL analysis of kernelshape and weight using recombinant inbred lines in wheat[J] .Euphytica, 2008,165:615-624; Gai Hongmei. the selection of China's wheat principal item involves utility analysis [doctorate paper]. Beijing: Chinese Academy of Agricultural Sciences's doctorate paper, 2007).Though in wheat, located some and QTLs (the Li S S wheat groat re-correlation by the method for linkage mapping, Jia J Z, Wei X Y, ZhangX C, Chen H M, Sun H Y, et al.An intervarietal genetic map and QTL analysisfor yield traits in wheat.Mol Breed, 2007,20:167-178; Kumar K, KulwalP L, Balyan H S, Gupta P K.QTL mapping for yield and yield contributingtraits in two mapping populations of bread wheat.Mol Breed, 2007,19:167-177), but because the wheat cdna group is huge, the research difficulty, the present report that does not still have the genes involved clone.
Summary of the invention
The purpose of this invention is to provide different thousand seed weight method of wheat of a kind of assisting sifting and special marker thereof.
Different thousand seed weight wheats of assisting sifting provided by the invention and/or the wide method of wheat of different grains are following (a) or (b):
(a) Nucleotide shown in the sequence 2 is A or G from 5 ' terminal the 2708th deoxyribonucleotide in the genomic dna of detection wheat to be measured, and the thousand seed weight of AA genotype (excellent allelic variation) wheat and/or grain are wide statistically greater than the GG genotype wheat; Described AA genotype is that Nucleotide shown in the sequence 2 is the homozygote of A from 5 ' terminal the 2708th deoxyribonucleotide; Described GG genotype is that Nucleotide shown in the sequence 2 is the homozygote of G from 5 ' terminal the 2708th deoxyribonucleotide;
(b) Nucleotide shown in the sequence 7 is A or G from 5 ' terminal the 254th deoxyribonucleotide in the genomic dna of detection wheat to be measured, and the thousand seed weight of AA genotype wheat and/or grain are wide statistically greater than the GG genotype wheat; Described AA genotype is that Nucleotide shown in the sequence 7 is the homozygote of A from 5 ' terminal the 254th deoxyribonucleotide; Described GG genotype is that Nucleotide shown in the sequence 2 is the homozygote of G from 5 ' terminal the 254th deoxyribonucleotide.
Nucleotide shown in the sequence 7 is white 5 ' the terminal 2455-2872 position Nucleotide of Nucleotide shown in the sequence 2.
Described method can comprise that extracting genome DNA, pcr amplification and enzyme cut.
Described method specifically comprises the steps:
(1) genomic dna of extraction wheat to be measured;
(2) genomic dna with step (1) is a template, with primer first is carried out pcr amplification; Described primer is that the primer that Nucleotide is formed shown in the sequence 4 of Nucleotide shown in the sequence 3 of sequence table and sequence table is right to first;
(3) the PCR product with step (2) is a template, carries out pcr amplification with primer pair B; Described primer pair B is that the primer that Nucleotide is formed shown in the sequence 6 of Nucleotide shown in the sequence 5 of sequence table and sequence table is right;
(4) cut the PCR product of step (3) with restriction enzyme Taq I enzyme; Cut product if obtain the enzyme of 167bp, wheat to be measured is the AA genotype; Cut product if obtain the enzyme of 218bp, wheat to be measured is the GG genotype.
In the described step (2): the used polysaccharase of pcr amplification specifically can be the pfu enzyme; The PCR response procedures specifically can be: 94 ℃ of 4min; 94 ℃ of 45sec, 64 ℃ of 45sec, 72 ℃ of 3min30sec, 32 circulations; 72 ℃ of 10min.
In the described step (3): the used polysaccharase of pcr amplification specifically can be the Taq enzyme; The PCR response procedures specifically can be: 94 ℃ of 4min; 94 ℃ of 45sec, 55 ℃ of 45sec, 72 ℃ of 50sec, 32 circulations; 72 ℃ of 10min.
In the described step (4), the enzyme tangent condition specifically can be: 65 ℃, 1h.
Available agarose gel electrophoresis detects the enzyme that described enzyme cuts and cuts product, as 2.0% (quality percentage composition) agarose gel electrophoresis.
