CN111321241B - Molecular marker of wheat thousand-grain weight and grain length gene TaGS3-4A and application thereof - Google Patents

Abstract

The invention discloses a molecular marker of a wheat thousand-grain weight and grain length gene TaGS3-4A and application thereof, wherein the molecular marker consists of a pair of complete molecular markers of a molecular marker CAPS-4A-357/713 and a molecular marker InDel-4A-351; the nucleotide sequence of the upstream primer CAPS-4A-357/713-F of the molecular marker CAPS-4A-357/713 is shown in SEQ ID NO. 2, and the nucleotide sequence of the downstream primer CAPS-4A-357/713-R is shown in SEQ ID NO. 3; the nucleotide sequence of the upstream primer InDel-4A-351-F of the molecular marker InDel-4A-351 is shown as SEQ ID NO. 4, and the nucleotide sequence of the downstream primer InDel-4A-351-R is shown as SEQ ID NO. 5. The invention disclosesTaGS3‑4AAllelic variation of the gene and the complete set of molecular markers CAPS-4A-357/713 and the molecular marker InDel-4A-351 can effectively screen out the geneTaGS3‑4AIn order to increase the thousand seed weight and the grain length of the wheat, the wheat variety or strain of excellent haplotype has a larger application space in auxiliary breeding of wheat molecules.

Description

A molecular marker for wheat thousand-grain weight and grain length gene TaGS3-4A and its application

Technical Field

The invention relates to the field of wheat molecular biotechnology and breeding applications, in particular to a molecular marker of wheat thousand-grain weight and grain length gene TaGS3-4A and application thereof.

Background Art

Wheat (Triticum aestivum L.) is one of the most important food crops in the world, and increasing yield is the most important breeding goal for wheat. The number of spikes per mu, the number of grains per spike, and grain weight together constitute the three elements of yield. Among them, grain weight has high heritability and stable phenotype. The selection of grain weight in high-yield breeding is the most effective (Alexander et al., 1984), and increasing grain length and width is an effective way to increase wheat grain weight. The increase in grain weight has played a key role in the increase of wheat yield in my country. The thousand-grain weight has increased from 31.5g in the 1940s to 44.64g in recent years, with an average increase of 2.19g every ten years (Wang et al., 2012; Zhang et al., 2012). Cloning the key genes that regulate grain size in wheat, discovering excellent alleles, and developing functional markers will effectively enrich the genetic resources of high-yield wheat and promote the development of molecular breeding for high-yield wheat.

Homologous cloning is an important way to discover new genes. The use of micro core germplasm (MCC) to screen for excellent allelic variation of genes has been widely used (Hao Chenyang et al., 2008). In recent years, multiple genes related to grain size have been homologously cloned, their excellent allelic variation has been analyzed, and molecular markers have been developed (Ma et al., 2012; Chang et al., 2013). Su et al. (2011) found that there were two haplotypes, Hap-6A-A and Hap-6A-G, in the promoter region of TaGW2-6A. Hap-6A-A increased the thousand-grain weight by more than 3g, which is an excellent haplotype and is strongly selected in breeding. Jiang et al. (2011) reported that the two haplotypes of TaSus2, Hap-H and Hap-L, were associated with thousand-grain weight, and the haplotype Hap-H associated with high thousand-grain weight was strongly selected in the breeding varieties. Zhang et al. (2012) identified five haplotypes of TaCKX6-D1, among which Hap-a was significantly associated with high 1000-grain weight and was an excellent haplotype. Zhang et al. (2014) conducted marker/trait association analysis on TaGS-D1 and detected that TaGS-D1a was significantly associated with high 1000-grain weight in 175 bred varieties. Jiang et al. (2015) conducted marker/trait association analysis on TaCWI-4A and TaCWI-5D and detected that Hap-5D-C was significantly associated with high 1000-grain weight in 348 major bred varieties in my country. Ma (2016) used the allelic variation of TaGS5 to develop CAPS markers to distinguish TaGS5-3A-T and TaGS5-3A-G. Association analysis found that TaGS5-3A-T was associated with grain size and grain weight. In short, relevant researchers in the industry are actively cloning genes related to high yield and developing related molecular markers to use molecular marker-assisted selection breeding to improve target trait selection and achieve the purpose of improving crops. However, to date, the industry has not reported excellent haplotypes of wheat gene TaGS3-4A for thousand-grain weight and grain length. Therefore, cloning the key gene for wheat grain size regulation, discovering excellent allelic variation, and developing functional markers to increase wheat thousand-grain weight and grain length will add a new and effective way for wheat molecular-assisted breeding and breeding more high-yield wheat varieties.

