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CN-121592806-B - KASP molecular marker linked with wheat-wheatgrass distant hybridization offspring powdery mildew resistance locus PmYZW-6A-2 and application thereof

CN121592806BCN 121592806 BCN121592806 BCN 121592806BCN-121592806-B

Abstract

The invention relates to the technical field of biotechnology and molecular breeding, and provides a KASP molecular marker linked with a wheat-wheatgrass distant hybridization offspring powdery mildew resistance site PmYZW-6A-2 and application thereof, wherein the physical position of a SNP site of the KASP molecular marker linked with the wheat-wheatgrass distant hybridization offspring powdery mildew resistance site PmYZW-6A-2 corresponds to 95180338bp on a 6A chromosome of a common ice 3504 genome, the nucleotide sequence of the SNP site is shown as SEQ ID NO.4, and M in the nucleotide sequence is A or G. By the technical scheme, the problems that the screening period of the wheat-wheatgrass distant hybridization offspring powdery mildew resistance is long, the working amount is large, the accurate screening cannot be carried out in early generation, and the breeding difficulty is large in the related technology are solved.

Inventors

  • LI LIHUI
  • LING HONGQING
  • ZHENG LI
  • ZHENG LIYAN

Assignees

  • 崖州湾国家实验室
  • 海南大学三亚南繁研究院

Dates

Publication Date
20260508
Application Date
20260128

Claims (9)

  1. 1. The KASP molecular marker is linked with a wheat-wheatgrass distant hybridization offspring powdery mildew resistance site PmYZW-6A-2, and is characterized in that the nucleotide sequence of the KASP molecular marker is shown as SEQ ID NO.4, and M in the nucleotide sequence is A or G.
  2. 2. A primer set for amplifying the KASP molecular marker according to claim 1, wherein the primer set comprises an upstream primer F1 having a nucleotide sequence shown in SEQ ID No.1, an upstream primer F2 having a nucleotide sequence shown in SEQ ID No.2, and a downstream primer R having a nucleotide sequence shown in SEQ ID No. 3.
  3. 3. The primer set of claim 2, wherein the 5 'end of the upstream primer F1 and the 5' end of the upstream primer F2 each contain a different fluorescent detection sequence.
  4. 4. A kit for detecting powdery mildew resistance of wheat-wheatgrass distant hybridization offspring, characterized in that the kit comprises the primer set of claim 2 or 3.
  5. 5. Use of a primer set according to claim 2 or 3 or a kit according to claim 4, for any one of the following applications: a1, identifying or assisting in identifying powdery mildew resistance of wheat-wheatgrass distant hybridization offspring; a2, breeding powdery mildew resistant wheat-wheatgrass distant hybridization offspring single plants or strains or varieties; a3, preparing a product of a wheat-wheatgrass distant hybridization offspring single plant or strain or variety for breeding powdery mildew resistance; a4, screening wheat-wheatgrass distant hybridization offspring single plants from which powdery mildew infection is eliminated; In application, if the genotype is determined to be AA genotype, the wheat-wheatgrass distant hybridization offspring to be detected is judged to be powdery mildew resistance, if the genotype is determined to be GG genotype, the wheat-wheatgrass distant hybridization offspring to be detected is judged to be powdery mildew susceptibility, and the wheat-wheatgrass distant hybridization offspring are common ice 3504 and She Kaola hybridization derivative offspring.
  6. 6. A method for identifying powdery mildew resistance of wheat-wheatgrass distant hybridization offspring, comprising the steps of: Taking genome DNA of a wheat-agropyron aegerita distant hybridization offspring sample to be detected as a template, carrying out PCR amplification on the template by using the primer set according to claim 2 or 3 or the kit according to claim 4, determining genotyping after carrying out fluorescence detection by using an amplification result, and judging the resistance of the wheat-agropyron aegerita distant hybridization offspring to be detected to powdery mildew; If the genotype is determined to be AA genotype, judging that the wheat-wheatgrass distant hybridization to be detected represents powdery mildew resistance, and if the genotype is determined to be GG genotype, judging that the wheat-wheatgrass distant hybridization offspring to be detected represents powdery mildew infection; The wheat-wheatgrass distant hybridization offspring are common ice 3504 and She Kaola hybridization derivative offspring.
  7. 7. The method according to claim 6, wherein the PCR amplification reaction system comprises 2.5. Mu.L of 2X KASP MASTER Mix, 0.07. Mu.L of a primer mixture comprising the upstream primer F1, the upstream primer F2 and the downstream primer R in a volume ratio of 1:1:3, and 50ng of template DNA and ddH 2 O are complemented to 5. Mu.L.
  8. 8. The method of claim 7, wherein the concentration of the upstream primer F1, the upstream primer F2 and the downstream primer R is 100. Mu.M.
  9. 9. A method for breeding powdery mildew resistant wheat is characterized by comprising the steps of extracting genomic DNA of a wheat sample to be tested, carrying out PCR (polymerase chain reaction) amplification on the genomic DNA by using the primer set according to claim 2 or 3, determining genotyping after fluorescent detection by using an amplification result, and if the determined genotype is AA type, retaining the wheat to be tested for breeding, wherein the wheat is a progeny derived by hybridization of common ice 3504 and She Kaola.