The present invention also protects the primer special of different thousand seed weight wheats of assisting sifting and/or the wide wheat of different grains, primer first and primer pair B is made of; Described primer is that the primer that Nucleotide is formed shown in the sequence 4 of Nucleotide shown in the sequence 3 of sequence table and sequence table is right to first; Described primer pair B is that the primer that Nucleotide is formed shown in the sequence 6 of Nucleotide shown in the sequence 5 of sequence table and sequence table is right.
The application of described primer special in the test kit of different thousand seed weight wheats of preparation assisting sifting and/or the wide wheat of different grains also belongs to protection scope of the present invention.
The present invention protects the test kit of different thousand seed weight wheats of a kind of assisting sifting and/or the wide wheat of different grains simultaneously, comprises described primer special.
Described method, described primer special, described test kit all can be applicable in the wheat breeding, specifically the genotypic wheat of AA can be used for breeding.The thousand seed weight of described AA genotype wheat and/or grain are wide statistically greater than the GG genotype wheat, specifically can be difference and reach utmost point conspicuous level (P＜0.01).
The invention discloses following content: thousand grain weight of wheat candidate gene TaGW2-A1 cDNA encoding sequence, and this assignment of genes gene mapping is on wheat 6A karyomit(e); The promoter region sequence of the about 3.3kb of TaGW2-A1 upstream of coding region, the position of having predicted the core promoter functional element have been obtained by this gene; Find that different grains weigh (grain is wide) storeroom TaGW2-A1 gene-593 (upstream from start codon) and locate to exist single nucleotide polymorphism (SNP) site, constitute two different genotype, this genotype and seed width are good corresponding relationship; According to TaGW2-A1 upstream region of gene promoter region mononucleotide site (593 places, difference G/A) has been developed the SNP mark, called after SNP593; SNP593 mark scanning China 255 parts of wheat micro core germplasm of application and development, grain characters data in conjunction with all material, utilize mark/proterties association analysis to find, SNP593-A allelic variation and the high grain of the wheat nearly significant correlation of reclosing (P＜0.0528), with a wide utmost point significant correlation (P＜0.004).This SNP is the codominant marker, and good reproducibility, cost are low, has application promise in clinical practice in molecular marker assisted selection that improves thousand grain weight of wheat (grain is wide) and molecular designing breeding.
Description of drawings
Fig. 1 is the amplification of Chinese spring seed cDNA GW2; The 1-4:PCR amplified production; M:Marker.
Fig. 2 is main grass GW2 coding region sequence homology relatively (a DNAMAN software); Other sequences all come from ncbi database except that TaGW2-A1.
Fig. 3 is the amplification of TaGW2-A1 upstream promoter region sequence; The 1-3:PCR amplified production; M:Marker.
Fig. 4 is a TaGW2-A1 core promoter original paper synoptic diagram.
Fig. 5 is different grain wide material promoter region single nucleotide polymorphism (SNP) sites.
Fig. 6 is the chromosomal localization of TaGW2-A1 SNP593 mark; NT6A6B, NT6A6D, NT6B6A, NT6D6B represent wheat 6A respectively, 6A, 6B, the chromosomal disappearance of 6D; H
2O and China spring (CS) are represented feminine gender and positive control respectively.
Fig. 7 is a SNP mark secondary PCR amplification; M:Marker; 1: Bai Mangmai; 2: March Huang; 3: red winter wheat; 4: Lankao 906; 5: Xuzhou 22; 6: Laizhou 953.
Fig. 8 is that big grain detects with granule kind SNP mark sepharose; M:Marker; 1,2: Bai Mangmai; 3,4: March Huang; 5,6: red winter wheat; 7,8: Lankao 906; 9,10: Xuzhou 22; 11,12: Laizhou 953.
Fig. 9 is the thousand seed weight of various years improved variety, the analysis of trend of the wide and SNP593-A frequency of grain; A: thousand seed weight; B: grain is wide; C allelic variation SNP593_A distributes.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.
Wheat breed among the embodiment is all available from Chinese crop germplasm Information Network, and being numbered in the net in table 1 and the table 2 numbered.Network address: http://www.cgris.net/zhongzhidinggou/index.php.
Experiment material sees Table 1.