Summary of the invention

The purpose of the present invention is to provide a molecular marker of wheat thousand-grain weight and grain length gene TaGS3-4A and its application, which is used to efficiently identify whether the gene TaGS3-4A in wheat varieties or strains carries an excellent haplotype that increases wheat thousand-grain weight and grain length, so as to provide a scientific means for enriching wheat high-yield gene resources and promoting the development of wheat high-yield molecular breeding.

The present invention is realized by the following method: a molecular marker of wheat thousand-grain weight and grain length gene TaGS3-4A, which is a molecular marker of excellent haplotype of wheat with increased thousand-grain weight and grain length, and is composed of a pair of molecular markers, namely, molecular marker CAPS-4A-357/713 and molecular marker InDel-4A-351;

The molecular marker CAPS-4A-357/713 is the nucleotide sequence of TaGS3-4A in the wheat genome corresponding to the 357th and 713th nucleotides in SEQ ID NO:1. The upstream primer CAPS-4A-357/713-F of the molecular marker CAPS-4A-357/713 is shown in SEQ ID NO:2, and the downstream primer CAPS-4A-357/713-R is shown in SEQ ID NO: As shown in NO:3, PCR amplification of wheat genomic DNA was performed with the upstream and downstream primers, and the amplified products were digested with Hpy188I and BsrI, respectively. The digested products were detected by electrophoresis, and the amplified products obtained two target bands with molecular weights of 537 bp and 265 bp or two target bands of 540 bp and 265 bp after Hpy188I digestion, and two target bands with molecular weights of 181 bp and 621 bp or two target bands of 184 bp and 621 bp after BsrI digestion.

The molecular marker InDel-4A-351 is the 351st nucleotide corresponding to the nucleotide sequence SEQ ID NO:1 of TaGS3-4A in the wheat genome. The upstream primer InDel-4A-351-F of the molecular marker InDel-4A-351 is shown in SEQ ID NO:4, and the downstream primer InDel-4A-351-R is shown in SEQ ID NO:5. The above and downstream primers are used to perform PCR amplification on wheat genomic DNA, and the amplified products are separated by electrophoresis to obtain a band of any one of the amplified products with a molecular weight of 198bp, 200bp or 201bp.

The invention clones the nucleotide sequence of gene TaGS-4A in wheat KN9204, as shown in SEQ ID NO:1; wherein the primers of molecular marker CAPS-4A-357/713 are designed according to the nucleotide at position 357 of wheat genomic DNA corresponding to sequence table SEQ ID NO:1 being G or missing and the nucleotide at position 713 being T or A; the primers of molecular marker InDel-4A-351 are designed according to the 3bp ACT tandem insertion/deletion (Insertion/Deletion) of the nucleotide at position 351 of wheat genomic DNA corresponding to sequence table SEQ ID NO:1; PCR amplification is performed on wheat genomic DNA using the upper and downstream primers of molecular marker CAPS-4A-357/713 and the upper and downstream primers of molecular marker InDel-4A-351 to determine the gene sequence SEQ ID In NO:1, ACT deletion (351-0) or ACT (351-1ACT) is at 351bp, G (357-G) is at 357bp, and T (713-T) is at 713bp, which is a wheat variety or strain with an excellent haplotype for increasing thousand-grain weight and grain length, and is a molecular marker of the present invention.

