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CN-122012778-A - Construction of identification primer and fingerprint of panus giganteus strain InDel and liquid strain of panus giganteus strain InDel

CN122012778ACN 122012778 ACN122012778 ACN 122012778ACN-122012778-A

Abstract

The invention discloses a pig tripe mushroom strain InDel identification primer, construction of a fingerprint and a liquid strain thereof, belonging to the field of molecular markers and edible fungus detection. The invention provides an InDel marked primer combination (SEQ ID NO: 1-14) consisting of 7 pairs of primers, and a method for constructing a DNA fingerprint by using the primer combination and rapidly identifying the mushroom strains of the Gastrodia elata Blume (P. Yoshi) No. 1 and the mushroom strain of the P. Yoshi (P. Yoshi) No. 2. Meanwhile, the invention also provides a special culture medium for the liquid strain suitable for the strain and an optimized culture method thereof. The technology combines molecular identification with liquid strain production, forms a complete technical system from strain identity verification to high-quality strain preparation, has the advantages of accurate identification, high efficiency and strong repeatability, and provides an effective technical means for the protection of the variety rights of the Gastrodia elata, the quality control of the strain and industrial production.

Inventors

  • YU HAILONG
  • LI QIAOZHEN
  • ZHANG MEIYAN
  • Luan Tengye
  • JIANG NING
  • TANG GUIRONG
  • SONG CHUNYAN
  • SHANG XIAODONG

Assignees

  • 上海市农业科学院

Dates

Publication Date
20260512
Application Date
20260209
Priority Date
20251203

Claims (10)

  1. 1. An InDel-labeled primer combination for identifying a strain of panus giganteus 1 and 2, characterized in that the primer combination consists of 7 pairs of InDel-labeled primer pairs, the nucleotide sequences of which are as follows: Lefp _id001 forward primer is shown as SEQ ID NO.1, and reverse primer is shown as SEQ ID NO. 2; lefp _id002 forward primer is shown as SEQ ID NO. 3, and reverse primer is shown as SEQ ID NO. 4; Lefp _id003 forward primer is shown as SEQ ID NO. 5, and reverse primer is shown as SEQ ID NO. 6; lefp _id004 forward primer is shown as SEQ ID NO. 7, and reverse primer is shown as SEQ ID NO. 8; Lefp _id005 forward primer is shown as SEQ ID NO. 9, and reverse primer is shown as SEQ ID NO. 10; Lefp _id006 forward primer is shown as SEQ ID NO. 11, and reverse primer is shown as SEQ ID NO. 12; lefp _id007 the forward primer is shown as SEQ ID NO. 13, and the reverse primer is shown as SEQ ID NO. 14.
  2. 2. A kit for identifying a strain of clitocybe maxima, clitocybe maxima No. 1 and clitocybe maxima No. 2, comprising the 7 pairs of InDel marker primer pairs of claim 1.
  3. 3. The method for constructing the InDel marker fingerprint of the panus giganteus strain is characterized by comprising the following steps of: (1) Extracting genome DNA of a to-be-detected pig tripe mushroom strain; (2) Amplifying the DNA to be tested by a PCR reaction using the InDel labeled primer combination of claim 1; (3) And (3) carrying out electrophoresis detection and analysis on the PCR amplification product, thereby obtaining a DNA fingerprint consisting of 7 InDel marked allelic fragments.
  4. 4. A standard InDel marker fingerprint of a mushroom 1 strain of a panus giganteus is characterized in that the fingerprint consists of 7-site allelic fragment bands obtained by amplification by using the primer combination of claim 1, wherein the band combinations are (1+2), (1+2), 2, (1+2), 2, 1 and (1+2), and the fragment lengths corresponding to the allelic fragment numbers are respectively: lefp _id001:1 fragment 234 bp,2 fragment 217 bp; Lefp _id002 the fragment No.1 239 bp, the fragment No.2 212 bp; Lefp _id003:1 fragment 285 bp,2 fragment 264 bp; lefp _id004 fragment 154 bp, fragment 127 bp; lefp _id005 fragment 134 bp, fragment 109 bp; Lefp _id006:180 bp of fragment 1, 161: 161 bp of fragment 2; Lefp _id007 fragment 208 bp, fragment 191 bp.
  5. 5. A standard InDel-marked fingerprint of a strain of clitocybe maxima, clitocybe maxima No. 2, characterized in that the fingerprint consists of 7-locus allelic fragment patterns obtained by amplification using the primer combination of claim 1, the pattern combinations being (1+2), 2,2, 1, 1, 2, (1+2), the allelic fragment numbers being the same as in claim 4.
  6. 6. A method for identifying a fungus strain of a panus giganteus 1 or 2, which is characterized in that, (1) Constructing a fingerprint of a to-be-detected pig tripe mushroom strain by using the method of claim 3; (2) Comparing the fingerprint obtained in the step (1) with the fingerprint of claim 4 or 5; If the fingerprint of the strain to be detected is consistent with the fingerprint of claim 4, the strain is identified as the mushroom 1 strain of the panus giganteus, and if the fingerprint of the strain to be detected is consistent with the fingerprint of claim 5, the strain is identified as the mushroom 2 strain of the panus giganteus.
  7. 7. A special culture medium for the fermentation of liquid spawn of the panus giganteus 1 or 2, which is characterized in that the culture medium comprises the following components in concentration: 15-20 g/L of sucrose, 5-10 g/L of glucose, 20-30 g/L of poplar wood chip leaching solution, 4-7 g/L of yeast extract and oat leaching solution 5 ~ 10 g/L,MgSO 4 1.0 ~ 2.0 g/L,KH 2 PO 4 3.0 ~ 5.0 g/L,MnSO 4 0.01~ 0.02g/L.
  8. 8. A method for culturing liquid strain of Hypsizygus marmoreus (Armillariella) No.1 or Armillariella 2 is characterized by comprising the following steps: bacterial strain activation, namely inoculating the bacterial strain to a PDA plate, and culturing until hyphae grow on the plate; Seed liquid preparation, namely inoculating the activated mycelium blocks into a triangular flask filled with the liquid culture medium of claim 7, and culturing in a shaking table to prepare primary seed liquid; fermenting in a fermentation tank, namely inoculating the first-stage seed liquid into the fermentation tank according to the inoculation amount of 0.05v/v% -1 v/v%, and performing fermentation culture, wherein the fermentation medium is the liquid culture medium; the first-stage culture conditions are as follows: Bottling volume is 250-300 mL/500 mL triangular bottles; Initial pH is natural; the culture temperature is 24-26 ℃; the rotating speed of the shaking table is 100-120 r/min; the culture time is 5-6 days; the key culture parameters of the liquid fermentation tank are as follows: tank pressure 0.03 MPa~0.05 MPa; The ventilation ratio is 0.2-0.3 vvm; Culturing at 24-26 ℃; the culture time is 5-6 days.
  9. 9. The molecular identity verified liquid spawn product of the panus giganteus is characterized by comprising a liquid spawn of the panus giganteus 1 or 2, which is produced and verified by a method comprising the following steps: (a) Culturing using the special culture medium of claim 7 and the culture method of claim 8; (b) Performing PCR amplification and fingerprint construction on the obtained mycelium by using the InDel marked primer combination of claim 1; (c) When the constructed fingerprint is completely consistent with the fingerprint of claim 4 or 5, the liquid strain is confirmed to be the corresponding real strain.
  10. 10. Use of the InDel-labeled primer combination of claim 1 and/or the kit of claim 3 and/or the fingerprint of claim 4 or 5 and/or the special culture medium of claim 7 and/or the culture method of claim 8 and/or the liquid spawn product of claim 9 in the identification of the authenticity of a porcine tripe-mushroom spawn, the protection of the spawn, or the breeding process.