Used wheat lines among table 1 embodiment 1
| Wheat breed | Numbering | Primary source |
| China spring | ??ZM005452 | The Sichuan local variety |
| Bai Mangmai | ??ZM000215 | Ji County, Hebei (farm variety) |
| March Huang | ??ZM002685 | Originate not quite clear (farm variety) |
| Red winter wheat | ??ZM005188 | Tacheng (farm variety) |
| Lankao 906 | ??ZM025358 | Lankao County kind exhibition centre, Henan |
| Xuzhou 22 | ??ZM022308 | Academy of agricultural sciences, Xuzhou City, Jiangsu Province |
| Laizhou 953 | ??ZM022727 | Laizhou City, Shandong institute of agricultural sciences |
One, the acquisition of TaGW2-A1 cDNA and promoter region nucleotide sequence thereof
1, the clone of TaGW2-A1 cDNA
With paddy rice GW2 sequence is target, BLAST homologous sequence in the est database of wheat, and the wheat est sequence in the comparison carries out electronic splicing, is template design primer with the electronic splicing sequence.With Chinese spring seed cDNA is template, with Auele Specific Primer (cDNA amplimer) is carried out pcr amplification, obtains the target fragment (the amplified production electrophorogram is seen Fig. 1) about 1.2kb.Through sequence verification, amplified production length is 1275bp, names for TaGW2-A1 (nucleotide sequence is seen the sequence 1 of sequence table).There is higher homology (homology is relatively seen Fig. 2) in the gene that TaGW2-A1 and NCBI go up paddy rice, barley, false bromegrass, corn and the Chinese sorghum of announcing.Therefore can be clear and definite, the cDNA clone who obtains in wheat is the homologous gene of paddy rice GW2.
CDNA amplimer: GW2F:5 '-ATGGGGAACAGAATAGGAGGGAGGA-3 ';
GW2R：5′-TTACAACCATGCCAACCCTTGCGTG-3′。
The Pfu enzyme of PCR polysaccharase: TRANSGENE.
PCR response procedures: 94 ℃ of 4min; 94 ℃ of 45sec, 60 ℃ of 30sec, 72 ℃ of 2min, 32 circulations; 72 ℃ of 10min.
2, Chinese spring BAC library screening
Get 5ul behind the bacterial classification mixing that China spring BAC mixing pit is preserved and be inoculated in the LB substratum shaking speed 220rpm, 37 ℃ of overnight incubation.Alkaline lysis method of extracting plasmid DNA increases according to TaGW2-A1 gene order primer, and positive mixing pit is carried out following screening:
1) after the preservation bacterium liquid dilution with positive BAC mixing pit, be coated on LB solid culture primary surface, 37 ℃ of incubated overnight, the mono-clonal of 5 times of clone's numbers in mixing pit of picking is inoculated in 384 orifice plates 37 ℃ of overnight incubation, the freezing preservation of substratum then;
2) be prepared into mixing pit with all mono-clonals in every 384 orifice plate, be inoculated in overnight incubation in the LB substratum, alkaline lysis method of extracting plasmid DNA, PCR detects;
3) to step 2) in the mixing pit that is positive carry out the positive monoclonal screening.16 clones that walk crosswise in (A-P) and 24 files (1-24) in 384 orifice plates are mixed respectively, obtain 40 littler clone's mixing pits altogether.Be template with its bacterium liquid respectively, use the allele specific PCR primer amplification, detect and whether contain positive colony.The point of crossing of walking crosswise with file of containing positive colony is exactly possible positive monoclonal; Mono-clonal to corresponding position in 384 orifice plates is verified with the PCR primer.
3, the extraction of BAC mono-clonal plasmid
Utilize Qiagen Large-Construct Kit (Germany) to extract the BAC plasmid DNA, the explanation of operation reference reagent box.
4, the end sequencing of BAC mono-clonal plasmid
Adopt ABI3730 analyser (Applied Biosystems) to check order.According to known array design sequencing primer, progressively check order to the upstream from start codon sequence.Order-checking BAC plasmid template concentration 〉=300ng.After finishing, order-checking utilize Seqman software to carry out sequence assembly, integration (sequence of TaGW2-A1 upstream of coding region 3kb is shown in the sequence 2 of sequence table).TaGW2-A1 upstream promoter region sequence amplification is seen Fig. 3.TaGW2-A1 core promoter original paper synoptic diagram is seen Fig. 4.
Two, the excavation in TaGW2-A1 promoter region SNP site between kind
Select the following wheat breed excavation material that serves as a mark: (narrow) grain kind that occupies a narrow space (thousand seed weight＜35g, grain is wide≤3.0mm): Bai Mangmai, March Huang, red winter wheat; (thousand seed weight＞45g, grain is wide＞3.3mm): Lankao 906, Xuzhou 22, Laizhou 953 to breed big (wide) grain kind.