The PCR amplification systems in the molecular marker CAPS-4A-357/713 and the molecular marker InDel-4A-351 were both 20 μL, including 10 μL of 2×Taq PCR MasterMix, 1 μL of upstream and downstream primers, 1 μL of DNA template, and the rest was supplemented with ddH ₂ O.

The PCR amplification procedure of the molecular marker CAPS-4A-357/713 is as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, 33 cycles; final extension at 72°C for 10 min, and storage at 4°C.

The PCR amplification procedure of the molecular marker InDel-4A-351 is as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 33 cycles; final extension at 72°C for 10 min, and storage at 4°C.

The present invention also provides the application of the molecular marker in molecule-assisted breeding.

The application refers to using the molecular marker to identify whether the gene TaGS3-4A is a wheat variety or strain with an excellent haplotype for increasing thousand-grain weight and grain length, and the method comprises the following steps:

(1) extracting genomic DNA of the wheat sample to be identified;

(2) PCR amplification of wheat genomic DNA was performed using the upstream and downstream primers of the molecular marker CAPS-4A-357/713, and the amplified product was digested with Hpy188I and detected by electrophoresis, so that two target bands with molecular weights of 537 bp and 265 bp or two target bands with molecular weights of 540 bp and 265 bp were obtained, or any one target band with molecular weights of 802 bp, 804 bp or 808 bp was obtained; the amplified product was digested with BsrI and detected by electrophoresis, so that two target bands with molecular weights of 181 bp and 621 bp or two target bands with molecular weights of 184 bp and 621 bp or two target bands with molecular weights of 187 bp and 621 bp were obtained, or one target band with molecular weight of 804 bp was obtained;

(3) PCR amplification of wheat genomic DNA was performed using the upstream and downstream primers of the molecular marker InDel-4A-351, and the amplified products were separated by electrophoresis, and a band with a molecular weight of 198 bp, 200 bp, 201 bp or 204 bp was obtained;

(4) In the amplification results, if the sample to be tested is detected by molecular marker CAPS-4A-357/713, and two target bands with molecular weights of 537 bp and 265 bp or two target bands with molecular weights of 540 bp and 265 bp are obtained after detection by Hpy188I enzyme digestion, and two target bands with molecular weights of 181 bp and 621 bp or two target bands with molecular weights of 184 bp and 621 bp are obtained after detection by BsrI enzyme digestion, and the molecular marker InDel-4A-351 is used to obtain any one of the target bands with molecular weights of 198 bp, 200 bp or 201 bp, then the sample is a wheat variety or strain with the excellent haplotype of gene TaGS3-4A for increasing thousand-grain weight and grain length.

In the application, the upstream primer CAPS-4A-357/713-F of the molecular marker CAPS-4A-357/713 in step (2) is shown in SEQ ID NO:2, and the downstream primer CAPS-4A-357/713-R is shown in SEQ ID NO:3.

In the application, the upstream primer InDel-4A-351-F of the molecular marker InDel-4A-351 in step (3) is shown as SEQ ID NO:4, and the downstream primer InDel-4A-351-R is shown as SEQ ID NO:5.

In the application, the PCR amplification systems in the molecular marker CAPS-4A-357/713 and the molecular marker InDel-4A-351 are both 20 μL, including 10 μL of 2×Taq PCR MasterMix, 1 μL of upstream and downstream primers, 1 μL of DNA template, and the rest is supplemented with ddH ₂ O.

In the application, the PCR amplification procedure of the molecular marker CAPS-4A-357/713 is: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 1 min, 33 cycles; 72°C final extension for 10 min, and storage at 4°C; the PCR amplification procedure of the molecular marker InDel-4A-351 is: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, 33 cycles; 72°C final extension for 10 min, and storage at 4°C.

The wheat variety Kenong 9204 in the present invention is a semi-winter, medium-maturing hybrid wheat variety, and its variety approval number is Guoshenmai 2003037.

