Molecular marker for wheat yellow mosaic disease resistance identification and application
By developing molecular markers based on KASP technology, and utilizing the specificity of primer terminal bases to detect key variant sites in the wheat yellow mosaic virus resistance gene TaRD21A, the problems of complex operation and low detection efficiency in existing technologies have been solved, enabling efficient and accurate screening and breeding of disease-resistant varieties.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-25
- Publication Date
- 2026-03-27
AI Technical Summary
Existing molecular marker technologies such as SSR, STS, and CAPS are cumbersome and complex to operate in wheat yellow mosaic virus resistance identification, have low detection efficiency, and are difficult to meet production needs.
Molecular markers based on competitive allele-specific PCR (KASP) technology were developed to detect key variant sites in the wheat yellow mosaic virus resistance gene TaRD21A by using specific matching of primer terminal bases. Combined with FAM and HEX fluorescent markers, high-throughput, low-cost, and low-error-rate genotyping was achieved.
This technology enables efficient and accurate identification of wheat yellow mosaic disease resistance, improves breeding efficiency, expands varietal genetic diversity, and has broad application prospects.
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Figure CN121737334A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular genetic breeding technology, and in particular to a molecular marker for identifying wheat yellow mosaic disease resistance and its application, especially a molecular marker based on competitive allele-specific PCR (KASP) technology and its application in wheat disease resistance breeding. Background Technology
[0002] Wheat is an important food crop, with 35%-40% of the world's population relying on it as a staple food. Wheat yellow mosaic virus (WYMV) is a global viral disease affecting wheat. Infected wheat plants typically exhibit yellowing leaves, stunted growth, and abnormal tillering, generally leading to yield losses of 10-30%, and in severe cases, losses exceeding 70%, posing a serious threat to wheat production in my country. Breeding and planting highly resistant varieties of wheat YMV is the most economical, safe, and effective approach. Currently, nine WYMV resistance loci have been identified in wheat, located on chromosomes 2A, 2DL, 3BS, 4B, 4D, 5AL, 6DS, and 7BS of common wheat, and chromosome 4V of the wild closely related species *Triticum aestivum*. Among them, the resistance gene TaRD21A located at the 2DL resistance locus has been successfully cloned, and this gene exhibits the highest resistance effect. Therefore, developing molecular markers closely linked to wheat yellow mosaic virus resistance genes based on key variant sites of these resistance genes is an important foundation for breeding disease-resistant materials.
[0003] Traditionally used molecular markers, such as SSR, STS, and CAPS, are cumbersome and complex to operate, have low detection efficiency, and are difficult to meet production needs. KASP (Kompetitive Allele-Specific PCR) markers are based on specific matching of primer terminal bases to genotype SNPs (single nucleotide polymorphisms) and detect InDels (insertions and deletions). This technology can accurately identify SNPs or InDels widely present in genomic DNA, and is a high-throughput, low-cost, and low-error-rate SNP genotyping technique. Furthermore, KASP markers do not require laboratory gel electrophoresis, enabling automated and platform-based operations, and are characterized by high throughput, low cost, and good stability. Given these characteristics, KASP markers play an important role in wheat gene mapping and marker-assisted selection breeding. Summary of the Invention
[0004] The purpose of this invention is to provide a molecular marker for identifying wheat yellow mosaic disease resistance and its application. This molecular marker and the primers for amplifying it can be used to assist in screening wheat varieties resistant to wheat yellow mosaic disease, providing a scientific basis for wheat improvement and breeding of new varieties.
[0005] To achieve the above objectives, this invention provides a primer set for amplifying molecular markers for identifying wheat yellow mosaic disease resistance. The primer set includes a first forward primer, a second forward primer, and a universal reverse primer, with the following sequences: The first forward primer is shown in SEQ ID NO.1: 5'-ctcgttggtgaggtcggC-3'; The second forward primer is shown in SEQ ID NO.2: 5'-ctcgttggtgaggtcggA-3'; The universal reverse primer is shown in SEQ ID NO.3: 5'- gccgcttcgaggtgttcc -3'; The molecular marker is located on the wheat 2DL chromosome. TaRD21A The 286th position in the gene coding region has a nucleic acid polymorphism of G / T.
[0006] Furthermore, the first forward primer, when combined with the FAM fluorescent binding characteristic sequence, appears blue in the KASP detection results, corresponding to the susceptible genotype; the FAM fluorescent binding characteristic sequence is: 5'-GAAGGTGACCAAGTTCATGCT-3', as shown in SEQ ID NO.4; The second forward primer, combined with the HEX fluorescent binding characteristic sequence, appears red in the KASP detection results, corresponding to the disease-resistant genotype; the HEX fluorescent binding characteristic sequence is 5'-GAAGGTCGGAGTCAACGGATT-3', as shown in SEQ ID NO.5.
[0007] Furthermore, the present invention also provides the application of the above primer set in the preparation of a kit for detecting wheat yellow mosaic virus resistance.
[0008] Furthermore, the present invention also provides the application of the above-mentioned primer set in the breeding of wheat varieties resistant to wheat yellow mosaic virus.