1, the discovery in SNP site
Termini of promoters sequences Design primer (promoter region amplimer, downstream primer comprise the sequence of ATG downstream 387bp) according to the step 1 acquisition.The genomic dna that extracts above-mentioned wheat breed respectively carries out pcr amplification as template with the promoter region amplimer.
Promoter region amplimer: F595:5 '-GTTTTCCTGGGCTGGCACAATCA-3 ';
R1781：5′-GCGGCACTCTACGGCAGAACAAAT-3′。
The pfu enzyme of PCR polysaccharase: TRANSGENE.
PCR response procedures: 94 ℃ of 4min; 94 ℃ of 45sec, 64 ℃ of 45sec, 72 ℃ of 3min30sec, 32 circulations; 72 ℃ of 10min.
Utilize the DNA recovery test kit of Bioteke company to reclaim behind the fragment amplification, reclaim fragment and utilize the P-easy Blunt Kit of TranGen company to connect conversion.Adopt alkaline lysis method of extracting to connect the positive carrier of mono-clonal of conversion, use the ABI3730 analyser and check order.Utilize the Seqman among the DNAStar to carry out sequence assembly, MegAlign carries out sequence alignment.Sequence alignment is found: a SNP site (SNP593 mark) (see figure 5) is arranged at wheat A genomic upstream of coding region-593 place between differing materials, little (narrow) grain kind is (593G), (wide) grain kind is (593A) greatly, big grain kind (TCGA) is compared with granule kind (TCGG), has a Taq I restriction enzyme site.Carry out the exploitation of SNP mark according to this difference.
2, chromosomal localization
According to the nucleotide sequence of SNP region design special primer (location primer), and use wheat and lack-limbs carry out the chromosomal localization of SNP mark, are located (see figure 6) on the 6A of wheat karyomit(e).
Location primer: LocAF3:5 '-CGTTACCTCTGGTTTGGGTGTCGTG-3 ';
LocAR3：5′-CACCTCTCGAAAATCTTCCCAATTA-3′。
PCR enzyme: the pfu enzyme of TRANSGENE company.
PCR response procedures: 94 ℃ of 4min; 94 ℃ of 45sec, 60 ℃ of 45sec, 72 ℃ of 1min30sec, 32 circulations; 72 ℃ of 10min.
3, the exploitation of SNP mark
Auele Specific Primer is to the principle of design of (molecule marker): 1) wheat A, B, three genomes of D can not design the sequence that a primer amplification goes out the A genome specific in that SNP site district similarity is higher near this; 2) the TaqI restriction endonuclease is only discerned 4 bases, and the restriction enzyme site in sequence is more; 3) restriction endonuclease is had relatively high expectations to the PCR product, should reduce the appearance of non-specific amplification and primer dimer in the PCR product as far as possible.
Adopt following flow process to carry out the exploitation of SNP mark:
1) genomic dna that extracts six grow wheat material seedling leaves respectively contains the dna fragmentation in SNP site as template with PCR primer amplification of SNP mark.
PCR primer: LocAF3:5 '-CGTTACCTCTGGTTTGGGTGTCGTG-3 ' of SNP mark;
LocAR3：5′-CACCTCTCGAAAATCTTCCCAATTA-3′。
The pfu enzyme of PCR polysaccharase: TRANSGENE.
PCR response procedures: 94 ℃ of 4min; 94 ℃ of 45sec, 64 ℃ of 45sec, 72 ℃ of 3min30sec, 32 circulations; 72 ℃ of 10min.
The purpose of a PCR is an amplification wheat A genome, to avoid the interference of other genomic similar sequences.
2) with after 100 times of the PCR product dilutions, get the template of 1ul as secondary PCR, SNP mark secondary PCR primer increases.
SNP mark secondary PCR primer: TAQ1F403:5 '-GAGAAAGGGCTGGTGCTATGGA-3 ';
TAQ1R820：5′-GTAACGCTTGATAAACATAGGTAAT-3′。
The Taq enzyme of PCR polysaccharase: Fermentas.
PCR response procedures: 94 ℃ of 4min; 94 ℃ of 45sec, 55 ℃ of 45sec, 72 ℃ of 50sec, 32 circulations; 72 ℃ of 10min.
