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CN-120400400-B - Indel molecular marker primer pair, kit and method for identifying Yichang orange

CN120400400BCN 120400400 BCN120400400 BCN 120400400BCN-120400400-B

Abstract

The invention provides an Indel molecular marker primer pair, a kit and a method for identifying Yichang orange, which belong to the technical field of biochemistry, wherein the Indel molecular marker primer pair comprises at least one of an InDel-2 primer pair and an InDel-3 primer pair. The Indel molecular marker primer pair or the kit can specifically identify Yichang orange, realize efficient and accurate identification of Yichang orange and filial generation thereof, and has wide application prospect in aspects of citrus germplasm resource protection, variety rights maintenance, breeding selection, market supervision and the like. The method for identifying Yichang orange can accurately identify Yichang orange and filial generation thereof, has high specificity, economy and high efficiency, is an accurate, economical and efficient hybrid identification means, can effectively identify Yichang orange varieties and hybrids thereof, and provides powerful technical support for scientific research and breeding work.

Inventors

  • CHAI LIJUN
  • YANG YANG
  • LI SHUNXIN
  • YE JUNLI
  • XIE ZONGZHOU
  • DENG XIUXIN

Assignees

  • 华中农业大学

Dates

Publication Date
20260508
Application Date
20250408

Claims (9)

  1. 1. The Indel molecular marker primer pair for identifying Yichang orange is characterized by comprising at least one of an InDel-2 primer pair and an InDel-3 primer pair, wherein the sequence of the InDel-2 primer pair is shown as SEQ ID No.1 and SEQ ID No.2, and the sequence of the InDel-3 primer pair is shown as SEQ ID No.3 and SEQ ID No. 4.
  2. 2. A kit for identifying yichang orange, said kit comprising the Indel molecular marker primer pair of claim 1.
  3. 3. A kit for identifying yichang orange as claimed in claim 2, wherein said kit further comprises PCR amplification reagents and double distilled water.
  4. 4. Use of the Indel molecular marker primer pair of claim 1 or the kit of claim 2 or 3 for identifying yichang orange.
  5. 5. A method for identifying yichang orange, comprising the steps of: extracting genome DNA of a sample to be detected; respectively carrying out PCR amplification by taking the extracted genome DNA of the sample to be detected as a template and establishing a PCR amplification system by using the Indel molecular marker primer pair according to claim 1 to obtain amplification products; respectively carrying out agarose gel electrophoresis on the amplification products to obtain an electrophoresis chart; and carrying out variety identification on the sample to be detected according to the strip information on the electrophoresis chart.
  6. 6. A method for identifying Yichang orange according to claim 5, wherein the PCR amplification system comprises 10. Mu.l 2 x Taq plus master mix,0.5. Mu.l forward primer, 0.5. Mu.l reverse primer, 150. 150 ng genomic DNA template of the sample to be tested and is complemented to 20. Mu.l with ddH 2 O.
  7. 7. The method for identifying Yichang orange according to claim 5, wherein the PCR amplification conditions are 95℃pre-denaturation 5min, 95℃denaturation 30 s,56℃annealing 30 s,72℃extension 50s,35 cycles, and 72℃extension 5 min.
  8. 8. The method for identifying Yichang orange according to claim 5, wherein the agarose gel electrophoresis is carried out by adopting 2.0% -4.0% of agarose gel by mass percent, the voltage is 100-150V, the current is 300-500 mA, and the electrophoresis time is 10-30 min.
  9. 9. The method for identifying Yichang orange according to claim 5, wherein said identifying the variety of the sample to be detected based on the band information on said electrophoretogram comprises the steps of: The Indel molecular marker primer pair is InDel-2 primer pair, when the agarose gel shows a band of 330bp, the sample to be detected is of other varieties, when the agarose gel shows a band of less than 330bp, the sample to be detected is Yichang orange; The Indel molecular marker primer pair is InDel-3 primer pair, when the agarose gel shows a 400bp band, the sample to be detected is of other varieties, and when the agarose gel shows a band smaller than 400bp, the sample to be detected is Yichang orange.