The invention provides a molecular marker of wheat thousand-grain weight and grain length gene TaGS3-4A and an application thereof. The molecular marker consists of a pair of complete molecular markers, namely, molecular marker CAPS-4A-357/713 and molecular marker InDel-4A-351; wherein the primer of the molecular marker CAPS-4A-357/713 is designed based on the fact that the nucleotide at position 357 is G or missing and the nucleotide at position 713 is T or A in the sequence table SEQ ID NO:1 corresponding to the gene TaGS3-4A in the wheat genomic DNA; and the primer of the molecular marker InDel-4A-351 is designed based on the fact that the nucleotide at position 357 is G or missing and the nucleotide at position 713 is T or A in the sequence table SEQ ID NO:1 corresponding to the gene TaGS3-4A in the wheat genomic DNA. The design was carried out by 3bp ACT tandem insertion/deletion (Insertion/Deletion) of the 351st nucleotide of NO:1. The association analysis of PCR amplification results and phenotypic identification proved that the molecular marker CAPS-4A-357/713 and the molecular marker InDel-4A-351 were used in combination to effectively screen out wheat varieties or strains with excellent haplotypes (HAP-4A-1 and HAP-4A-2) of gene TaGS3-4A that increase wheat thousand-grain weight and grain length. Therefore, the allelic variation of the TaGS3-4A gene disclosed in the present invention and the set of molecular markers CAPS-4A-357/713 and InDel-4A-351 have a large application space in wheat molecular assisted breeding.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of expression specificity analysis of gene TaGS3-4A in different wheat tissues.

Figure 2 is a partial sequence alignment of sites 351 and 357 of the wheat gene TaGS3-4A.

FIG. 3 is a partial sequence alignment of position 713 of the wheat gene TaGS3-4A.

FIG. 4 shows the molecular marker detection result of site 351 of wheat gene TaGS3-4A.

FIG. 5 shows the molecular marker detection results of site 357 of wheat gene TaGS3-4A.

FIG. 6 shows the molecular marker detection result of site 713 of wheat gene TaGS3-4A.

DETAILED DESCRIPTION

The following examples are used to further illustrate the present invention, but are not intended to limit the present invention in any form.

Example 1 Cloning of gene TaGS3

The genomic DNA of the hexaploid wheat variety KN9204 (Kenon 9204) was extracted by conventional CTAB method, and the TaGS3 gene region was amplified. The amplification primers used were upstream primer TaGS3-F and downstream primer TaGS3-R, and their nucleotide sequences were:

Upstream primer TaGS3-F: 5′-CAATGGCGGCGCCCAGGC-3′;

Downstream primer TaGS3-R: 5’-TGGAACACAGGCAGCAGCGAGG-3’.

After amplification, agarose gel electrophoresis was performed, and the amplified fragment was recovered, connected to the T vector and sequenced; the nucleotide sequence of TaGS3-4A in KN9204 was determined by comparison with three TaGS3 homologous gene sequences in hexaploid wheat Chinese Spring, TraesCS7A02G017700, TraesCS7D02G015000, and TraesCS4A02G474000, and the sequence is shown in SEQ ID NO: 1.

Example 2 Analysis of TaGS3-4A gene expression pattern and expression level differences

The expression pattern and expression level of TaGS3-4A gene were analyzed by quantitative PCR. Wheat roots, stems, flag leaves, ears and other tissues and organs, as well as ears and grains at different developmental stages (7 days intervals) were collected to extract RNA, DNase digested DNA contamination, and cDNA reverse transcription was performed. Specific primers were designed, and the primers and nucleotide sequences were as follows:

Upstream primer TaGS3-4A-mF: 5′-GATCTGCGGCGGCGGGTGCTCT-3′;

Downstream primer TaGS3-4A-mR: 5’-CGAGCAGCCACACGAGGGCCCG-3’.

The above pair of primers was used for fluorescence quantitative PCR amplification to detect the relative expression of TaGS3-4A gene; at the same time, GADPH was used as the internal reference gene, and the upstream primer GAPDH-F: 5'-TTAGACTTGCGAAGCCAGCA-3 and the downstream primer GAPDH-R: 5'-AAATGCCCTTGAGGTTTCCC-3' were used for fluorescence quantitative PCR amplification. The amplification results are shown in Figure 1. The expression level of TaGS3-4A gene in KN9204 is relatively high in developing seeds.