The electrophorogram of amplified production (secondary PCR product) is seen Fig. 7.
3) getting the secondary PCR product uses Taq I restriction endonuclease (Fermentas company) at 65 ℃ of following complete degestion 1h.
4) enzyme is cut product carries out endonuclease bamhi on 2.0% agarose detection, seen Fig. 8.(GG SNP593), has obtained the band of 218bp to three granule kinds; And three big grain kinds (AA SNP593), has obtained the band of 167bp.
The functional verification of embodiment 2, TaGW2-A1 gene SNP 593
Respectively 255 parts of wheat lines are carried out following operation:
1, extracts genomic dna.
2, the genomic dna that extracts with step 1 is a template, with primer first is carried out pcr amplification.
Primer is to first: LocAF3:5 '-CGTTACCTCTGGTTTGGGTGTCGTG-3 ';
LocAR3：5′-CACCTCTCGAAAATCTTCCCAATTA-3′。
The pfu enzyme of PCR polysaccharase: TRANSGENE.
PCR response procedures: 94 ℃ of 4min; 94 ℃ of 45sec, 64 ℃ of 45sec, 72 ℃ of 3min30sec, 32 circulations; 72 ℃ of 10min.
3, the PCR product with step 2 is a template, carries out pcr amplification with primer pair B.
Primer pair B: TAQ1F403:5 '-GAGAAAGGGCTGGTGCTATGGA-3 ';
TAQ1R820：5′-GTAACGCTTGATAAACATAGGTAAT-3′。
The Taq enzyme of PCR polysaccharase: Fermentas.
PCR response procedures: 94 ℃ of 4min; 94 ℃ of 45sec, 55 ℃ of 45sec, 72 ℃ of 50sec, 32 circulations; 72 ℃ of 10min.
4, with under 65 ℃ of the Taq I restriction endonucleases (Fermentas company) the PCR product enzyme of step 3 is cut 1h.
5, enzyme is cut product carries out endonuclease bamhi on 2.0% agarose detection.The genotype of sample with band of 218bp is GG (SNP593), and the genotype of sample with band of 167bp is AA (SNP593).
The thousand seed weight and the grain that detect each wheat breed respectively are wide.Wherein thousand seed weight is the average result of 3 measurements, and grain wide is 20 a width average.The PCR product of step 3 is carried out sequence verification, and it is consistent that sequencing result and enzyme are cut detected result.
Wheat sample and detected result see Table 2.
Used wheat lines and detected result among table 2 embodiment 2
Genotype and thousand grain weight properties, the wide proterties of grain of wheat sample are carried out association analysis, the results are shown in Table 3.
Table 3 association analysis result
| Genotype | The material number | Thousand seed weight (g) | Grain wide (mm) |
| ??GG | ??130 | ??35.08±6.34 | ??3.14±0.18 |
| ??AA | ??125 | ??36.69±6.93 | ??3.21±0.23 |
| The P value | ??0.0528 | ??0.004 |
Carrying out association analysis in conjunction with thousand seed weight, the wide phenotypic data of grain finds: AA genotypic mean thousand seed weight is 36.69g, and GG genotypic mean thousand seed weight is 35.08g, and difference is near conspicuous level; AA genotypic mean grain is wide to be 3.21mm, and GG genotypic mean grain is wide to be 3.14mm, and difference reaches utmost point conspicuous level (P＜0.01).
The analysis of trend of the thousand seed weight of embodiment 3, various years improved variety, the wide and SNP593-A frequency of grain
Thousand seed weight, grain variation tendency wide and the SNP593-A frequency to various years improved variety in the micro core germplasm are analyzed the discovery (see figure 9): along with the passing in age, the thousand seed weight of China's wheat improved variety and the wide trend (Fig. 9 A, B) that all is increase of grain, the frequency of occurrences of SNP593-A allelic variation in the various years kind also increases gradually, with thousand seed weight and grain wide variation tendency consistent (Fig. 9 C), this shows that modern breeding has carried out very strong selective action to this variation, and should variation, grain wide significant correlation heavy with grain.Therefore, this mark can be used as and improves the functional label that wheat groat heavy (grain is wide) carries out the improving yield of wheat molecular mark.
