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CN-122012798-A - Primer combination for identifying hardness of apple fruits, product and application of primer combination

CN122012798ACN 122012798 ACN122012798 ACN 122012798ACN-122012798-A

Abstract

The invention relates to the technical field of germplasm resource and F 1 group identification, in particular to a primer combination, a product and application thereof for identifying apple fruit hardness. The invention designs a specific primer (first primer pair) to carry out common PCR verification on a sample, and the result shows that 40F 1 filial generations with extremely high hardness and extremely low hardness can only amplify small bands, which is not in accordance with the expected result of parent genetics. Therefore, the invention creatively designs a second primer pair, judges whether the mutation site exists by judging whether a target band appears in a PCR product of the second primer pair, if the second PCR amplification does not have a band, the site can be judged to be a homozygous deletion genotype in a offspring genome, and if the second PCR amplification has a target band, the site can be judged to be a heterozygous genotype in the offspring genome. Therefore, the invention creatively provides the double PCR verification results of the two pairs of primers to jointly confirm the accurate genotype of the test sample.

Inventors

  • SU QIUFANG
  • KAN JIALIANG
  • AN YANRU
  • FAN YUE
  • LI XIAOGANG

Assignees

  • 江苏省农业科学院

Dates

Publication Date
20260512
Application Date
20260407

Claims (10)

  1. 1. A primer combination for identifying hardness of apple fruits, characterized in that the primer combination comprises a first primer pair and a second primer pair; The first primer pair comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.1 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 2; the second primer pair comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.3 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 4.
  2. 2. Use of a primer combination according to claim 1 for the preparation of a product for identifying apple fruit firmness.
  3. 3. The use according to claim 2, wherein the products comprise reagents, kits and chips.
  4. 4. A product for identifying fruit firmness, comprising the primer combination of claim 1.
  5. 5. The product of claim 4, wherein the product comprises reagents, kits and chips.
  6. 6. Use of the primer combination of claim 1 or the product of claim 4 or 5 for identifying apple fruit firmness.
  7. 7. A method for identifying apple fruit hardness, which is characterized in that the DNA of apples to be detected is used as a template, a first primer pair in the primer combination of claim 1 is adopted for PCR amplification, a second primer pair in the primer combination of claim 1 is adopted for PCR amplification, and the apple fruit hardness is judged according to the results of the two PCR amplification.
  8. 8. The method of claim 7, wherein the first round of PCR products exhibit a specific band at 1013bp and no band in the second round of amplification products are defined as homozygous deletion genotypes, and wherein the first round of PCR products exhibit a specific band at 2179bp and the second round of PCR products exhibit a specific band at 955 bp, and wherein the second round of PCR products exhibit a homozygous insertion genotype, and wherein the second round of PCR products exhibit a low hardness apple fruits.
  9. 9. The method of claim 7, wherein the PCR amplification reaction system is 5. Mu.L of a2 XPCR polymerase mixture, 3. Mu.L of sterile water, 0.5. Mu.L of an upstream primer, 0.5. Mu.L of a downstream primer, and 1. Mu.L of a template.
  10. 10. The method of claim 7, wherein the PCR amplification is performed for a period of about 5 minutes at 94 ℃, about 30 seconds at 60 ℃, about 1-2 minutes at 72 ℃, about 35 cycles, and about 10 minutes at 72 ℃.

Description

Primer combination for identifying hardness of apple fruits, product and application of primer combination Technical Field The invention relates to the technical field of germplasm resource and F 1 group identification, in particular to a primer combination, a product and application thereof for identifying apple fruit hardness. Background The fruit essence mainly comprises hardness, brittleness and juiciness, is a key index for evaluating the quality of fruits, and directly influences the purchase intention and market competitiveness of consumers. Higher fruit hardness helps to maintain good commodity appearance and storability, but too low hardness or too soft fruits easily cause pathogen infection, remarkably shortens shelf life and reduces economic value. In apple breeding, hardness in the mature period is one of five key traits, is closely related to hardness reduction in the mature period and postharvest storage period of fruits, and belongs to complex quantitative traits regulated by multiple genes. At present, the quality of fruit texture has become an important point in modern apple breeding and industry research. The genetic molecular mechanism of apple fruit hardness formation is deeply revealed, and key genes and functional genetic loci are excavated and utilized, so that the method has important theoretical and application values for guiding hybridization breeding practice, cultivating high-quality fruits and promoting industry quality improvement and efficiency improvement. Apples have long been developed mainly around the hormone ethylene as typical fruit with respiratory transitions, the mature softening studies of which have been carried out for a long time. Two key rate-limiting enzymes for ethylene biosynthesis, ACS synthase and ACC oxidase, directly regulate their biosynthesis. In breeding practice, the hardness-related molecular markers MdACS1 (166 bp) and MdACO1 (66 bp) are often used for breeding ideal fruit substance. However, although the above markers are closely related to ethylene synthesis, they are generally only capable of explaining limited phenotypic variation in the hard phenotype, and thus have significant limitations in application to molecular marker assisted breeding. In addition to the hormonal pathway, degradation of cell wall components and destruction of structures can reduce cell-to-cell adhesion, and are closely linked with the softening process of fruits, and generally involve metabolism of cell wall polysaccharides (pectin, cellulose hemicellulose), regulation of related cell wall degrading enzymes (polygalacturonase PG, pectin lyase PL, cellulase, etc.), synergistic effects of non-enzymatic substance-expanding proteins EXP, and the like. The hardness of the fruits in the mature period is closely linked with the storage characteristics after harvest. The molecular markers MdACS, mdACO, mdPG1, mdEXP, mdERF and the like which are known at present are obviously related to fruit maturity hardness, and mainly relate to an ethylene synthesis pathway. Because fruit hardness is regulated by multiple genes, a single genetic locus is difficult to accurately predict phenotypic traits, and therefore more major genes and functional genetic variations still need to be mined. With the development of high throughput sequencing, a rich high quality genome was constructed. Many studies have shown that Structural Variations (SVs) contribute to a large extent to diverse phenotypic variations. Therefore, next generation high throughput sequencing strategies should be utilized to identify those genetic variations that are more closely related to fruit hardness at maturity. And (3) identifying key genetic loci and variation which are specifically related to the hardness of fruits, separating the key genetic loci from a complex ethylene synthesis path, and developing a primer pair capable of being accurately identified, so as to realize accurate genetic improvement of the characteristics. Disclosure of Invention The invention aims to provide a primer combination for identifying hardness of apple fruits, a product and application thereof, so as to solve the problems of the prior art. In order to achieve the above object, the present invention provides the following solutions: The invention provides a primer combination for identifying hardness of apple fruits, wherein the primer combination comprises a first primer pair and a second primer pair; The first primer pair comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.1 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 2; the second primer pair comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.3 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 4. The invention provides application of the primer combination in preparing a product for identifying hardness of apple fruits. Optionally, the product includes reagents, kits and chips. The invention provi