Example 3 Analysis of TaGS3-4A gene nucleotide sequence polymorphism

The genomic DNA of wheat materials was extracted by conventional CTAB method, and the TaGS3-4A gene region of different wheat materials was amplified. The amplification primers used were:

Upstream primer TaGS3-4A-F: 5′-CTGCTGTTACTCTGTTATTATTAC-3′;

Downstream primer TaGS3-4A-R: 5’-CAAGAACTGCCTGTACAGTC-3’.

After amplification, agarose gel electrophoresis was performed, and the amplified fragments were recovered for sequencing; DNAMAN was used for sequence alignment, and the results are shown below.

First, there is a 3 bp tandem insertion/deletion at 351 bp in the first intron of the TaGS3-4A gene, forming three types of allelic variation. Part of the sequence is shown in Figure 2, which shows:

When the nucleotide at position 351 is missing, the allelic variation type of the TaGS3-4A gene is 351-0;

When the nucleotide at position 351 is ACT, the allelic variation type of the TaGS3-4A gene is 351-1ACT;

When the nucleotide at position 351 is ACTACT, the allelic variation type of the TaGS3-4A gene is 351-2ACT.

Second, there is a SNP at 357bp in the first intron of the TaGS3-4A gene, forming two allele types. Part of the sequence is shown in Figure 2, which shows:

When the nucleotide at position 357 is missing, the allelic variation type of the TaGS3-4A gene is 357-0;

When the nucleotide at position 357 is G, the allelic variation type of the TaGS3-4A gene is 357-G.

Third, there is a SNP at 713bp in the second intron of the TaGS3-4A gene, forming two allele types. Part of the sequence is shown in Figure 3, which shows:

When the nucleotide at position 713 is T, the allelic variation type of the TaGS3-4A gene is 713-T;

When the nucleotide at position 713 is A, the allelic variation type of the TaGS3-4A gene is 713-A.

The haplotype of TaGS3-4A gene was determined based on the allelic variation of loci 351, 357 and 713 as follows:

Table 1 Allelic variation at loci 351, 357 and 713 and haplotype of TaGS3-4A

Example 4 Development of functional molecular markers of TaGS3-4A gene

(1) The genomic DNA of wheat materials was extracted by conventional CTAB method, and the nucleotide regions at sites 713 and 357 of TaGS3-4A gene were amplified by PCR. The amplification primers were:

Upstream primer CAPS-4A-357/713-F: 5'-CTGCTGTTACTCTGTTATTATTAC-3' (SEQ ID NO: 2);

Downstream primer CAPS-4A-357/713-R 5’-CAAGAACTGCCTGTACAGTC-3’ (SEQ ID NO: 3).

The PCR amplification system was 20ul, including 2×Taq PCR MasterMix 10ul, 1μL of upstream and downstream primers, 1μL of DNA template, and the rest was supplemented with ddH ₂ O; the PCR amplification program was: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 1min/kb, 33 cycles; 72℃ final extension for 10 minutes, and storage at 4℃. The above amplification products were digested with Hpy188I and BsrI respectively. The digestion products were all detected by agarose gel electrophoresis with a mass percentage concentration of 1%, and the conditions of the wheat to be tested at the 713 and 357 sites were judged and recorded according to the digestion results, as shown in Figures 5 and 6.