[0009] Furthermore, the present invention also provides a kit for identifying wheat yellow mosaic disease resistance, comprising the above-mentioned primer set.
[0010] Furthermore, the present invention also provides a method for identifying resistance to wheat yellow mosaic virus, comprising the following steps: Step 1: Extract genomic DNA from wheat leaves; Step 2: Perform PCR amplification of genomic DNA using the primer set described above; Step 3: Genotyping based on PCR amplification results.
[0011] Furthermore, in step 2, the total reaction volume for PCR amplification is 5 μl, including 2.43 μl of genomic DNA, 2.5 μl of 2×KASP Master Mix, and 0.07 μl of primer mixing working solution.
[0012] Furthermore, the PCR amplification reaction program was as follows: 94℃, 15 min; 94℃, 20 s, 61~55℃ gradient PCR, 1 min, decreasing by 0.6℃ for each cycle, for 10 cycles; 94℃, 20 s, 55℃, 1 min, for 26 cycles; stored at 10℃.
[0013] Furthermore, the PCR results were run and analyzed using a Thermo QuantStudio 5 real-time PCR instrument.
[0014] Furthermore, in step 3, when the genotype is TT, it is a disease-resistant variety; when the genotype is GG, it is a disease-susceptible variety.
[0015] Furthermore, the aforementioned primer set can be used for haplotype analysis of individual plants in natural wheat populations.
[0016] The advantages and positive effects of the molecular marker for identifying wheat yellow mosaic disease resistance described in this invention are as follows: This invention targets the major gene for resistance to wheat yellow mosaic virus. TaRD21A A KASP molecular marker closely linked to wheat yellow mosaic virus resistance was developed based on key variant SNP sites. The T / G variation in the KASP molecular marker of this invention leads to a susceptibility-resistance phenotype for wheat yellow mosaic virus. KASP molecular markers designed based on these two variant sites can be used for marker-assisted selection of wheat yellow mosaic virus resistance in wheat germplasm resources, improving breeding efficiency, expanding varietal genetic diversity, and have broad application prospects.
[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description
[0018] Figure 1 This is the result of the nucleotide sequence comparison of the TaRD21A homologous gene between the wheat yellow mosaic disease resistant variety Fielder and the susceptible variety YM158 in this embodiment of the invention.
[0019] Figure 2 This is a genotyping diagram of the KASP marker in 162 wheat materials in an embodiment of the present invention, where blue represents the G / G genotype, red represents the T / T genotype, and black boxes represent negative controls using H2O as a template. Detailed Implementation
[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.
[0021] Unless otherwise defined, the technical or scientific terms used in this invention should be understood in their ordinary sense by those skilled in the art. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.
[0022] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.
[0023] Example 1: Major gene for resistance to wheat yellow mosaic virus TaRD21A Genotyping between disease-resistant and disease-susceptible wheat varieties Major gene for resistance to wheat yellow mosaic virus TaRD21A Located on chromosome 2DL, for determination TaRD21A The key variant sites related to resistance in the gene were identified, and the coding sequence (CDS) of this gene was compared between the resistant variety Fielder and the susceptible variety Yangmai 158 (YM158).
[0024] TaRD21A CDS sequence SEQ ID No. 8 in Fielder, a wheat variety resistant to yellow mosaic virus:
[0025] Using Fielder TaRD21A The sequence was found in the wheat yellow mosaic virus susceptible variety Yangmai 158 (YM158) through online BLAST alignment using WheatOmics 1.0. TaRD21A The CDS sequence of the homologous gene, SEQ ID No. 9:
[0026] The sequences of SEQ ID No. 8 and SEQ ID No. 9 were compared using DNAMAN software. The sequence differences are as follows: Figure 1 As shown. This nonsynonymous mutation causes the 96th amino acid in the TaRD21A-encoded protein to change from glycine to threonine.
[0027] Example 2 is based on TaRD21A Development and application of KASP molecular markers for key variant SNP sites TaRD21A A G→T mutation exists at position 286 of the nucleotide sequence encoding the protein. When the genotype at this site is G, the wheat plant exhibits susceptibility to the disease; when the genotype at this site is T, the wheat plant exhibits resistance. Primers were designed using the 100bp flanking sequences at both ends of the SNP marker site to develop a KASP marker. KASP marker primers for the SNP were developed by searching the Fielder wheat reference genome sequence (WheatOmics 1.0 accession number: TraesFLD2D01G567800.1, http: / / wheatomics.sdau.edu.cn / cgi-bin / get_fasta_bedtools.py?database=all_gene&ID=TraesFLD2D01G567800.1). The forward primer sequence SEQ ID NO.1 is 5'-ctcgttggtgaggtcggC-3', SEQ ID NO.2 is 5'-ctcgttggtgaggtcggA-3', and the universal reverse primer SEQ ID NO.3 is 5'-gccgcttcgaggtgttcc-3'.
