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CN-122012782-A - Specific primer pair and identification method for identifying mating type of stropharia rugoso-annulata mononuclear strain

CN122012782ACN 122012782 ACN122012782 ACN 122012782ACN-122012782-A

Abstract

The application discloses a specific primer pair and a method for identifying mating type single-core strain of stropharia rugoso-annulata, belonging to the field of molecular detection. The mating type identification primer based on stropharia rugoso-annulata can be used for carrying out mating type identification on single-core strains of stropharia rugoso-annulata, the identification method is simple, economical and efficient, the detection method can be used for successfully hybridizing the assembled hybridization combinations according to the mating type identification result of the single-core strains, and the detection of the locking combination is proved to have high detection accuracy when the primer is used for identifying the mating type of stropharia rugoso-annulata, and the mating type site-specific primer combination can be used for specifically detecting A1 or A2, B1 or B2, can clearly distinguish and judge the variety of the mating type and has stronger specificity.

Inventors

  • LIU XIAOHONG
  • LIU SHIYI
  • YU HONGXIAO
  • XIE KEXIN
  • LIN FUCHENG

Assignees

  • 浙江大学

Dates

Publication Date
20260512
Application Date
20260227

Claims (9)

  1. 1. A specific primer pair for identifying mating type of stropharia rugoso-annulata mononuclear strain is characterized by comprising a primer pair for specifically amplifying A1 mating type site of stropharia rugoso-annulata, a primer pair for specifically amplifying A2 mating type site of stropharia rugoso-annulata, a primer pair for specifically amplifying B1 mating type site of stropharia rugoso-annulata and a primer pair for specifically amplifying B2 mating type site of stropharia rugoso-annulata, The primer pair for specifically amplifying the A1 mating type locus of stropharia rugoso-annulata comprises the following primers: A1-F:5’-CGGCTCTACATCAACTACTC-3’, A1-R:5’-GTTCACGTCGTCTATGCATC-3’; The primer pair for specifically amplifying the A2 mating type locus of stropharia rugoso-annulata comprises the following primers: A2-F:5’-GGCTGTCGAAAGTAATGGAG-3’, A2-R:5’-CTCTTGCCTCAAAACTTGG-3’; the primer pair for specifically amplifying the B1 mating type locus of stropharia rugoso-annulata comprises the following primers: B1-F:5’-GACTAACCTCTGCCATGATG-3’, B1-R:5’-GTTCTTTTTGGCCTCTTCCG-3’; The primer pair for specifically amplifying the B2 mating type locus of stropharia rugoso-annulata comprises the following primers: B2-F:5’-GCCTTCATCGCGAATTTGTG-3’, B2-R:5’-CACCAGAAGAGCATCATAGC-3’。
  2. 2. a product for identifying mating types of monokaryostrains of stropharia rugoso-annulata, comprising a specific primer pair according to claim 1.
  3. 3. Use of a specific primer pair according to claim 1 or a reagent or kit according to claim 2 in mating type of stropharia rugoso-annulata mononuclear strains.
  4. 4. The method for identifying mating type of stropharia rugoso-annulata mononuclear strains is characterized in that the molecular identification of the stropharia rugoso-annulata mononuclear strains is carried out by adopting the specific primer pair as claimed in claim 1 or the reagent or the kit as claimed in claim 2.
  5. 5. The authentication method according to claim 4, comprising the steps of: (1) Extracting genome DNA of a single-core strain of stropharia rugoso-annulata to be detected; (2) Respectively carrying out PCR amplification on the genome DNA by adopting the specific primer pair of claim 1 to obtain an amplification product; (3) And (3) analyzing and identifying the mating type of the stropharia rugoso-annulata mononuclear strain by amplifying mating type sites corresponding to the fragment length.
  6. 6. The method according to claim 5, wherein in the step (2), the amplification conditions are a PCR amplification system of 25. Mu. L GREEN TAQ Mix, 2. Mu.L of 10. Mu.M upstream primer, 2. Mu.L of 10. Mu.M downstream primer, 1. Mu.L of genomic DNA, and sterile ultra pure water to 50. Mu.L; PCR amplification conditions were 95℃pre-denaturation for 3 min, 95℃denaturation for 15 s,58℃annealing for 15 s,72℃extension for 45 s,33 cycles, 72℃extension for 5min, and 4℃storage.
  7. 7. The method according to claim 5, wherein in the step (3), if the primers A1 to F and A1 to R are amplified to obtain a DNA fragment having a length of 998 bp, it is judged that the single-core strain of Stropharia rugoso-annulata to be tested has an A1 mating type site; if the primers A2-F and A2-R are amplified to obtain a DNA fragment with the length of 1096 bp, judging that the single-core strain of the stropharia rugoso-annulata to be detected has an A2 mating site; If the primers B1-F and B1-R are amplified to obtain DNA fragments with the length of 868 bp, judging that the single-core strain of the stropharia rugoso-annulata to be detected has B1 mating sites; if the primers B2-F and B2-R are amplified to obtain the DNA fragment with the length of 714 bp, the stropharia rugoso-annulata mononuclear strain to be detected can be judged to have the B2 mating type site.
  8. 8. The method according to claim 5, wherein in the step (3), the mating type of the monokaryon strain of stropharia rugoso-annulata to be tested is a mating type site combination corresponding to the length of the amplified fragment of the specific primer pair.
  9. 9. Use of a specific primer pair according to claim 1, a reagent or kit according to claim 2 or an identification method according to any one of claims 4-8 in stropharia rugoso-annulata breeding and/or strain identification.

