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CN-122012796-A - Mustard KASP molecular marker combination and application thereof in germplasm resource identification and breeding

CN122012796ACN 122012796 ACN122012796 ACN 122012796ACN-122012796-A

Abstract

The invention discloses a mustard KASP molecular marker combination and application thereof in germplasm resource identification and breeding, and belongs to the technical field of molecular markers. The invention develops the mustard KASP molecular marker set from 193 mustard germplasm resources based on whole genome resequencing, and the mustard KASP molecular marker can rapidly, accurately and effectively identify and evaluate germplasm resource materials or breed varieties, and can solve the problems of resource redundancy, inconvenient preservation and management, intellectual property disputes and the like caused by mass germplasm accumulation. Provides a molecular marker for genetic map construction, gene positioning and molecular auxiliary breeding.

Inventors

  • ZHANG YAN
  • YANG HAO
  • ZHONG YANTING
  • LI TINGYAO
  • YANG YI
  • ZHOU XUAN
  • WU ZENGXIANG

Assignees

  • 广东省农业科学院蔬菜研究所

Dates

Publication Date
20260512
Application Date
20260402

Claims (9)

  1. 1. The KASP molecular marker combination for constructing the leaf mustard fingerprint is characterized by comprising KASPJC, KASPJC 4-10, KASPJC 12-14, KASPJC 17-18, KASPJC 22-27, KASPJC and KASPJC 33; KASPJC2 is located on the AA01 chromosome of the mustard genome, and a base polymorphism A/G exists at 21508882; KASPJC4 is located on the AA02 chromosome of the mustard genome, and the 9149701 th nucleotide polymorphism G/A exists; KASPJC5 is located on the AA02 chromosome of the mustard genome, and a base polymorphism T/A exists at 25375102; KASPJC.sup.6 is located on the AA03 chromosome of the mustard genome, and there is a base polymorphism T/C at 20858767; KASPJC7 is located on the AA03 chromosome of the mustard genome, and a base polymorphism T/C exists at 1207962; KASPJC8 is located on the AA06 chromosome of the mustard genome, and the 33425368 th nucleotide polymorphism T/G exists; KASPJC9 is located on the AA06 chromosome of the mustard genome, and a base polymorphism A/T exists at 4179480; KASPJC 10A is located on the AA06 chromosome of the mustard genome, and a base polymorphism A/G exists at 10059237; KASPJC 12A/C is located on AA08 chromosome of mustard genome, and base polymorphism A/C is located at 10191874; KASPJC 13A is located on the AA08 chromosome of the mustard genome, and G/A of the base polymorphism exists at 10553690; KASPJC14 is located on the AA08 chromosome of the mustard genome, and a base polymorphism T/G exists at 16488896; KASPJC 17A 17 is located on the AA09 chromosome of the mustard genome, and a base polymorphism G/T exists at 52646724; KASPJC18 is located on the BB01 chromosome of the mustard genome, and there is a base polymorphism T/G at 7218713; KASPJC 22A/T is located on BB01 chromosome of mustard genome, and base polymorphism A/T is located at 46667455; KASPJC23 is located on the BB02 chromosome of the mustard genome, and a base polymorphism G/A exists at 44834278; KASPJC24 is located on the BB04 chromosome of the mustard genome, and a nucleotide polymorphism C/G exists at 11524175; KASPJC25 is located on the BB04 chromosome of the mustard genome, and a base polymorphism T/C exists at 9110022; KASPJC 26A/G was located on the BB05 chromosome of the mustard genome and at position 1462186; KASPJC27 is located on the BB05 chromosome of the mustard genome, and a base polymorphism G/T exists at 1462170; KASPJC 31A is located on the BB07 chromosome of the mustard genome, and a base polymorphism T/A exists at 12663845; KASPJC33 is located on the BB07 chromosome of the mustard genome, and a nucleotide polymorphism C/G is present at 28738996.
  2. 2. The primer group for specifically detecting the KASP molecular marker combination according to claim 1 is characterized by comprising primers shown as SEQ ID NO. 4-6, SEQ ID NO. 10-30, SEQ ID NO. 34-42, SEQ ID NO. 49-54, SEQ ID NO. 64-81, SEQ ID NO. 91-93 and SEQ ID NO. 97-99.
  3. 3. A reagent, kit or chip for specifically detecting the KASP molecular-labelled combination according to claim 1, comprising the primer set according to claim 2.
  4. 4. Use of the KASP molecular marker combination of claim 1 in germplasm resource identification or variety identification of brassica juncea.
  5. 5. Use of a KASP molecular marker combination according to claim 1 for the construction of a mustard genetic map.
  6. 6. Use of the KASP molecular marker combination of claim 1 in mustard gene mapping or molecular marker assisted breeding.
  7. 7. The method for constructing the leaf mustard fingerprint is characterized by comprising the following steps of: (1) Collecting a tissue sample of a sample, and extracting total DNA of the sample; (2) Performing PCR amplification using the primer set of claim 2, and sequencing the amplified product; (3) Identifying the genotype of the locus of the KASP molecular marker combination according to claim 1 on the genome of the sample, and constructing the mustard fingerprint by using the identified genotype.
  8. 8. A method for identifying a mustard variety, comprising the steps of: (1) Collecting a tissue sample of a sample to be identified, and extracting total DNA of the sample; (2) Identifying the genotype of the locus of the KASP molecular marker combination according to claim 1 on the genome of the sample to be identified; (3) Comparing the genotype of the sample to be identified with the fingerprint constructed in the claim 7, and judging the variety of the sample to be identified according to the comparison result.
  9. 9. Use of a KASP molecular marker combination according to claim 1, a primer set according to claim 2 and a reagent, kit or chip according to claim 3 for identifying mustard varieties.