Sequence table
＜110〉Chinese Academy of Agricultural Sciences's crop science institute
＜120〉different thousand seed weight method of wheat of a kind of assisting sifting and special marker thereof
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atatgtacag?agtgctttct?tcaaatgaaa?ccaactcata?ctgctcgacc?tacacaatgc????300
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agaacaatcg?ctccaacaac?agaagtggag?tatcgagata?tttgcagcac?atcctattca????540
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gaagtggtaa?gagaggaagg?agagtgctca?accgaccact?tgtcagaggg?tgcagaggcc??1080
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tttgctgaca?attacagtat?ggttgcaagc?catttccgtc?ctgagagcat?cgaagaacaa??1200
atgatgtatt?ccatggctgt?ttctttagca?gaagctcatg?gtagaacgca?cacgcaaggg??1260
ttggcatggt?tgtaa???????????????????????????????????????????????????1275
<210>2
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＜213〉Chinese spring (Triticum aestivum L)
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tcttgggggg?gggggtcgta?ccgctctata?caggataatt?cctagatgtg?agcacgagat????60
gaacttgggg?gcagaccaac?tcgaaatttt?agaccaaatc?gaaggggaaa?ataaaatccg????120
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atcagcaaaa?actcacaaac?aacaaaatgc?aagaaatttt?ggggggctct?tttcatggat????240
tttttttgaa?atttagaaga?aaattcgaac?taaaggctag?aacatggggg?ggggggtagg????300
gctccaaatt?cgtgtcaacg?tggctcatga?taccaagatg?atataaggca?cgggccaaag????360
tgccctgatt?tgtcccaatg?ttcacgaaat?tggaggtgga?ttcgaggagg?aacacgaagg????420
gaaaacatgg?agaaatcaaa?gggggaaaca?ctctattgga?caagattcac?tcccggcact????480
cctcaaatca?caagcaatac?aagaggcgca?tcaagatcca?agaacaacaa?gggaaataaa????540
gatacaaggt?agttcatcca?aatatctagg?agagatggct?cttgatgatt?cacttgggga????600
atccttctcc?acatgtggag?gtcttgcatc?cactagggat?cttctccata?ggaggccttg????660
cactccaatg?gagtcttctc?tctcacaaga?ggaatcccac?aaagggagat?acatgagcta????720
agctctatga?tatgagttct?aatcttagct?aaccctagaa?atgaggtggg?gaaagagtat????780
atatagtcct?agccaggaag?gggtaagtga?ggggcagaat?gggaaagata?gaaggccact????840
tggccaactc?tacgtgcaga?cagaccggtt?gtaccggttg?tactataact?atcgggtggt????900
accggtcttg?taccgctcga?ttacagtaaa?gtgtccaagc?gaagcagtag?tggaccggtt????960
gtaccggtgg?accggttgtt?gaccggtagt?ggactagttg?taccagtttt?cctgggctgg????1020
cacaatcatc?ttcttccttc?tcttccatgc?ttccctcccg?gatggtgtag?ttgtactctt????1080
gtgtttggac?tcctcgtcat?ccataaagct?atgacaatac?ctatatatgc?atacaagagg????1140
agtgtcaagt?agtataccat?cctcgaaggg?gtcaagtgaa?cacgtgtaaa?tgagatgagt????1200
cacctttatg?tacgtgaagt?agatgttgca?tgtgtcactt?gccaaacgaa?ctcttgacat????1260
ggtgatgtct?gtaggatgct?ccacatcaca?aacacattag?tcccttaacc?catttgtctt????1320
caatactcca?aaaccactta?ggggcactag?atgcacttac?atttatacct?ggaattttgc????1380
ttgcaaagta?acatatggta?tctacctatc?ttgctcatcc?ccttgatgca?ccactctgtt????1440
agcgatactt?cctagagtgt?ttatctagtt?cctctagcaa?tggtaagtta?agaagaactt????1500
aagaactcct?tcaattgtac?gatcaaaatg?ttggtgtcat?ttgtaaagcc?cttggacccc????1560
caagttctga?caagccttca?ttaggaaatt?gctttttgaa?gcatgtagga?gcaacaaagg????1620
gggttctttt?cgtaccttag?atggatcatt?ctgagccttc?ttgaagagct?aggcgccgaa????1680
gctcccggcg?attggcgatt?gatccccgcc?ggcatctgga?tcttgctgtt?tcagcaacca????1740
agactagatc?cagttcagta?caaagaatgg?aagaacaaaa?aaacatgaca?tgattcgatc????1800
cttgcgctga?ttttcttacc?cgtcgaggat?gtgggtgtgg?tcacatggac?aatgctgccg????1860
atgtccccga?caacccgcat?gtttcctcct?accttctgct?tccatgtcgg?gcccgcggca????1920
catgcacatc?atgaacgaga?aacacgagta?aaagcactac?atgaacccct?tatgaacgat????1980
ttagcaatga?acacaaaatt?ctagtattta?gacatgagaa?tgccttgtac?agaatttaaa????2040