Description

KASP molecular marker linked with wheat-wheatgrass distant hybridization offspring powdery mildew resistance locus PmYZW-6A-2 and application thereof Technical Field The invention relates to the field of biotechnology and molecular breeding, in particular to a KASP molecular marker linked with a wheat-wheatgrass distant hybridization offspring powdery mildew resistance site PmYZW-6A-2 and application thereof. Background Wheat powdery mildew is a worldwide serious leaf disease caused by obligate parasitic fungi (Blumeria graminis f. Sp.tritici), seriously threatens the safe production of wheat worldwide, often causes the yield loss to reach 10% -30%, and is even in serious cases of harvest. At present, the cultivation and planting of disease-resistant varieties is the most economical, effective and environment-friendly strategy for controlling the diseases. The wheatgrass is used as a kindred wild species of wheat, has rich genetic diversity, contains high resistance and even immunity genes against various diseases such as powdery mildew, rust disease and the like, and is an important gene library for widening the genetic basis of the wheat. The excellent disease resistance gene in the wheatgrass is introduced into the common wheat by the distant hybridization technology, so that the wheatgrass is an effective way for creating a new resistance source. However, in distant filial offspring, identification of the trait of interest typically relies on field artificial inoculation or natural onset phenotype identification. The method is greatly influenced by environmental conditions, has long period and large workload, and cannot be accurately screened in early generation. In addition, exogenous chromosomal fragments often accompany poor agronomic traits, which can present difficulties in breeding due to linkage drag. Molecular marker assisted selection techniques provide a solution to the above-mentioned problems. Compared with the traditional morphological markers and biochemical markers, SNP (single base nucleotide polymorphism) markers have become the most ideal marker type in modern molecular breeding due to the advantages of high distribution density in genome, good genetic stability, easy realization of high-throughput automatic detection and the like. KASP (Kompetitive Allele-SPECIFIC PCR) marker, namely competitive allele specific PCR, is characterized in that different fluorescent groups are added at the tail ends of the primers, a target sequence is typed based on reading judgment of fluorescent signals of a PCR terminal, specific SNP contained in the target allele can be identified, the identification process has the advantages of high efficiency, low cost, rapidness and convenience, high throughput analysis can be carried out, the molecular marker assisted selection process is greatly accelerated, and the method has wide application prospects in crop breeding. Therefore, developing KASP markers for identifying wheat powdery mildew resistance provides an effective detection means for breeding new varieties of powdery mildew resistant wheat, and has important significance for realizing rapid and accurate screening of antipathogenic materials and accelerating positioning, cloning and breeding processes of new varieties of antipathogenic genes. Disclosure of Invention The invention provides a KASP molecular marker linked with a wheat-wheatgrass distant hybridization offspring powdery mildew resistance site PmYZW-6A-2 and application thereof, and solves the problems that the wheat-wheatgrass distant hybridization offspring powdery mildew resistance screening period is long, the working amount is large, accurate screening cannot be carried out in early generation, and the breeding difficulty is large in the related technology. The technical scheme of the invention is as follows: The invention provides a KASP molecular marker linked with a wheat-wheatgrass distant hybridization offspring powdery mildew resistance locus PmYZW-6A-2, wherein the physical position of a SNP locus of the KASP molecular marker corresponds to 95180338bp on a 6A chromosome of a genome of common ice 3504, the nucleotide sequence of the SNP locus is shown as SEQ ID NO.4, and M in the nucleotide sequence is A or G. The invention also provides a primer group for amplifying the KASP molecular marker, which comprises an upstream primer F1 with a nucleotide sequence shown as SEQ ID NO.1, an upstream primer F2 with a nucleotide sequence shown as SEQ ID NO.2 and a downstream primer R with a nucleotide sequence shown as SEQ ID NO. 3. As a further technical scheme, the 5 'end of the upstream primer F1 and the 5' end of the upstream primer F2 respectively contain different fluorescence detection sequences. The 5 'end of the upstream primer F1 contains a fluorescence detection sequence FAM, and the 5' end of the upstream primer F2 contains a fluorescence detection sequence VIC. The invention also provides a kit for detecting the powdery mildew r