Description

Mustard KASP molecular marker combination and application thereof in germplasm resource identification and breeding Technical Field The invention relates to the technical field of molecular markers, in particular to a mustard KASP molecular marker combination and application thereof in germplasm resource identification and breeding. Background Mustard (Brassica juncea (l.) czern), an annual herb of brassica genus of cruciferae, is a major edible vegetable variety in our country. Molecular markers (RAPD, SSR, indel, SNP and the like) are widely applied to the aspects of genetic diversity evaluation, genetic map construction, gene positioning, molecular marker assisted breeding and the like of mustard germplasm resources. Among them, the RAPD technique is more susceptible to various factors. Whether the quality and concentration of the template, the short primer sequences, the number of PCR cycles, the complexity of the genomic DNA, the technical equipment, etc., can lead to unstable and difficult reproducibility of the RAPD results. SSR markers have been widely used in genetic breeding of vegetable crops in China because of the advantages of high polymorphism, simple operation and the like. But it does not meet the large-scale, high-throughput, automated detection requirements. SNP markers are used as a third generation molecular marker technology, and have the advantages of high density and more uniform distribution in genome, high flux, detection sites up to millions, and easy realization of automation of data statistics and data integration comparison. Among them, competitive allele PCR (kompetitive ALLELE SPECIFICPCR, KASP) is a genotyping technique based on fluorescence detection. The technology is mainly applied to SNP or Indel gene parting research, and gradually becomes a main technical means of genetic map construction, gene positioning, molecular auxiliary breeding and germplasm resource evaluation. Disclosure of Invention The invention aims to provide a mustard KASP molecular marker combination and application thereof in germplasm resource identification and breeding so as to solve the problems in the prior art. The invention provides a new path for genetic map construction, gene positioning, molecular auxiliary breeding and other works of the leaf mustard based on the leaf mustard core KASP molecular marker developed by whole genome resequencing. In order to achieve the above object, the present invention provides the following solutions: According to one of the technical schemes, the KASP molecular marker combination for constructing the leaf mustard fingerprint is composed of KASPJC, KASPJC 4-10, KASPJC-14, KASPJC 17-18, KASPJC 22-27, KASPJC and KASPJC; KASPJC2 is located on the AA01 chromosome of the mustard genome, and a base polymorphism A/G exists at 21508882; KASPJC4 is located on the AA02 chromosome of the mustard genome, and the 9149701 th nucleotide polymorphism G/A exists; KASPJC5 is located on the AA02 chromosome of the mustard genome, and a base polymorphism T/A exists at 25375102; KASPJC.sup.6 is located on the AA03 chromosome of the mustard genome, and there is a base polymorphism T/C at 20858767; KASPJC7 is located on the AA03 chromosome of the mustard genome, and a base polymorphism T/C exists at 1207962; KASPJC8 is located on the AA06 chromosome of the mustard genome, and the 33425368 th nucleotide polymorphism T/G exists; KASPJC9 is located on the AA06 chromosome of the mustard genome, and a base polymorphism A/T exists at 4179480; KASPJC 10A is located on the AA06 chromosome of the mustard genome, and a base polymorphism A/G exists at 10059237; KASPJC 12A/C is located on AA08 chromosome of mustard genome, and base polymorphism A/C is located at 10191874; KASPJC 13A is located on the AA08 chromosome of the mustard genome, and G/A of the base polymorphism exists at 10553690; KASPJC14 is located on the AA08 chromosome of the mustard genome, and a base polymorphism T/G exists at 16488896; KASPJC 17A 17 is located on the AA09 chromosome of the mustard genome, and a base polymorphism G/T exists at 52646724; KASPJC18 is located on the BB01 chromosome of the mustard genome, and there is a base polymorphism T/G at 7218713; KASPJC 22A/T is located on BB01 chromosome of mustard genome, and base polymorphism A/T is located at 46667455; KASPJC23 is located on the BB02 chromosome of the mustard genome, and a base polymorphism G/A exists at 44834278; KASPJC24 is located on the BB04 chromosome of the mustard genome, and a nucleotide polymorphism C/G exists at 11524175; KASPJC25 is located on the BB04 chromosome of the mustard genome, and a base polymorphism T/C exists at 9110022; KASPJC 26A/G was located on the BB05 chromosome of the mustard genome and at position 1462186; KASPJC27 is located on the BB05 chromosome of the mustard genome, and a base polymorphism G/T exists at 1462170; KASPJC 31A is located on the BB07 chromosome of the mustard genome, and a base polymorphism T/A exists at 1266