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CN-121992133-A - DCAPs molecular marker related to early ripening property of citrus unshiu fruits and application thereof

CN121992133ACN 121992133 ACN121992133 ACN 121992133ACN-121992133-A

Abstract

The invention relates to the technical field of plant molecular breeding and biology, in particular to a dCAPs molecular marker related to the precocity trait of citrus unshiu fruits and application thereof. A dCAPs molecular marker associated with the precocity trait of citrus unshiu fruits, said molecular marker comprising a Single Nucleotide Polymorphism (SNP) site located at chromosome 39493125 of citrus unshiu genome, said SNP site having a nucleotide of T or a, wherein said citrus unshiu genome has an accession number of GWHERCA00000000 at the national genomics data center. The molecular marker comprises a nucleotide sequence shown as SEQ ID NO. 1. The invention is based on the key SNP mutation causing the CLASP gene function loss, can be directly used for early screening of the Mizhou mandarin orange premature bud mutation resource, auxiliary selection of filial generation premature single plants and identification of variety authenticity and purity, can obviously shorten the breeding period and improve the breeding efficiency.

Inventors

  • WU JUXUN
  • WANG LIXIN
  • HE CHAO
  • HE ZHENCHENG
  • YI HUALIN

Assignees

  • 华中农业大学

Dates

Publication Date
20260508
Application Date
20260205

Claims (8)

  1. 1. A dCAPs molecular marker associated with the precocity trait of citrus unshiu fruits, wherein said molecular marker comprises a SNP site located at chromosome 39493125 of citrus unshiu genome, said SNP site having a nucleotide of T or a, wherein said citrus unshiu genome has an accession number of GWHERCA00000000 at the national genomics data center.
  2. 2. The dCAPs molecular marker according to claim 1, wherein the molecular marker comprises a nucleotide sequence as shown in seq id No. 1.
  3. 3. A primer pair for detecting the dCAPs molecular marker according to claim 1 or 2, comprising a forward primer comprising a nucleotide sequence as shown in seq id No. 2 and a reverse primer comprising a nucleotide sequence as shown in seq id No. 3.
  4. 4. Use of the primer pair of claim 3 in the preparation of a kit for identifying the ripening period of citrus unshiu fruits.
  5. 5. A method for identifying the ripening period of citrus unshiu fruits, comprising the steps of: step (1), extracting genome DNA of a citrus unshiu sample to be detected; Step (2), using the genome DNA extracted in the step (1) as a template, and carrying out PCR amplification by using the primer pair of claim 3 to obtain an amplification product; step (3), performing enzyme digestion treatment on the amplification product obtained in the step (2) by using restriction enzyme HindIII to obtain an enzyme digestion product; And (4) detecting the enzyme digestion product obtained in the step (3), wherein if the enzyme digestion product shows a single DNA fragment of about 259bp, the condition that the citrus unshiu sample is homozygous at the SNP locus is judged to belong to a late maturing variety, and if the enzyme digestion product shows two DNA fragments with about 25bp different lengths, the condition that the citrus unshiu sample is heterozygous at the SNP locus is judged to belong to an early maturing variety.
  6. 6. The method according to claim 5, wherein the PCR amplified product in step (2) has a length of 259bp.
  7. 7. The method according to claim 5, wherein in step (4), the cleavage product is detected by agarose gel electrophoresis or capillary electrophoresis.
  8. 8. A kit for identifying the ripening period of citrus unshiu fruits, comprising the primer pair of claim 3 and restriction enzyme HindIII.

Description

DCAPs molecular marker related to early ripening property of citrus unshiu fruits and application thereof Technical Field The invention relates to the technical field of plant molecular breeding and biology, in particular to a dCAPs molecular marker related to the precocity trait of citrus unshiu fruits and application thereof. Background Citrus is one of the global bulk fruits, and the scale of the citrus industry in China is the first place in the world. Through long-term development, the citrus variety improvement in China has significantly progressed in the aspects of application, maturity and variety diversification. However, the citrus fruit ripening period in China is still concentrated, the medium-ripening variety accounts for over 50%, and the high-quality super-early-ripening variety and the late-ripening variety are relatively deficient. Therefore, the method for breeding early maturing varieties and accurately regulating and controlling the mature period of fruits has key effects on preempting market precedent, improving planting benefits and realizing annual balanced supply of fresh fruits, and is an important target for citrus breeding. The citrus unshiu is one of main varieties of citrus in China, and has higher natural mutation rate. Through continuous multi-generation bud mutation seed selection, china, japan and other countries have bred hundreds of bud mutation varieties such as 'tail strain', 'Gong Chuan' and the like from primary strains such as Yi mu Li, early-stage strain and the like, so that a satsuma mandarin bud mutation product population with large scale and rich genetic background is formed. The population shows remarkable difference in the fruit ripening period characters, and the fruit ripening period between the extremely early-maturing variety and the late-maturing variety can be different by more than 50 days. The method not only greatly prolongs the market supply period of the citrus unshiu, but also provides valuable genetic materials for analyzing the genetic mechanism of the fruit ripening period regulation and developing directional molecular breeding. Along with the development of high-throughput sequencing technology, single Nucleotide Polymorphism (SNP) markers have become one of core tools for molecular marker assisted breeding due to the advantages of large number, wide distribution, good stability and the like. CAPS markers were developed based on restriction enzyme cleavage polymorphisms of PCR amplification products, but their use was limited by the presence of natural cleavage sites. The derived CAPS (dCAPS) technology creates or destroys a restriction enzyme recognition site by artificially introducing mismatched bases into a specific primer, so that almost any SNP site can be converted into dCAPS markers which are easy to detect by conventional electrophoresis, and the application range of the SNP typing technology is greatly expanded. At present, no functional dCAPs mark aiming at the precocity trait of citrus unshiu has been reported yet. The development of the mark has important theoretical and application values for accelerating the breeding process of the citrus unshiu in the ripening period and realizing variety diversity layout. Disclosure of Invention The invention aims to solve the defects existing in the prior art. One of the purposes of the present invention is to propose a dCAPs molecular marker related to the precocity trait of citrus unshiu fruits, wherein the molecular marker comprises a Single Nucleotide Polymorphism (SNP) site located at 39493125 th chromosome of citrus unshiu genome, and the SNP site has a nucleotide of T or A, wherein the accession number of the citrus unshiu genome at the national genomics data center is GWHERCA00000000. Further, the molecular marker comprises a nucleotide sequence shown as SEQ ID NO. 1. The second object of the invention is to provide a primer pair for dCAPs molecular marker, comprising a forward primer and a reverse primer, wherein the forward primer comprises a nucleotide sequence shown as SEQ ID NO. 2, and the reverse primer comprises a nucleotide sequence shown as SEQ ID NO. 3. The third purpose of the invention is to provide the application of the primer pair in preparing a kit for identifying the ripening period of citrus unshiu fruits. The fourth object of the invention is to provide a method for identifying the ripening period of citrus unshiu fruits, which comprises the following steps: step (1), extracting genome DNA of a citrus unshiu sample to be detected; Step (2), using the genome DNA extracted in the step (1) as a template, and carrying out PCR amplification by using the primer pair of claim 3 to obtain an amplification product; step (3), performing enzyme digestion treatment on the amplification product obtained in the step (2) by using restriction enzyme HindIII to obtain an enzyme digestion product; And (4) detecting the enzyme digestion product obtained in the step (3), wherein if the