[0028] Adding the FAM fluorescent binding characteristic sequence SEQ ID NO.4: 5'-GAAGGTGACCAAGTTCATGCT-3' to the 5' end of SEQ ID NO.1 resulted in a blue color in the KASP results; adding the HEX fluorescent binding characteristic sequence SEQ ID NO.5: 5'-GAAGGTCGGAGTCAACGGATT-3' to the 5' end of SEQ ID NO.2 resulted in a red color in the KASP results. Complete primers were synthesized by Youkang Biotechnology Co., Ltd. (Table 1).
[0029] Table 1 Complete Primers
[0030] To verify the accuracy of the KASP markers developed in this invention and demonstrate their application potential, markers that have been clearly identified through resequencing were selected. TaRD21AWheat varieties with different genotypes were used as controls, including the disease-resistant variety Jimai 44 (genotype T / T) and the disease-susceptible variety Zhengmai 379 (genotype G / G). At the same time, 160 wheat cultivars with diverse genetic backgrounds were selected as natural populations.
[0031] Genomic DNA was extracted from all materials (162 samples in total), and KASP reactions were performed on a Thermo QuantStudio 5 real-time PCR instrument using the primers described above. The reaction volume (5 μL) consisted of: 2.43 μL genomic DNA, 2.5 μL 2× KASP MasterMix, and 0.07 μL primer mixing working solution. The reaction program was as follows: 94℃ for 15 min; 10 cycles of gradient amplification (94℃, 20 s; 61-55℃, 1 min; decreasing by 0.6℃ per cycle); and 26 cycles of amplification (94℃, 20 s; 55℃, 1 min).
[0032] The typing results show that ( Figure 2 As shown in the figure, the fluorescence signal of Jimai 44 clearly clustered in the red cluster (HEX signal, corresponding to the T / T resistant genotype), while the fluorescence signal of Zhengmai 379 clearly clustered in the blue cluster (FAM signal, corresponding to the G / G susceptible genotype). The KASP genotyping results of the control varieties were completely consistent with the known resequencing genotype results, which fully demonstrates that the KASP marker provided by this invention has high accuracy and specificity and can be used for subsequent genotyping work.
[0033] The remaining 160 wheat accessions were also clearly divided into two clusters based on their genotypes, perfectly matching the clustering regions of the two control varieties. This indicates that this marker can reliably and accurately genotype wheat materials from different genetic backgrounds. The genotyping results of the natural population are shown in Table 2.
[0034] Table 2 Genotypes corresponding to the 286th SNP of TaRD21A in wheat cultivars.
[0035] Therefore, this invention targets the major gene for resistance to wheat yellow mosaic virus. TaRD21A A KASP molecular marker tightly linked to wheat yellow mosaic virus resistance was developed at a key variant SNP site. Specific KASP primer sequences and genotyping methods for detecting this site are also provided. The T / G variation in the KASP molecular marker of this invention leads to a susceptibility-resistance phenotype for wheat yellow mosaic virus. KASP molecular markers designed based on these two variant sites can be used for marker-assisted selection of wheat yellow mosaic virus resistance in wheat germplasm resources, improving breeding efficiency, expanding varietal genetic diversity, and have broad application prospects.
[0036] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.
Claims
1. A primer set for amplifying molecular markers for identifying resistance to wheat yellow mosaic virus, characterized in that: The primer set includes a first forward primer, a second forward primer, and a universal reverse primer, with the following sequences: The first forward primer is shown in SEQ ID NO.1: 5'-ctcgttggtgaggtcggC-3'; The second forward primer is shown in SEQ ID NO.2: 5'-ctcgttggtgaggtcggA-3'; The universal reverse primer is shown in SEQ ID NO.3: 5'-gccgcttcgaggtgttcc -3'; The molecular marker is located on the wheat 2DL chromosome. TaRD21A The 286th position in the gene coding region has a nucleic acid polymorphism of G / T.
2. The primer set according to claim 1, characterized in that: The first forward primer, combined with the FAM fluorescent binding characteristic sequence, appears blue in the KASP detection results, corresponding to the susceptible genotype; the FAM fluorescent binding characteristic sequence is 5'-GAAGGTGACCAAGTTCATGCT-3', as shown in SEQ ID NO.4; The second forward primer, combined with the HEX fluorescent binding characteristic sequence, appears red in the KASP detection results, corresponding to the disease-resistant genotype; the HEX fluorescent binding characteristic sequence is 5'-GAAGGTCGGAGTCAACGGATT-3', as shown in SEQ ID NO.
5.
3. The use of the primer set according to claim 1 or 2 in the preparation of a kit for detecting wheat yellow mosaic virus resistance.
4. The application of the primer set described in claim 1 or 2 in the breeding of wheat varieties resistant to wheat yellow mosaic virus.
5. A kit for identifying resistance to wheat yellow mosaic virus, characterized in that: It includes the primer set as described in claim 1 or 2.
6. A method for identifying resistance to wheat yellow mosaic virus, characterized in that, Includes the following steps: Step 1: Extract genomic DNA from wheat leaves; Step 2: Perform PCR amplification of genomic DNA using the primer set described in claim 1 or 2; Step 3: Genotyping based on PCR amplification results.
7. The method according to claim 6, characterized in that: In step 3, if the genotype is TT, it is a disease-resistant variety; if the genotype is GG, it is a disease-susceptible variety.