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CN-122012769-A - Wheat powdery mildew resistance major QTL and closely linked KASP molecular marker and application thereof

CN122012769ACN 122012769 ACN122012769 ACN 122012769ACN-122012769-A

Abstract

The invention belongs to the technical field of molecular genetics, and particularly relates to a wheat powdery mildew resistance major QTL and a KASP molecular marker closely linked with the wheat powdery mildew resistance major QTL and application of the wheat powdery mildew resistance major QTL. The invention provides a wheat powdery mildew resistance main effect QTL, wherein the main effect QTL site is positioned at the short arm end part of a wheat 1A chromosome, is positioned in a 4.4 cM genetic interval between flanking KASP markers Chr1A_6275522_K2 and Chr1A_11524652_K9, develops 9 KASP molecules, can perform auxiliary selective breeding, screens powdery mildew resistance wheat strains, and provides a new available molecular marker for screening powdery mildew resistance materials of wheat.

Inventors

  • WANG XIAOLU
  • SUI XINXIA
  • ZHUANG YAMEI
  • LI YONGBO
  • CUI DEZHOU
  • LIU CHENG
  • HAN RAN
  • CHEN GANG
  • Xue Chenjing
  • XU WENJING
  • QI GUANG
  • WANG KAI
  • FAN QINGQI

Assignees

  • 山东省农业科学院作物研究所

Dates

Publication Date
20260512
Application Date
20260120

Claims (7)

