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CN-121975967-A - Wheat thousand seed weight related gene TaMADS-A haplotype molecular marker and application thereof

CN121975967ACN 121975967 ACN121975967 ACN 121975967ACN-121975967-A

Abstract

The invention relates to the technical field of genetic engineering, in particular to a wheat thousand-grain weight related gene TaMADS-A haplotype molecular marker and application thereof. The SNP locus is positioned at 553039901bp of a 6A chromosome, and the polymorphism is C/T. The phenotype difference analysis of the analyzed grain type of TaMADS-A haplotype is carried out to obtain a haplotype related to thousand grain weight of wheat grains, and a corresponding KASP primer combination is designed aiming at SNP loci in the haplotype. The KASP primer combination provided by the invention can accurately and rapidly identify the thousand grain weight of wheat grains, and has important significance in breeding of high-yield wheat.

Inventors

  • MAO LONG
  • LIU SHAOSHUAI
  • LI AILI
  • GENG SHUAIFENG
  • Deng Zhongyin
  • CHE YUQING
  • CHEN QIHANG

Assignees

  • 中国农业科学院作物科学研究所

Dates

Publication Date
20260505
Application Date
20250909

Claims (10)

  1. 1. The wheat thousand-grain-weight related gene TaMADS-A haplotype SNP locus is characterized in that the SNP locus is positioned at 553039901bp of a 6A chromosome, and the polymorphism is C or T.
  2. 2.A KASP primer combination for amplifying SNP sites, comprising 553039901-F1,553039901-F2 and 553039901-R; the nucleotide sequence of 553039901-F1 is shown as SEQ ID NO. 2; the nucleotide sequence of 553039901-F2 is shown as SEQ ID NO. 3; the nucleotide sequence of 553039901-R is shown as SEQ ID NO. 4.
  3. 3. The KASP primer combination according to claim 2, wherein 553039901-F1 and 553039901-F2 are linked to fluorescent tag sequences FAM and HEX, respectively, the nucleotide sequence of FAM is shown in SEQ ID No. 5 and the nucleotide sequence of HEX is shown in SEQ ID No. 6.
  4. 4. A kit comprising the SNP locus of claim 1 or the KASP primer set of claim 2.
  5. 5. Use of the SNP molecular marker of claim 1 or the KASP primer combination of claim 2 or the kit of claim 3 for identifying, screening or breeding thousand kernel weight of wheat kernels.
  6. 6. A method for identifying thousand kernel weight of a plant, comprising: PCR amplification is carried out by taking DNA of a plant sample to be detected as a template and adopting the KASP primer combination of claim 2 or the kit of claim 4, and the thousand seed weight of the plant to be detected is judged according to the amplification result.
  7. 7. The method of claim 6, wherein the plant to be tested is one or more of wheat, corn or rice.
  8. 8. The method of claim 6, wherein the step of providing the first layer comprises, The PCR amplification system comprises, based on 10. Mu.L of the total system: KASP Mix (2X), 4 to 6. Mu.L, primer Mix,0.12 to 0.16. Mu. L, DNA 25 to 35ng, the balance being water.
  9. 9. The method of claim 6, wherein the reaction procedure for PCR amplification comprises: 95 ℃ 8-12 min, 95 ℃ 15-25 s,61 ℃ 60s, 8-12 cycles, annealing temperature reduction of 0.5-0.7 ℃ in each cycle, 95 ℃ 15-25 s,55 ℃ 35-45 s, 32-36 cycles, 25 ℃ 10-20 min.
  10. 10. The method according to any one of claims 6 to 9, wherein the determining thousand kernel weight, kernel length, kernel width, kernel thickness of the wheat kernel to be tested according to the amplification result comprises: In the amplification result, the SNP locus identifies plants with genotype TT, and has higher thousand grain weight, grain length, grain width and grain thickness than plants with genotype CC.

