Detection primer group of stropharia rugosoannulata cfcc5619 strain and application thereof

Abstract

This invention discloses a primer set for detecting *Agaricus bisporus* strain CFCC5619 and its application, relating to the field of molecular detection technology. Using the primer set shown in SEQ ID NO.1-SEQ ID NO.22 of this invention, PCR amplification of *Agaricus bisporus* strain CFCC5619 was performed, yielding a banding combination of 21121111121. This method is highly specific for identifying *Agaricus bisporus* strain CFCC5619, requiring only 3-4 days for detection. Compared with existing conventional morphological detection, antagonism tests, and fruiting tests, it has the advantages of short detection time, high accuracy, and good repeatability, showing promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology, and more specifically to a detection primer set for the *Agaricus macrocarpa* strain CFCC5619 and its application. Background Technology

[0002] Giant king mushroom ( Stropharia rugosoannulata Farl. ex Murrill Also known as the wrinkled cap mushroom or wine-red cap mushroom, it is a highly sought-after emerging rare edible mushroom variety in recent years due to its thick, tender flesh, sweet and delicious taste, and rich content of protein, dietary fiber, polysaccharides, and various vitamins and minerals. It has both high nutritional value and certain health benefits.

[0003] However, with the rapid expansion of the *Agaricus bisporus* industry, problems at the strain level have gradually become prominent, among which the phenomenon of "synonyms for the same species and names for different species" is particularly prominent. On the one hand, some breeding units or strain distributors, in pursuit of market profits, give different names to the same *Agaricus bisporus* strain and promote it repeatedly, making it difficult for producers to distinguish between genuine and counterfeit strains. On the other hand, strains from different sources have little difference in morphological characteristics, and traditional identification methods based on fruiting body morphology and culture characteristics are difficult to accurately distinguish. Some inferior or mixed strains enter the market, which not only leads to large fluctuations in the yield and inconsistent quality of *Agaricus bisporus*, damaging the economic interests of producers, but also infringes on the intellectual property rights of breeders of high-quality *Agaricus bisporus* strains, seriously disrupting market order.

[0004] Establishing a simple, rapid, and accurate method for identifying the specific characteristics of *Agaricus bisporus* strains has become an urgent need for the healthy, stable, and sustainable development of the *Agaricus bisporus* industry. Summary of the Invention

[0005] In view of this, the present invention provides a detection primer set for the Pleurotus ostreatus strain CFCC5619 and its application. Compared with existing morphological detection, antagonism test and fruiting test, the fingerprint spectrum constructed using this detection primer set has the advantages of short detection time, high accuracy and good repeatability.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A primer set for detecting a strain of *Agaricus macrocarpa* CFCC5619, comprising 11 primer pairs including SEQ ID NO.1-SEQ ID NO.22.

[0008] Another object of the present invention is to provide a detection kit for the *Agaricus macrocarpa* strain CFCC5619, the detection kit comprising the above-mentioned detection primer set.

[0009] Another objective of this invention is to provide an InDel-labeled fingerprint of the *Agaricus bisporus* strain CFCC5619. PCR amplification of the *Agaricus bisporus* strain CFCC5619 using the aforementioned primer set or kit yielded fragments of sizes 125bp, 112bp, 198bp, 131bp, 211bp, 172bp, 162bp, 223bp, 176bp, 176bp, and 167bp, respectively, with the resulting banding combination being 21121111121.

[0010] Another object of the present invention is to provide the application of the above-mentioned detection primer set, the above-mentioned detection kit, or the above-mentioned fingerprint spectrum in the identification of Pleurotus ostreatus CFCC5619 strain.

[0011] Another object of the present invention is to provide a method for identifying the germplasm of the *Agaricus bisporus* strain CFCC5619, comprising the following steps:

[0012] S1: PCR amplification of the genome of the target *Agaricus bisporus* was performed using the above-mentioned detection primer set or the above-mentioned detection kit.

[0013] S2: Perform electrophoresis on the PCR amplification product and compare the result with the fingerprint pattern above. If they match, it is the *Agaricus bisporus* strain CFCC5619.

[0014] Beneficial effects: The InDel-labeled fingerprint spectrum of the *Agaricus bisporus* strain CFCC5619 of this invention can be used for the identification of *Agaricus bisporus* strain CFCC5619. It has high specificity and the detection time is only 3-4 days. Compared with the existing conventional morphological detection, antagonism test (which takes at least two weeks), and fruiting test (which takes at least 3 months), it has the advantages of short detection time, high accuracy, and good repeatability, and has good application prospects. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0016] Figure 1 This is the InDel-marked fingerprint of the *Agaricus bisporus* strain CFCC5619 of the present invention, where M is a D2000 bp DNA Marker, numbers 1-11 represent the 11 pairs of InDel marker primers used, and the arrows indicate the specific InDel allelic fragment combinations of *Agaricus bisporus* strain CFCC5619.