① If the PCR product is electrophoresed after Hpy188I digestion to obtain two target bands of 537 bp and 265 bp or two target bands of 540 bp and 265 bp, the allelic variation type of the 713 site of the TaGS3-4A gene is 713-T; if the PCR product is electrophoresed after Hpy188I digestion to obtain any one target band of 802 bp, 804 bp or 808 bp, the allelic variation type of the 713 site of the TaGS3-4A gene is 713-A, as shown in Figure 6. The 16 wheat material samples in Figure 6 are: 1. Jinchun No. 3 (Xichun Dwarf No. 2); 2. Liang Laiyou white-skinned wheat; 3. Shanxi white wheat; 4. Niu Zhijia; 5. Mahuaban; 6. Fake red wheat; 7. Hongjin wheat; 8. Fengkang No. 2 (5248); 9. Changzhi 6406; 10. Beijing No. 8; 11. Mingxian 169; 12. Dongfanghong No. 3; 13. Jinghong No. 5; 14. Neimai 11; 15. Dingxingzhai; 16. Dahongmai.

② If the PCR product is electrophoresed after BsrI digestion to obtain two target bands of 181bp and 621bp, or two target bands of 184bp and 621bp, or two target bands of 187bp and 621bp, the allelic variation type of the 357 site of the TaGS3-4A gene is 357-G; if the PCR product is electrophoresed after BsrI digestion to obtain a single target band of 804bp, the allelic variation type of the 357 site of the TaGS3-4A gene is 357-0, as shown in Figure 5. The 22 wheat material samples in Figure 5 are: 1. Shanxi white wheat; 2. Fengkang 2 (5248); 3. Changzhi 6406; 4. Beijing 8; 5. Mingxian 169; 6. Dongfanghong 3; 7. Xianmai; 8. Jiangdongmen; 9. Jinghong 5; 10. Neimai 11; 11. Jinchun 3 (Xichunai 2); 12. Lianglaiyou white wheat; 13. Dingxingzhai; 14. Dahongmai; 15. Niuzhijia; 16. Mahuaban; 17. Jiahongmai; 18. Hongjinmai; 19. Yuandong 822; 20. Lyuhan 328; 21. Jiangxizao; 22. Honghuazao.

(2) Development of molecular marker InDel-4A-351: The genomic DNA of wheat materials was extracted using the conventional CTAB method, and the nucleotide region where the 351 site of the TaGS3-4A gene was located was amplified by PCR. The amplification primers were:

Upstream primer InDel-4A-351-F: 5'-TGCTGTTACTCTGTTATTATTA-3' (SEQ ID NO: 4);

Downstream primer InDel-4A-351-R: 5’-TAACACTTAGAATCTACTAC-3’ (SEQ ID NO: 5).

The PCR amplification system is 20 μL, including 10 μL of 2×Taq PCR MasterMix, 1 μL of upstream and downstream primers, 1 μL of DNA template, and the rest is supplemented with ddH ₂ O; the PCR amplification program is: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, 33 cycles; 72°C final extension for 10 min, and storage at 4°C. The above amplified products were subjected to polyacrylamide gel electrophoresis with a mass percentage concentration of 12%, a voltage of 140 volts, and 3.5 hours. The size of the PCR product fragment was identified by silver staining, and the situation of the wheat to be tested at the 351 site was judged and recorded according to the following method, as shown in Figure 4. The 12 wheat material samples in Figure 4 are: 1. Jinchun No. 3 (Xichun Ai No. 2), 2. Liang Laiyou white-skinned wheat; 3. Dingxingzhai; 4. Dahongmai; 5. Jinghong No. 5; 6. Neimai 11; 7. Shanxi white wheat; 8. Mingxian 169; 9. Niuzhijia; 10. Mahuaban; 11. Jiahongmai; 12. Hongjinmai.

① If the size of the PCR product of the wheat to be tested is a fragment of 204 bp (maximum), the allelic variation type of the TaGS3-4A gene in the genome of the plant to be tested is 351-2ACT;

② If the size of the PCR amplification product of the wheat to be tested is a fragment of 200 bp or 201 bp (centered), the allelic variation type of the TaGS3-4A gene in the genome of the plant to be tested is 351-1ACT;

③ If the size of the PCR amplification product of the wheat to be tested is a fragment of 198 bp (minimum), the allelic variation type of the TaGS3-4A gene in the genome of the plant to be tested is 351-0.