agtcatgaaa?aacgttacct?ctggtttggg?tgtcgtgcaa?ataaaacaga?gataataaat????2100
tccttatagc?caatgtggtg?aacatagcaa?attgattccc?ccgggtttga?ttccatgtgc????2160
tctagccaaa?atagatcaaa?tcagcaagat?atcttatgct?atgaatggtg?atagtggtcg????2220
ctcatgttca?tctcgactgc?cgaaatcatg?tgcccttagc?ggacgttgta?ctcctcggca????2280
gtggccacta?tcaacgagcg?gcggcagcca?aggcgaaggg?atccatgcag?atcgtgagca????2340
accgtccatg?cgtggctgcg?gcagaccgga?gcacgagtag?gaggcggcag?attccaccat????2400
ggaaggccga?ggagtggcaa?gggtagaggt?ggatgcagga?gggagggggg?gggggagaaa????2460
gggctggtgc?tatggaccgc?gggaggggag?gacgtgccag?tgacgaggga?agcgaagggc????2520
ggagcggcag?gaggcctgtc?gggtcgatga?gatcccgtac?agcagctcgc?aacaaaccct????2580
agctcgcgcg?agaagagaga?ggggatgttc?ggatcaaaga?gaggacgaga?gaaaaccggc????2640
gtggtaagaa?aaatcgataa?ggaaagaaca?tcgtatgagt?ggagaagggt?gagacgaaaa????2700
taaatcgaac?gaaaataatc?ataaagtgaa?agctaccaag?tccttcttta?aaagtagaga????2760
tcacatattc?gcttagagga?aagatgaagg?ggtaggtgat?gcgcccgcgg?tgatgcactc????2820
atcatgtcgc?ttcccattta?cgaaagcatt?acctatgttt?atcaagcgtt?acatgggata????2880
ggggatatac?acaaacatgt?tcctaaatta?attaaaaaaa?acatgtttct?aaattgtgac????2940
acagatcgat?ggtccagaat?ctacgtgtca?caaaactaat?tgggaagatt?ttcgagaggt????3000
gtatctcttg?cagcgcagcg?ggccagcggc?agagaagccg?aagcctagtg?agctgtgttg????3060
tcgtcgagtg?tcgcgtgcgc?cttctctttt?gctttgcctc?cctgttctcc?caccccgagg????3120
ggaaatgcat?ttttaaaaac?agggtaatcc?cacctcgcct?cggcgctccc?caagccccgc????3180
agcatcgcgt?acgagtgcgt?ataccgtatg?tatctacagt?gaggcggcgc?ggggcaaggg????3240
tttcgtctcc?ggcggcggcg?acggcggcgg?cggcggcgga?ggaggctaga?tctgggaggg????3300
atg??????????????????????????????????????????????????????????????????3303
<210>3
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
cgttacctct?ggtttgggtg?tcgtg??????????????????????????????????????????25
<210>4
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
cacctctcga?aaatcttccc?aatta????????????????????????????????????????????25
<210>5
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>5
gagaaagggc?tggtgctatg?ga???????????????????????????????????????????????22
<210>6
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>6
gtaacgcttg?ataaacatag?gtaat????????????????????????????????????????????25
<210>7
<211>418
<212>DNA
＜213〉Chinese spring (Triticum aestivum L)
<400>7
gagaaagggc?tggtgctatg?gaccgcggga?ggggaggacg?tgccagtgac?gagggaagcg????60
aagggcggag?cggcaggagg?cctgtcgggt?cgatgagatc?ccgtacagca?gctcgcaaca????120
aaccctagct?cgcgcgagaa?gagagagggg?atgttcggat?caaagagagg?acgagagaaa????180
accggcgtgg?taagaaaaat?cgataaggaa?agaacatcgt?atgagtggag?aagggtgaga????240
cgaaaataaa?tcgaacgaaa?ataatcataa?agtgaaagct?accaagtcct?tctttaaaag????300
tagagatcac?atattcgctt?agaggaaaga?tgaaggggta?ggtgatgcgc?ccgcggtgat????360
gcactcatca?tgtcgcttcc?catttacgaa?agcattacct?atgtttatca?agcgttac??????418
Claims (10)
1. different thousand seed weight wheats of an assisting sifting and/or the wide method of wheat of different grains are following (a) or (b):
(a) Nucleotide shown in the sequence 2 is A or G from 5 ' terminal the 2708th deoxyribonucleotide in the genomic dna of detection wheat to be measured, and the thousand seed weight of AA genotype wheat and/or grain are wide statistically greater than the GG genotype wheat; Described AA genotype is that Nucleotide shown in the sequence 2 is the homozygote of A from 5 ' terminal the 2708th deoxyribonucleotide; Described GG genotype is that Nucleotide shown in the sequence 2 is the homozygote of G from 5 ' terminal the 2708th deoxyribonucleotide;