  1. 1. A wheat powdery mildew resistance main effect QTL, characterized in that the main effect QTL is located at the short arm end of the wheat 1A chromosome and is located within the 4.4 cM genetic interval between the flanking KASP markers chr1a_6275522_k2 and chr1a_11524652_k9.
  2. 2. A KASP molecular marker closely linked to the wheat powdery mildew resistance main QTL of claim 1, characterized in that the KASP molecular marker is KASP-2, KASP-4, KASP-5, KASP-9, KASP-12, KASP-17, KASP-22, KASP-24, KASP-25; the 5 'ends of the two KASP forward primers are respectively added with fluorescent tag sequences to modify FAM 5' -GAAGGTGACCAAGTTCATGCT-3; HEX 5’-GAAGGTCGGAGTCAACGGATT-3’。
  3. 3. The molecular marker of KASP according to claim 1 or 2, wherein the specific sequences of KASP-2, KASP-4, KASP-5, KASP-9, KASP-12, KASP-17, KASP-22, KASP-24, KASP-25 are as follows: ①KASP-2 FAM primer: GAAGGTGACCAAGTTCATGCTCCTCAACATGTGTCACCCG as shown in SEQ ID NO. 1; HEX primer: GAAGGTCGGAGTCAACGGATTCCTCAACATGTGTCACCCA as shown in SEQ ID NO. 2; reverse primer: CCTTACATTCCTGGCATGCA as shown in SEQ ID NO. 3; ②KASP-4 the FAM primer GAAGGTGACCAAGTTCATGCTACAACAATTTTTTTCATAGTGCC is shown as SEQ ID NO. 4; GAAGGTCGGAGTCAACGGATTACAACAATTTTTTTCATAGTGCT of HEX primer, shown as SEQ ID NO. 5; reverse primer: TCCGTCAACAAATCTCAAGC as shown in SEQ ID NO. 6; ③KASP-5 the FAM primer GAAGGTGACCAAGTTCATGCTCTACACCCGGTTTTTTCTCAA is shown as SEQ ID NO. 7; GAAGGTCGGAGTCAACGGATTCTACACCCGGTTTTTTCTCAG of HEX primer, shown as SEQ ID NO. 8; GCTTACACTCCTCGTTGATGTC as shown in SEQ ID NO. 9; ④KASP-9 The FAM primer GAAGGTGACCAAGTTCATGCTTAGGAAATGCCCCGTGAACA is shown as SEQ ID NO. 10; GAAGGTCGGAGTCAACGGATTTAGGAAATGCCCCGTGAACG of HEX primer, shown as SEQ ID NO. 11; reverse primer: ATCGGATGCTACCACTGGAC as shown in SEQ ID NO. 12; ⑤KASP-12 the FAM primer GAAGGTGACCAAGTTCATGCTCTATGGACAAGAAGAACAACATTCA is shown as SEQ ID NO. 13; GAAGGTCGGAGTCAACGGATTCTATGGACAAGAAGAACAACATTCG of HEX primer, shown as SEQ ID NO. 14; reverse primer: TCCGTCAACAAATCTCAAGC as shown in SEQ ID NO. 15; ⑥KASP-17 FAM primer: GAAGGTGACCAAGTTCATGCTCTGACCCACAGGCAATTGC as shown in SEQ ID NO. 16; HEX primer: GAAGGTCGGAGTCAACGGATTCTGACCCACAGGCAATTGT as shown in SEQ ID NO. 17; reverse primer: AGTGTGGTGCAGCAAATCAG as shown in SEQ ID NO. 18; ⑦KASP-22 FAM primer: GAAGGTGACCAAGTTCATGCTGCCGCGTTCCTTGATCTCG as shown in SEQ ID NO. 19; HEX primer: GAAGGTCGGAGTCAACGGATTGCCGCGTTCCTTGATCTCA as shown in SEQ ID NO. 20; reverse primer: GGAAGATGGAAGAACACAACAATTT as shown in SEQ ID NO. 21; ⑧KASP-24 FAM primer: GAAGGTGACCAAGTTCATGCTGCCGCGTTCCTTGATCTCG as shown in SEQ ID NO. 22; HEX primer: GAAGGTCGGAGTCAACGGATTGCCGCGTTCCTTGATCTCA as shown in SEQ ID NO. 23; reverse primer: TGGGAAGATGGAAGAACACAAC as shown in SEQ ID NO. 24; ⑨KASP-25 FAM primer: GAAGGTGACCAAGTTCATGCTGCCGCGTTCCTTGATCTCG as shown in SEQ ID NO. 25; HEX primer: GAAGGTCGGAGTCAACGGATTGCCGCGTTCCTTGATCTCA as shown in SEQ ID NO. 26; reverse primer: TCATTTCCTGGGAAGATGGAAGAA as shown in SEQ ID NO. 27.
  4. 4. A genotyping method of wheat powdery mildew resistance major QTL according to claim 1, comprising the steps of extracting wheat genomic DNA and PCR amplifying the extracted genomic DNA with a primer sequence labeled with KASP-2, KASP-4, KASP-5, KASP-9, KASP-12, KASP-17, KASP-22, KASP-24, KASP-25.
  5. 5. The genotyping method according to claim 4, wherein the PCR amplification method comprises the steps of 2. Mu.L of 2x HiGeno reagent, 0.056. Mu.L of primer mixture and 1. Mu.L of LDNA template (100 ng) in a total volume of 4. Mu.L.
  6. 6. The genotyping method according to claim 4 or 5, wherein the PCR amplification is performed by pre-denaturing at 94℃for 15min, denaturing at 94℃for 5min, annealing at 62-55℃for 30S, 10 cycles, annealing at 72℃for 57℃for 30S, annealing at 72℃for 10min, reading fluorescence values on Phestar instrument after completion of the reaction, analyzing genotyping results with KLUSTER software, amplifying products with FAM adapter and HEX adapter and generating red fluorescence, and the heterozygote corresponds to green fluorescence.
  7. 7. Use of a KASP molecular marker closely linked to a wheat powdery mildew resistance major QTL according to any of claims 1-3 in wheat disease-resistant breeding.

