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CN-118638959-B - Molecular marker and primer closely related to purity of capsicum seed and application of molecular marker and primer in identifying purity of hybrid seed

CN118638959BCN 118638959 BCN118638959 BCN 118638959BCN-118638959-B

Abstract

The invention provides a molecular marker closely related to the purity of pepper seeds, a primer and application thereof in identifying the purity of hybrid seeds, wherein the molecular marker comprises a molecular marker 1 and a molecular marker 2, the molecular marker 1 is the variation of C to T at the 1150917bp position on chromosome Capsicum annuum L of a pepper genome, and the molecular marker 2 is the variation of T to C at the 30093662bp position on chromosome Capsicum annuum L of the pepper genome. According to the invention, 2 SNP loci different between two parents are screened, SNP markers are successfully developed, and two pairs of KASP primer combinations are designed, so that the method can be directly used for identifying a variety of 'Xingshui spicy No. 1' of a commercial variety of early-maturing capsicum by utilizing a fluorescence detection technology.

Inventors

  • YANG SHA
  • WU ZHUOXUAN
  • CUI QINGZHI
  • ZHANG ZHUQING
  • LIANG CHENGLIANG
  • CHEN WENCHAO
  • DONG ZHIXUE
  • ZOU XUEXIAO

Assignees

  • 湖南农业大学
  • 湖南省蔬菜研究所

Dates

Publication Date
20260505
Application Date
20240628

Claims (5)

  1. 1. The application of the primer closely related to the purity of the capsicum seed in the identification of the purity of the hybrid seed of Xingshui No. 1 is characterized in that the primer is a primer group of Chr01-1150917 and a primer group of Chr 06-30093662; The primer group of the Chr01-1150917 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 C with a nucleotide sequence shown as SEQ ID NO. 3; The primer group of the Chr06-30093662 comprises an upstream primer F1 with a nucleotide sequence shown as SEQ ID NO. 4, an upstream primer F2 with a nucleotide sequence shown as SEQ ID NO. 5 and a downstream primer C with a nucleotide sequence shown as SEQ ID NO. 6.
  2. 2. The use of claim 1, wherein the 5 'ends of the upstream primer F1 in the Chr01-1150917 primer set and the Chr06-30093662 primer set are respectively connected with a FAM fluorescent linker, the 5' ends of the upstream primer F2 are respectively connected with a HEX fluorescent linker, and the nucleotide sequences of the FAM and HEX fluorescent linkers are respectively shown in SEQ ID No. 7 and SEQ ID No. 8.
  3. 3. The use according to claim 1, wherein the specific step of identifying the purity of the hybrid seed of the hot, spicy, 1 st vegetable species comprises: (1) Taking genomic DNA of a sample to be detected as a template, and carrying out PCR (polymerase chain reaction) amplification by using the primer marked by the capsicum molecules to obtain an amplification product; (2) Performing fluorescence detection on the amplified product, wherein if HEX fluorescence signals corresponding to primer groups of Chur 01-1150917 or Chur 06-30093662 are detected, the corresponding detection site is A or C, the genotype is judged to be a male parent false hybrid, if FAM fluorescence signals corresponding to primer groups of Chur 01-1150917 or Chur 06-30093662 are detected, the corresponding detection site is G or T, the genotype is judged to be a female parent false hybrid, if both FAM and HEX fluorescence signals are detected, the detection site is G, A or T, the genotype is judged to be a true hybrid, and if no fluorescence signal is detected, the combination of other genotypes is judged to be other types of hybrid.
  4. 4. The use according to claim 3, wherein the purity of the pepper hybrid seed is calculated by counting the ratio of true hybrid seeds to total test sample by the method of identifying the authenticity of the variety of the early-matured pepper of the "Xingshui No. 1" through the molecular marker of the pepper.
  5. 5. The use according to claim 3, wherein the method of PCR amplification specifically comprises: The Touchdown PCR amplification procedure was carried out using Touchdown PCR cycles of 94℃for 15min, 95℃for 20s, 65℃to 56℃for 60s,10 cycles of annealing extension temperature decrease of 0.8℃per cycle, 94℃for 20s, 57℃for 60s,26 cycles.

Description

Molecular marker and primer closely related to purity of capsicum seed and application of molecular marker and primer in identifying purity of hybrid seed Technical Field The invention belongs to the technical field of molecular breeding, and particularly relates to a molecular marker and a primer closely related to the purity of capsicum seed and application thereof in identifying the purity of hybridized seed. Background The country uses the agriculture as the root and the agriculture uses the seed as the first. Seed quality is the life and basis of seed production and sales, and is the key to control production risk, and cultivation and production of good seeds are core tasks of the planting industry. The four general indexes of seed quality are seed purity, germination rate and water content, and the purity is the most core index for seed purity. Because of this, seed purity detection is an important link for seed quality internal control and is also a main quality index for market supervision. Common methods for purity identification of general varieties include seed morphology identification, seedling morphology identification, protein (isozyme) electrophoresis method, DNA molecular marker identification and field cell planting identification. The seed morphology identification is carried out according to morphological characteristics of seed grain type, grain color, shape, size, embryo size, endosperm powder quality and the like, standard samples or patterns of the variety and related data are needed to be prepared during identification, the identification accuracy is low, the seedling morphology identification is carried out according to the bud sheath color (green, red, purple), leaf color, leaf shape, growth vigor and the like of the seedling, the seedling morphology identification is generally carried out by taking the bud sheath color as the main morphological characteristics of purity identification, the identification time is long, the identification efficiency is low, the protein (isoenzyme) electrophoresis method is carried out according to the difference of genetic basis of different varieties of seeds, the types and the quantity of synthesized proteins (isoenzymes) are different, the number of the seeds of other varieties can be judged after electrophoresis separation is different, the purpose of identifying the purity of the seeds is finally achieved after comparison with a comparison (standard) variety, the electrophoresis time is long and the operation is complex, the DNA molecular marker identification is carried out by taking a variety DNA fragment as a detection object, and the polymorphism DNA structure and composition of the variety are detected by using an electrophoresis method, and polymorphism analysis is carried out on the variety DNA sequence difference. The DNA molecular markers used in the current identification of variety authenticity and variety purity mainly comprise SSR, AFLP, RAPD technology based on PCR amplification and RFLP technology based on molecular hybridization. The method is based on electrophoresis to distinguish bands so as to distinguish true species from false species, and large-scale operation is not utilized. The field community planting and identification is the most reliable method for identifying the authenticity and purity of the current variety, is particularly suitable for identifying the hybrid seeds, has accurate and reliable result, is the most legal efficacy detection method for solving the seed quality dispute at present, has more observable and comparative characters, has the defects of labor and time consumption, long identification period and high cost, is greatly influenced by environment and human, and restricts the identification accuracy by the observation experience of an identifier. The existing variety identification methods have the defects. With the development of genome sequencing technology, researchers have developed detection of SNP marker loci for parents, and at present, SNP detection has been proposed as a method for identifying varieties on the DNA level by a number of international organizations, such as International Seed Testing Association (ISTA), international new variety protection Association (UPOV), international seed Union (ISF), and the like, as a latest molecular marker detection method. The method mainly comprises an SNP chip platform, a sample high-throughput in-situ scanning platform (LGC company KASP technology, life company Taqman technology), a site and sample high-throughput targeted sequencing technology and the like, and the technology platforms have higher cost and high identification cost. The Xingshui spicy No. 1 is a representative variety of high-taste quality peppers, the accumulated popularization area exceeds 5.0 mu in the provinces of Hunan, jiangxi and the like, and the variety identification is important for the popularization and application of the variety. However, currently, purity identif