(b) Nucleotide shown in the sequence 7 is A or G from 5 ' terminal the 254th deoxyribonucleotide in the genomic dna of detection wheat to be measured, and the thousand seed weight of AA genotype wheat and/or grain are wide statistically greater than the GG genotype wheat; Described AA genotype is that Nucleotide shown in the sequence 7 is the homozygote of A from 5 ' terminal the 254th deoxyribonucleotide; Described GG genotype is that Nucleotide shown in the sequence 2 is the homozygote of G from 5 ' terminal the 254th deoxyribonucleotide.
2. method according to claim 1 is characterized in that: described method comprises that extracting genome DNA, pcr amplification and enzyme cut.
3. method according to claim 2 is characterized in that: described method comprises the steps:
(1) genomic dna of extraction wheat to be measured;
(2) genomic dna with step (1) is a template, with primer first is carried out pcr amplification; Described primer is that the primer that Nucleotide is formed shown in the sequence 4 of Nucleotide shown in the sequence 3 of sequence table and sequence table is right to first;
(3) the PCR product with step (2) is a template, carries out pcr amplification with primer pair B; Described primer pair B is that the primer that Nucleotide is formed shown in the sequence 6 of Nucleotide shown in the sequence 5 of sequence table and sequence table is right;
(4) cut the PCR product of step (3) with restriction enzyme Taq I enzyme; Cut product if obtain the enzyme of 167bp, wheat to be measured is the AA genotype; Cut product if obtain the enzyme of 218bp, wheat to be measured is the GG genotype.
4. method as claimed in claim 3 is characterized in that:
In the described step (2): the used polysaccharase of pcr amplification is the pfu enzyme; The PCR response procedures is: 94 ℃ of 4min; 94 ℃ of 45sec, 64 ℃ of 45sec, 72 ℃ of 3min30sec, 32 circulations; 72 ℃ of 10min.
5. as claim 3 or 4 described methods, it is characterized in that: in the described step (3): the used polysaccharase of pcr amplification is the Taq enzyme; The PCR response procedures is: 94 ℃ of 4min; 94 ℃ of 45sec, 55 ℃ of 45sec, 72 ℃ of 50sec, 32 circulations; 72 ℃ of 10min.
6. as arbitrary described method in the claim 3 to 5, it is characterized in that: in the described step (4), the enzyme tangent condition is: 65 ℃, 1h; Detect the enzyme that described enzyme cuts with agarose gel electrophoresis and cut product.
7. the primer special of different thousand seed weight wheats of assisting sifting and/or the wide wheat of different grains is made up of first and primer pair B primer; Described primer is that the primer that Nucleotide is formed shown in the sequence 4 of Nucleotide shown in the sequence 3 of sequence table and sequence table is right to first; Described primer pair B is that the primer that Nucleotide is formed shown in the sequence 6 of Nucleotide shown in the sequence 5 of sequence table and sequence table is right.
8. the application of the described primer special of claim 7 in the test kit of different thousand seed weight wheats of preparation assisting sifting and/or the wide wheat of different grains.
9. the test kit of different thousand seed weight wheats of assisting sifting and/or the wide wheat of different grains comprises the described primer special of claim 7.
10. arbitrary described method, the described primer special of claim 7, the application of the described test kit of claim 9 in wheat breeding in the claim 1 to 6.
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