Description

Construction of identification primer and fingerprint of panus giganteus strain InDel and liquid strain of panus giganteus strain InDel Technical Field The invention belongs to the field of molecular markers and edible fungi detection, and particularly relates to a pig tripe mushroom strain InDel identification primer, a fingerprint construction and a liquid strain thereof. Background The disclosure of this background section is only intended to increase some understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art. The Gastrodia elata (Pleurotus giganteus) belongs to basidiomycetes (Basidiomycota) and Pleurotus (Pleurotus) and is a large fungus with high nutrition and medicinal value. The fruit body is rich in protein, polysaccharide, trace elements and various bioactive substances, and is favored by the market. In recent years, with the rapid development of industrial and large-scale production of domestic edible mushrooms, the cultivation area of the pig tripe mushrooms is continuously enlarged. However, not matching with the development of the industry, the germplasm resource identification, evaluation and new variety breeding work of the pig tripe mushroom are relatively lagged. At present, identification and differentiation of the pig tripe mushroom strains in production and scientific research still mainly depend on traditional morphological observation, mycelium antagonism test and fruiting cultivation test. These methods have inherent drawbacks that are difficult to overcome: the period is long, the antagonism test takes 1-2 weeks, and the fruiting test takes 3 months from inoculation to completion of biological characteristic identification, thus seriously delaying the breeding and production processes. The accuracy is poor, morphological characteristics are easily affected by environmental conditions, subjectivity is strong, and antagonism reaction may not be obvious among some near-edge strains, so that misjudgment or missed judgment is caused. The efficiency is low, and quick screening of a large number of samples cannot be realized. Particularly, with the popularization of the edible fungus liquid fermentation technology, liquid strains become the first choice for industrial production due to the advantages of consistent fungus age, short fermentation period, high inoculation efficiency, suitability for automatic control and the like. However, liquid species appear as suspended mycelium pellets or mycelium fragments, completely losing the macroscopic morphological features of fruiting bodies and solid mycelium, which renders all of the above conventional identification methods completely ineffective in the presence of this morphology of liquid species. This results in the risk of strain confusion, degradation, and even malicious counterfeiting occurring very easily in the production, distribution, and use links of liquid strains. Once a problem occurs, not only is huge economic loss brought to a production enterprise, but also market order is seriously disturbed, and tracing and maintaining rights are extremely difficult due to the lack of a rapid and effective identification means. Therefore, the technical scheme capable of breaking through morphological limitation and directly identifying the specific form of the pig tripe mushroom strain, especially the liquid strain, rapidly and accurately is urgently needed in industry. The development of molecular marking technology provides an ideal solution to the above problems. The insertion/deletion (InDel) marker is used as a molecular marker based on genome sequencing, and has the advantages of abundant quantity, co-dominance, high accuracy, good stability, simplicity and convenience in operation and the like. It is particularly useful for developing identification markers for specific species at the whole genome level. However, up to now, in the field of pig tripe mushroom research, there has not been disclosed report on InDel marker fingerprint patterns and identification methods thereof, which are developed based on the whole genome level and can be used for specifically identifying important cultivated strains (such as Shenshi mushroom No. 1 and Shenshi mushroom No. 2). More importantly, the prior art is completely lack of a complete quality control system capable of combining the production and preparation of liquid spawn with the molecular level identity authentication thereof, and the purity and the authenticity of the liquid spawn product cannot be ensured from the source, which becomes a key bottleneck for restricting the healthy, stable and sustainable development of the pig tripe mushroom industry. Disclosure of Invention Aiming at the defects of the prior art, in particular the problems that the traditional method can not identify liquid strains and lacks a specif