Description

Wheat powdery mildew resistance major QTL and closely linked KASP molecular marker and application thereof Technical Field The invention belongs to the technical field of molecular genetics, and particularly relates to a wheat powdery mildew resistance major QTL and a KASP molecular marker closely linked with the wheat powdery mildew resistance major QTL and application of the wheat powdery mildew resistance major QTL. Background Wheat is one of important grain crops for human beings, is also the main grain crop with the widest global planting area and highest yield, and has important strategic significance for world grain safety and social stability. Wheat growth is subject to many external abiotic and biotic stresses, among which powdery mildew of wheat caused by obligate parasitic powdery mildew of the family gramineae (Bgt) is one of the major diseases worldwide, also in front of the global crop yield-reducing factors. The method has wide popular range, high popular speed and huge harm, and seriously threatens the global food safety. Competitive allele-specific PCR (Kompetitive ALLELE SPECIFIC PCR, KASP) is a novel typing technique based on single nucleotide polymorphisms. The KASP technology can accurately judge the double alleles of the SNPs and InDels on specific sites in a wide genomic DNA sample, has good result accuracy and high repeatability, and is suitable for large-scale population identification and screening. To date, 130 or more powdery mildew resistance genes have been explored from wheat and its closely related species, distributed over 70 or more loci. There are 69 wheat powdery mildew resistance genes (Pm 1-Pm 69) formally named. Wherein Pm1, pm2, pm3, pm4, pm5, pm8 and Pm24 contain multiple resistance sites, namely Pm1 (Pm 1a-Pm1 e), pm2 (Pm 2a, pm2 b), pm3 (Pm 3a-Pm3 k), pm4 (Pm 4a-Pm4 d), pm5 (Pm 5a-Pm5 e), pm8 (Pm 8-Pm 17) and Pm24 (Pm 24a, pm24 b). In addition, in formally named wheat powdery mildew resistance genes Pm8=Pm17, pm18=pm1c, pm22=pm1e, pm22 = the first time of the process. Although many wheat powdery mildew resistance genes have been developed and located, only 17 genes have been cloned up to the present time, pm1, pm2, pm3, pm4, pm5, pm6Sl, pm8, pm13a, pm17, pm21, pm24, pm26, pm36, pm38, pm41, pm46, pm57, pm60 and PmAeu1, respectively. Disclosure of Invention Aiming at the problems in the prior art, the invention provides a wheat powdery mildew resistance major QTL. The invention further provides a KASP molecular marker closely linked with the wheat powdery mildew resistance major QTL. The invention also aims to provide application of the KASP molecular marker, and the molecular marker can be used for auxiliary selective breeding to screen powdery mildew resistant wheat strains. The invention carries out powdery mildew resistance identification and molecular marker detection of known disease resistance genes on 55 parts of imported American wheat materials, and screens and discovers that the PI601806 materials in the seedling stage and the adult stage have high powdery mildew resistance, have broad-spectrum resistance and possibly contain unknown powdery mildew resistance genes. By constructing genetic population with Jimai 22 and identifying powdery mildew resistance, PI601806 is determined to carry a dominant disease-resistant single gene, which is temporarily named Pm601806. Using cluster isolation analysis in combination with wheat exon trap sequencing (BSE-seq), it was speculated that powdery mildew resistance gene Pm601806 might be located on the wheat 1AS chromosome. The study is to locate the Pm601806 gene by genetic segregation population and using BSE-seq sequencing result and newly released wheat reference genome sequence development marker on the basis. In order to achieve the above purpose, the present invention provides the following technical solutions: The invention provides a wheat powdery mildew resistance main effect QTL, which is positioned at the end part of a short arm of a wheat 1A chromosome and is positioned in a 4.4 cM genetic interval between flanking KASP markers Chr1A_6275522_K2 and Chr1A_11524652_K9. The invention provides a KASP molecular marker closely linked with the wheat powdery mildew resistance main effect QTL, wherein the KASP molecular marker is KASP-2, KASP-4, KASP-5, KASP-9, KASP-12, KASP-17, KASP-22, KASP-24 and KASP-25; the 5 'ends of the two KASP forward primers are respectively added with fluorescent tag sequences to modify FAM 5' -GAAGGTGACCAAGTTCATGCT-3; HEX 5’-GAAGGTCGGAGTCAACGGATT-3’。 Preferably, the specific sequences of KASP-2, KASP-4, KASP-5, KASP-9, KASP-12, KASP-17, KASP-22, KASP-24, KASP-25 are as follows: ①KASP-2 The FAM primer GAAGGTGACCAAGTTCATGCTCCTCAACATGTGTCACCCG is shown as SEQ ID NO. 1; GAAGGTCGGAGTCAACGGATTCCTCAACATGTGTCACCCA of HEX primer, shown as SEQ ID NO. 2; CCTTACATTCCTGGCATGCA as shown in SEQ ID NO. 3; ②KASP-4 the FAM primer GAAGGTGACCAAGTTCATGCTACAACAATTTTTTTCATAGTGCC is shown as SEQ ID NO. 4; GAAG