Example 5 Functional verification application of TaGS3-4A gene polymorphism site

Using the gene haplotype loci and molecular marker primers obtained in Examples 3 and 4, and using the amplification system and amplification conditions described above, the function of the TaGS3-4A gene was verified in the Chinese wheat micro-core germplasm, and the phenotypic data of 1,000-grain weight, grain length, and grain width for many years and multiple points were analyzed for marker/trait association. The haplotype identification data of the TaGS3-4A gene of all wheat micro-core germplasm materials in this example are shown in Table 2.

Table 2 Genotype identification data of TaGS3-4A in wheat mini-core germplasm materials

The specific results of the association analysis are shown in Table 3. Table 3 shows that the differences in thousand-grain weight between HAP-4A-1 and HAP-4A-2 and HAP-4A-3 in 2002, 2005, 2006, 2010 and 2016 were 5.33-5.47 g, 3.79-3.98 g, 3.23-3.57 g, 6.17-3.26 g and 5.12-4.82 g, respectively, and the differences reached significant and extremely significant levels (P < 0.05 or P < 0.01); compared with HAP-4A-3, the HAP-4A-1 haploid significantly increased the grain length by 0.30-0.43 mm in four of the five years (P < 0.05 or P < 0.01). It can be seen that the allelic variations HAP-4A-1 and HAP-4A-2 are excellent allelic variations, and it is concluded that the molecular markers of wheat thousand-grain weight and grain length gene TaGS3-4A are composed of a pair of molecular markers, namely molecular marker CAPS-4A-357/713 and molecular marker InDel-4A-351.

Table 3 Association analysis between TaGS3-4A gene and grain-related traits in wheat mini-core germplasm materials

Note: 02LY means Luoyang (2002); 05LY means Luoyang (2005); 06LY means Luoyang (2006); 10SY means Beijing Shunyi (2010); 16LC means Luancheng (2016). Capital letters indicate P < 0.01, and lowercase letters indicate P < 0.05. Therefore, the combined use of molecular markers CAPS-4A-357/713 and InDel-4A-351 can effectively screen out the excellent haplotypes HAP-4A-1 and HAP-4A-2 of the wheat TaGS3-4A gene that can significantly improve the thousand-grain weight and grain length of wheat, and can be used as functional markers to improve the thousand-grain weight and grain length of wheat and conduct molecular marker-assisted breeding for high-yield wheat.

The above embodiments are optimized implementation schemes of the present invention, which are only used to illustrate the present invention but not to limit the present invention. Any modification or equivalent substitution made by those skilled in the art without departing from the purpose and principle of the implementation scheme of the present invention shall fall within the scope of protection claimed by the present invention.

SEQUENCE LISTING

<110> Agricultural Resources Research Center, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

Hebei University of Economics and Business

<120> A molecular marker for wheat thousand-grain weight and grain length gene TaGS3-4A and its application

<130>

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 1936

<212> DNA

<213> TaGS3-4A

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gtagattcta agtgttagaa tatcccacac agcagcctga aaagttggca catgccctct 420

cctccttgta ctctagcttg tagctactgc ttttttttca gtaaaaaata atttctctgg 480

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ggaagtttag caaatccggt aatgattgaa aatagtatac acgtacagtt gaacattatg 840

caaatctggt aacgatagtt taggaggttt ggttatacttga aatttgatta ggatgattga 900

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actgtacagg cagttcttgt atttctgtct ccagtcttag tgccactgtt tactttatattc 1020

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ctctctttac aagcttgcag ttcatctgag aaggggaacg ccgatcaatc tcatcgctcc 1380