Description

Wheat thousand seed weight related gene TaMADS-A haplotype molecular marker and application thereof Technical Field The invention relates to the technical field of genetic engineering, in particular to a wheat thousand-grain weight related gene TaMADS-A haplotype molecular marker and application thereof. Background About 20% of the world population is based on wheat, whose yield directly affects the dietary safety of 35 million people. Three elements of wheat (Triticum aestivum l.) yield are spike number per unit area, spike number and thousand kernel weight, which have relatively stable genetic effects. On the premise of keeping the effective spike number and spike number in unit area stable, the improvement of thousand grain weight of wheat has remarkable effect on increasing yield. MADS-box is a family of proteins encoding transcription factors, and the earliest studies on MADS-box genes were started from flower morphology mutants of Goldfish and Arabidopsis thaliana, the names of which are derived from Saccharomyces cerevisiae transcription factor MINICH ROMOSOME MAINTENANCE (MCM 1), arabidopsis thaliana flower homeogene AGAMOUS (AG), goldfish flower homeogene DEFICIENS (DEF) and human serum response factor SERUM RESPONSE FACTOR (SRF). MADS-box gene families can be classified into type I and type II, plant-specific MIKC-type genes and animal-and fungal-type MEF 2-type genes all belong to type II genes. The MIKC genes of plants are named as conserved MIKC structure of the coded proteins, namely MADS, INTERVENING, keratin-like and C-terminal 4 protein domains. MAD S box family proteins play an important role in various aspects of plant growth and development, including participation in plant organ development, flowering phase control, hormone signaling, and the like. SNP (Single Nucleotide Polymorphism ) refers to DNA sequence polymorphism caused by single nucleotide variation, and has the advantages of wide genome distribution, good genetic stability, suitability for high-throughput analysis and detection and the like. SNP chip types of 9K, 55K, 90K, 660K and the like have been developed on wheat and have been used for genetic research such as genetic diversity and high-density linkage map construction. However, chip-based genotyping platforms can only be used for sample whole genome scanning, which is costly when used for large sample volumes. KASP (Kompetitive ALLELE SPECIFIC PCR, competitive allele-specific PC R), which is a molecular marker developed based on SNP loci, has the characteristics of high stability, accuracy, low cost and the like, has been widely applied to high-throughput SNP typing, and is more remarkable in application of KAS P especially when the number of SNP loci is small in a large sample size. The KASP is used for molecular marking to screen varieties with high thousand grain weight, which provides scientific basis for researching high-yield breeding. In view of this, the present application has been proposed. Disclosure of Invention In order to solve the problems in the prior art, the invention provides a wheat thousand-grain weight related gene TaMADS-A haplotype molecular marker and application thereof. In a first aspect, the invention provides a SNP site located at the 553039901 base pair of the 6A chromosome, the polymorphism being C or T. In a second aspect, the invention provides a KASP primer combination for amplifying SNP sites, comprising 553039901-F1,553039901-F2 and 553039901-R; the nucleotide sequence of 553039901-F1 is shown as SEQ ID NO. 2; the nucleotide sequence of 553039901-F2 is shown as SEQ ID NO. 3; the nucleotide sequence of 553039901-R is shown as SEQ ID NO. 4. Furthermore, 553039901-F1 and 553039901-F2 in the KASP primer combination are respectively connected with fluorescent marker sequences FAM and HEX, the nucleotide sequence of FAM is shown as SEQ ID NO. 5, and the nucleotide sequence of HEX is shown as SEQ ID NO. 6. In a third aspect, the invention provides a kit comprising a SNP site as described above, or a KASP primer set as described above. The invention further provides applications of the SNP locus, or the KASP primer combination, or the kit in identifying thousand kernel weight of wheat kernels. The invention further provides application of the SNP locus, or the KASP primer combination, or the kit in screening or cultivating high thousand kernel weight wheat kernels. In a fourth aspect, the invention provides a method of identifying thousand kernel weight of a plant comprising: And (3) taking DNA of a plant sample to be detected as a template, adopting the KASP primer combination or the kit to carry out PCR amplification, and judging thousand seed weight of the wheat seeds to be detected according to an amplification result. Further, the PCR amplification system comprises, based on 10. Mu.L of the total system: KASP Mix (2X), 4 to 6. Mu.L, primer Mix,0.12 to 0.16. Mu. L, DNA 25 to 35ng, the balance being water. Further, the reaction procedure o