Description

Specific primer pair and identification method for identifying mating type of stropharia rugoso-annulata mononuclear strain Technical Field The invention belongs to the field of molecular detection, and particularly relates to a specific primer pair combination and an identification method for identifying mating type single-core strain of stropharia rugoso-annulata. Background Stropharia rugoso-annulata (Stropharia rugosoannulata) is an important fungus with edible, medicinal and ecological values, and has rich nutrition and unique flavor, so that the stropharia rugoso-annulata is an important economic species in the edible fungus industry. In the production of stropharia rugoso-annulata, cross breeding is a key means for cultivating new varieties. Stropharia rugoso-annulata belongs to a quadripolar foreign-mating fungus, and the mating type is controlled by two mating type factors A and B, and the factors follow the independent separation and free combination rules during meiosis, so that 4 different mating types can be generated. Therefore, obtaining stropharia rugoso-annulata monokaryons and accurately identifying mating types thereof is an important basis for developing cross breeding. At present, hybridization breeding of stropharia rugoso-annulata mainly adopts multi-round pairing hybridization on single-core strains, and whether hybridization is successful or not is judged by examining lock-shaped combination through a microscope. The stropharia rugoso-annulata mononuclear body is usually obtained by adopting methods such as protoplast mononuclear formation or basidiomycete single spore separation. However, pairing tests with monokaryostrains of unknown mating type are cumbersome, time consuming and inefficient. Along with the development of genome sequencing technology, mating type gene sequences of various edible fungi have been analyzed. Research shows that a large number of Single Nucleotide Polymorphisms (SNP) exist in mating type site areas of A and B, and specific primers can be designed based on the sites, so that the precise identification of mating type is realized. For example, the invention patent with publication number of CN120310957A discloses a molecular marker for identifying mating type of agrocybe aegerita and application thereof, and the molecular marker provided by the invention can be used for identifying mating type and typing by a molecular detection means in agrocybe aegerita breeding, so that the breeding process is shortened, and the selective breeding of excellent germplasm is quickened. The invention has the advantages of short time consumption, high accuracy, simple and convenient operation, low cost, no need of enzyme cutting and no need of fruiting compared with the conventional method for identifying the mating type of the agaricus bisporus single-spore strain. The method can rapidly identify heteronuclear or homonuclear sterile strains and mating strains thereof in the single spore strains of the agaricus bisporus W192 strain, and can greatly improve the efficiency of agaricus bisporus cross breeding. The method is the most accurate mode for the current edible fungus mating typing identification, and lays a foundation for guiding the molecular breeding of stropharia rugoso-annulata. Disclosure of Invention Based on the defects in the prior art, the invention aims to provide a specific primer pair combination for identifying mating type single-core strain of stropharia rugoso-annulata, an identification method and application. The specific technical scheme of the invention is as follows: The invention provides a specific primer pair for identifying mating type of single-core strain of stropharia rugoso-annulata, which comprises a primer pair for specifically amplifying A1 mating type site of stropharia rugoso-annulata, a primer pair for specifically amplifying A2 mating type site of stropharia rugoso-annulata, a primer pair for specifically amplifying B1 mating type site of stropharia rugoso-annulata, a primer pair for specifically amplifying B2 mating type site of stropharia rugoso-annulata, The primer pair for specifically amplifying the A1 mating type locus of stropharia rugoso-annulata comprises the following primers: A1-F:5’-CGGCTCTACATCAACTACTC-3’, A1-R:5’-GTTCACGTCGTCTATGCATC-3’; The primer pair for specifically amplifying the A2 mating type locus of stropharia rugoso-annulata comprises the following primers: A2-F:5’-GGCTGTCGAAAGTAATGGAG-3’, A2-R:5’-CTCTTGCCTCAAAACTTGG-3’; the primer pair for specifically amplifying the B1 mating type locus of stropharia rugoso-annulata comprises the following primers: B1-F:5’-GACTAACCTCTGCCATGATG-3’, B1-R:5’-GTTCTTTTTGGCCTCTTCCG-3’; The primer pair for specifically amplifying the B2 mating type locus of stropharia rugoso-annulata comprises the following primers: B2-F:5’-GCCTTCATCGCGAATTTGTG-3’, B2-R:5’-CACCAGAAGAGCATCATAGC-3’。 The invention also provides a product for identifying mating type single-core strain of stropharia ru