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gtagtctcgt tgtaatgaac tgactgaggg gaatgttaat gtttgtgcct gcagaacccg 1680

gtgggcgtgt ttgagctgct tcccgtggat ctgcggcggc gggtgctctg ccgtccagct 1740

caaggggccg agctgctgcc gcggatgccc ccgctgctgc gcggggagcg ggggctgcgg 1800

cggcggcggg ccctcgtgtg gctgctcgtg ctcctgcgcc ggctgctcct cctcttgcgc 1860

gtgccctgcc tgtgccggct gcggccccgt gtgctgcggc ggtgtccctc gccctcgctg 1920

ctgcctgtgt tcctga 1936

<210> 2

<211> 24

<212> DNA

<213> Upstream primer CAPS-4A-357/713-F

<400> 2

ctgctgttac tctgttatta ttac 24

<210> 3

<211> 20

<212> DNA

<213> Downstream primer CAPS-4A-357/713-R

<400> 3

caagaactgc ctgtacagtc 20

<210> 4

<211> 22

<212> DNA

<213> Upstream primer InDel-4A-351-F

<400> 4

tgctgttact ctgttattat ta 22

<210> 5

<211> 20

<212> DNA

<213> Downstream primer InDel-4A-351-R

<400> 5

taacacttag aatctactac 20

Claims (3)

1. A method for identifying whether wheat is a haplotype wheat variety or strain with excellent gene TaGS3-4A for increasing thousand-grain weight and grain length, which is characterized by including the following steps: (1) Extract the genomic DNA of the wheat sample to be identified; (2) Use the upstream and downstream primers of molecular marker CAPS-4A-357/713 to perform PCR amplification of wheat genomic DNA. The amplified product was digested with Hpy188I and detected by electrophoresis to obtain two entries with molecular weights of 537bp and 264bp respectively. or two target bands of 540bp and 264bp, or obtain one target band with a molecular weight of 801bp, 803bp or 807bp; digest the amplification product with BsrI and detect it by electrophoresis to obtain a target band with a molecular weight of 181bp and 620bp. Two target bands or two target bands of 184bp and 620bp or two target bands of 187bp and 620bp, or obtain one target band with a molecular weight of 803bp; the molecular marker CAPS-4A-357/713 The upstream primer CAPS-4A-357/713-F is shown in SEQ ID NO:2, and the downstream primer CAPS-4A-357/713-R is shown in SEQ ID NO:3; (3) Use the upstream and downstream primers of the molecular marker InDel-4A-351 to PCR amplify wheat genomic DNA, separate the amplified products by electrophoresis, and obtain any one of the amplified products with a molecular weight of 198bp, 200bp, 201bp or 204bp. The target band; the upstream primer InDel-4A-351-F of the molecular marker InDel-4A-351 is as shown in SEQ ID NO: 4, and the downstream primer InDel-4A-351-R is as shown in SEQ ID NO: 5; (4) In the amplification results, if the sample to be tested is detected using the molecular marker CAPS-4A-357/713, two target bands with molecular weights of 537bp and 264bp or two target bands of 540bp and 264bp will be obtained after digestion with Hpy188I. The bands of the entry were detected by BsrI digestion and two target bands with molecular weights of 181bp and 620bp or two target bands of 184bp and 620bp were obtained, and amplification was obtained after detection by the molecular marker InDel-4A-351. If the molecular weight of the product is a single band of 198 bp or 201 bp, then the sample is a wheat variety or strain with excellent haplotypes for increased 1000-grain weight and grain length due to the gene TaGS3-4A. 2. The method of identifying whether wheat is a haplotype wheat variety or strain with genes TaGS3-4A that increases thousand-grain weight and grain length according to claim 1, characterized in that the molecular markers described in steps (2) and (3) The PCR amplification systems in CAPS-4A-357/713 and the molecular marker InDel-4A-351 are both 20 µL, including 2×Taq PCR MasterMix 10 µL, 1 µL each of upstream and downstream primers, 1 µL DNA template, and ddH ₂ for the rest. O complement. 3. The method of identifying whether wheat is a wheat variety or strain with excellent haplotypes whose gene TaGS3-4A increases thousand-grain weight and grain length according to claim 2, characterized in that the molecular marker CAPS-4A-357/ in step (2) The PCR amplification program of 713 is: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, 33 cycles; final extension at 72°C for 10 minutes, and storage at 4°C; molecules in step (3) The procedure for PCR amplification of labeled InDel-4A-351 is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 33 cycles; final extension at 72°C for 10 min, and storage at 4°C.