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CN-121992144-A - SNP molecular marker for identifying south-peak Honghuang, method and application

CN121992144ACN 121992144 ACN121992144 ACN 121992144ACN-121992144-A

Abstract

The invention provides a SNP molecular marker for identifying Nanfeng honey, a method and application thereof, belonging to the technical field of genetic breeding and molecular biology, wherein the SNP molecular marker is positioned on a No. 6 chromosome of a Nanfeng honey orange reference genome, and a base G is mutated into A at 10668240 bp. The result shows that the genotype of the Nanfeng Honguang is heterozygous G/A, and the Hongfeng is homozygous G/G, the invention provides two specific methods which rely on the first-generation sequencing or the enzyme digestion amplification polymorphism sequence (CAPS), can accurately and rapidly identify the Nanfeng Hongfeng, and is beneficial to the popularization of the variety and the protection of the new plant variety right.

Inventors

  • YANG HUIDONG
  • WANG YUTING
  • XU QIANG
  • LIU XINCHENG
  • HU ZHONGDONG

Assignees

  • 江西省农业科学院园艺研究所

Dates

Publication Date
20260508
Application Date
20260401

Claims (7)

  1. 1. The PCR amplification primer for identifying the SNP molecular marker of the Nanfeng orange is characterized in that the SNP molecular marker is positioned on chromosome 6 of a Nanfeng orange reference genome, the base G is mutated into A at 10668240 bp, the PCR amplification primer comprises an upstream primer NF68240F and a downstream primer NF68240R, the nucleotide sequence of the upstream primer NF68240F is shown as SEQ ID NO. 2, and the nucleotide sequence of the downstream primer NF68240R is shown as SEQ ID NO. 3.
  2. 2. A first-generation sequencing method for identifying the Hongguang, which is characterized by comprising the steps of carrying out PCR amplification on genomic DNA of a material to be identified by adopting the PCR amplification primer of claim 1, and carrying out first-generation sequencing on an amplified product by taking NF68240F as a sequencing primer, wherein the Hongguang shows double peaks of G and A at the site of Chr6: 10668240.
  3. 3. The method of claim 2, wherein the PCR amplification is performed in a reaction sequence of 94℃pre-denaturation of 2min, 94℃denaturation of 30s,58℃renaturation of 30s,72℃extension of 30s, 35 cycles total, and a final 72℃extension of 2 min.
  4. 4. A method for identifying the polymorphic sequence of the cut-and-amplified south-peak Hongjie is characterized by comprising the steps of carrying out PCR amplification on genomic DNA of a Hongjie material to be identified by adopting the PCR amplification primer of claim 1, and carrying out electrophoresis after cutting an amplification product by using restriction enzyme EcoRI, wherein the south-peak Hongjie generates bands at 341bp, 173bp and 514 bp, and other Hongjie strains only generate bands at 514 bp.
  5. 5. The method for amplifying the polymorphic sequence by enzyme digestion according to claim 4, wherein the PCR amplification reaction program is 94 ℃ pre-denaturation for 2min, 94 ℃ denaturation for 30s,58 ℃ renaturation for 30s and 72 ℃ extension for 30s, which is 35 cycles, and finally 72 ℃ extension for 2 min.
  6. 6. The method for amplifying a polymorphic sequence according to claim 4, wherein the other Hongkong plant lines comprise Mianju, honggong, guizhi No.1, mianguang No.1 and Mianguang No. 2.
  7. 7. The use of the PCR amplification primer of claim 1 for identifying south mountain Hongguan.

Description

SNP molecular marker for identifying south-peak Honghuang, method and application Technical Field The invention belongs to the technical fields of genetic breeding and molecular biology, and particularly relates to a molecular marker, a method and application for identifying SNP (single nucleotide polymorphism ) with broad south peak honey. Background South Feng Miju (Citrus reticulata 'Kinokuni') is a local characteristic citrus variety in China, has long cultivation history, and forms abundant Nanfeng orange variation resources in surrounding areas in a long-term cultivation process. Honey guang (Miguang tangerine) is a natural hybrid of Nanfeng tangerines and fire tangerines, belongs to a precocious large fruit system, and is deeply favored by consumers because of golden color, fresh, tender and succulent, and rich flavor. However, the peel of the pepper has strong pepper smell, is easy to cause unpleasant feeling of eaters, has a maturation period of 11 months, belongs to medium-maturing varieties, and has the problems of concentrated maturation period and high market competition pressure. Bud mutation is a form of somatic mutation that occurs in the meristematic cells of a plant bud, and refers to a phenomenon that the bud grows into a shoot or an individual grown therefrom exhibits a different character from the original type. Typically in the form of shoot variation. The bud mutation breeding is a selective breeding method for breeding new varieties by utilizing mutated branches and buds to perform asexual propagation, fixing the characters and comparing and identifying. In the long-term citrus breeding process, the inventor discovers a bud variation single plant in a honey-guangzhangyuan, and finally breeds a bud variation variety with stable inheritance through continuous observation, identification and cultivation for many years, and names the bud variation variety as Nanfeng honey Guangdong, and the application number of Nanfeng honey Guangdong in a new variety protection office of agricultural rural plants for accepting protection of the new variety is 20261001807. The bud variety inherits the excellent characters of fresh, tender and succulent pulp and rich flavor of honey, realizes remarkable improvement on a plurality of key characters, effectively improves the eating experience and commodity quality of fruits, advances the mature period to the last 10 months each year, belongs to a premature variety, can avoid the peak period of 11 month citrus concentrated marketing, effectively relieves market competition pressure, and is most critical that the peel of the variety basically has no pepper smell, thoroughly overcomes the core defect of the bad smell of honey peel and remarkably improves the market acceptance of fruits. Meets the development trend and market demand of the current citrus industry of high quality, early curing and no nucleation, and has great market potential, popularization value and economic benefit. However, the south peak honey and other honey and honey plant lines are difficult to distinguish in morphology, the genetic background between the germinated varieties and the original varieties is very similar, and the method for accurately and rapidly identifying the south peak honey and the honey plant lines has important significance for protecting and popularizing new varieties. Disclosure of Invention The invention aims to provide SNP molecular markers, methods and applications for identifying Hongshuang, which can be used for identifying Hongshuang sprout-transformed varieties obtained through sprout transformation breeding, namely, namodal Hongshuang and other Hongshuang varieties. In order to achieve the above purpose, the invention adopts the following technical scheme: the invention provides a SNP molecular marker for identifying Nanfeng orange, which is positioned on chromosome 6 of Nanfeng orange reference genome and is mutated into A at 10668240 bp th base G. The invention also provides a PCR amplification primer for identifying the south Feng Miguang, which comprises an upstream primer NF68240F and a downstream primer NF68240R, wherein the nucleotide sequence of the upstream primer NF68240F is shown as SEQ ID NO. 2, and the nucleotide sequence of the downstream primer NF68240R is shown as SEQ ID NO. 3. The invention also provides a first generation sequencing method for identifying the Hongguang, which comprises the steps of carrying out PCR amplification on genome DNA of a Hongguang material to be identified by adopting the PCR amplification primer, and carrying out first generation sequencing on an amplified product by taking NF68240F as a sequencing primer, wherein the Hongguang shows double peaks of G and A at a Chr6:10668240 locus. Further, the PCR amplification reaction program is 94 ℃ pre-denaturation for 2min, 94 ℃ denaturation for 30s,58 ℃ renaturation for 30s,72 ℃ extension for 30s, which is 35 cycles, and finally 72 ℃ extension for 2 min. The invention a