Description

Indel molecular marker primer pair, kit and method for identifying Yichang orange Technical Field The invention belongs to the technical field of biochemistry, and particularly relates to an Indel molecular marker primer pair, a kit and a method for identifying Yichang orange. Background Citrus is the first major fruit in the world and has great industrial and economic value. Citrus is an important economic fruit tree, wherein Yichang orange is named as being discovered in Yichang mountain county of Hubei province at the earliest time, is distributed in the North subtropical and middle subtropical areas of China, and is a special wild citrus seed in China. Yichang orange is a single embryo citrus resource with cold resistance, barren resistance and shade resistance, which is less in citrus, and is a precious material in aspects of conventional hybridization breeding, cell fusion breeding and the like of citrus. Cross breeding is a main means of germplasm innovation, in the aspect of citrus variety identification, morphological, cytology, molecular marking and other means are often combined, however, the existing morphological marking is less, the citrus child-phase is longer, and in addition, in the seedling stage, the varieties of the same citrus or the same or similar parent sources are difficult to accurately distinguish. Along with the continuous enrichment of citrus varieties and the diversification of market demands, the problem of accurately and rapidly identifying the citrus varieties is needed to be solved, and the method has important significance in aspects of germplasm resource protection, variety rights and interests maintenance, market supervision and the like. Because the childhood period of fruit trees such as citrus is long, the rapid progress in terms of citrus resource development, breeding and the like is difficult to achieve in a short time by only means of biochemical analysis, phenotype evaluation and the like. With the development of molecular biology techniques, DNA molecular markers are widely used in plant variety identification. The SSR, RAPD, AFLP and other markers are applied to citrus variety identification, but the methods have the problems of more or less complicated operation, poor repeatability, high cost and the like. In recent years, with the rapid development of high throughput sequencing technology, the development of molecular markers based on whole genome resequencing data has become possible. Among them, indel (Indel) markers show great potential in plant variety identification due to their high polymorphism, co-dominance, good stability and other advantages. However, specific Indel markers for Yichang orange have not been reported. Therefore, it is of great importance to develop an Indel molecular marker based on resequencing data, which is capable of specifically recognizing Yichang orange. Disclosure of Invention The invention aims to provide an Indel molecular marker primer pair, a kit and a method for identifying Yichang orange, so as to specifically identify the Yichang orange and realize efficient and accurate identification of Yichang orange and filial generation thereof. To achieve the above object, in a first aspect of the present invention, there is provided an Indel molecular marker primer pair for identifying Yichang orange, the Indel molecular marker primer pair including at least one of an Indel-2 primer pair and an Indel-3 primer pair, the sequence of the Indel-2 primer pair being shown as SEQ ID No.1 and SEQ ID No.2, and the sequence of the Indel-3 primer pair being shown as SEQ ID No.3 and SEQ ID No. 4. The InDel-2 primer pair and the InDel-3 primer pair can be used for effectively identifying Yichang orange, and one of the two primer pairs can be selected at will for identification when the kit is applied. In a second aspect of the invention, there is provided a kit for identifying Yichang orange, said kit comprising an Indel molecular marker primer pair as described above. Further, the kit also comprises PCR amplification reagents and double distilled water. In a third aspect, the invention provides an Indel molecular marker primer pair or application of the kit in the identification of Yichang orange. In a fourth aspect of the present invention, there is provided a method of identifying Yichang orange comprising the steps of: extracting genome DNA of a sample to be detected; Taking the extracted genome DNA of the sample to be detected as a template, and establishing a PCR amplification system by using the Indel molecular marker primer pair to respectively carry out PCR amplification to obtain an amplification product; respectively carrying out agarose gel electrophoresis on the amplification products to obtain an electrophoresis chart; and carrying out variety identification on the sample to be detected according to the strip information on the electrophoresis chart. Further, the PCR amplification system included 10. Mu.l 2 x Taq plus master mix