[0017] Figure 2Allelic fragments obtained by amplifying InDel-labeled primers (pairs 1-11) were used as controls.

[0018] Figure 3 The amplification patterns of primers SEQ ID NO.1 and SEQ ID NO.2 in eight *Agaricus bisporus* materials are shown. The numbers 1-8 represent strains Xingnong No.1, Xingnong-6, *Agaricus bisporus*-1, Q1, CFCC 5619, CICC14086, D17, and P122, respectively.

[0019] Figure 4 The image shows the amplification patterns of primers SEQ ID NO.3 and SEQ ID NO.4 in eight selected *Agaricus bisporus* materials, where the numbers correspond to the strains and... Figure 3 same;

[0020] Figure 5 The amplification patterns of primers SEQ ID NO.5 and SEQ ID NO.6 in eight selected *Agaricus bisporus* materials are shown, where the numbers correspond to the strains and... Figure 3 same;

[0021] Figure 6 The image shows the amplification patterns of primers SEQ ID NO.7 and SEQ ID NO.8 in eight selected *Agaricus bisporus* materials, where the numbers correspond to the strains and... Figure 3 same;

[0022] Figure 7 The amplification patterns of primers SEQ ID NO. 9 and SEQ ID NO. 10 in the selected eight *Agaricus bisporus* materials are shown, where the numbers correspond to the strains and... Figure 3 same;

[0023] Figure 8 The image shows the amplification patterns of primers SEQ ID NO.11 and SEQ ID NO.12 in eight selected *Agaricus bisporus* materials, where the numbers correspond to the strains and... Figure 3 same;

[0024] Figure 9 The amplification patterns of primers SEQ ID NO.13 and SEQ ID NO.14 in the selected eight *Agaricus bisporus* materials are shown, where the numbers correspond to the strains and... Figure 3 same;

[0025] Figure 10 The amplification patterns of primers SEQ ID NO.15 and SEQ ID NO.16 in the selected eight *Agaricus bisporus* materials are shown, where the numbers correspond to the strains and... Figure 3 same;

[0026] Figure 11The amplification patterns of primers SEQ ID NO.17 and SEQ ID NO.18 in eight selected *Agaricus bisporus* materials are shown, where the numbers correspond to the strains and... Figure 3 same;

[0027] Figure 12 The amplification patterns of primers SEQ ID NO.19 and SEQ ID NO.20 in the selected eight *Agaricus bisporus* materials are shown, where the numbers correspond to the strains and... Figure 3 same;

[0028] Figure 13 The amplification patterns of primers SEQ ID NO.21 and SEQ ID NO.22 in the selected eight *Agaricus bisporus* materials are shown, where the numbers correspond to the strains and... Figure 3 same. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0030] The strains used in this invention are sourced from: Xingnong No. 1 strain, from Henan Shangqiu Jinlong Mushroom Industry Co., Ltd.; Xingnong-6 strain, from Henan Shangqiu Jinlong Mushroom Industry Co., Ltd.; Giant King Mushroom-1 strain, from Jiangdu Tianda Edible Fungus Research Institute; Q1 strain, from Ningxia Academy of Agricultural and Forestry Sciences; CFCC 5619 strain, from China Forestry Microbial Culture Collection Center; CICC14086 strain, from China Industrial Microbial Culture Collection Center; D17 strain, from Guizhou Academy of Agricultural Sciences; and P122 strain, from Guizhou Academy of Agricultural Sciences.

[0031] 1. Based on the whole-genome resequencing data of the above strains, this invention screened 11 InDel markers and constructed an InDel marker fingerprint of *Agaricus macrocarpa* CFCC 5619. InDel marker information and detection primers are shown in Table 1. The reference genome for designing the InDel primers is GenBank ID GCA_028532985.1 (link: https: / / ftp.ncbi.nlm.nih.gov / genomes / all / GCA / 028 / 532 / 985 / GCA_028532985.1_ASM2853298v1 / GCA_028532985.1_ASM2853298v1_genomic.fna.gz).

[0032] Table 1

[0033]

[0034] Continued from table:

[0035]

[0036] Table 1 lists the allelic fragments amplified by each primer set, with the larger fragment defined as 1 and the smaller fragment as 2. The *Agaricus bisporus* CFCC 5619 strain was amplified using InDel1-11, and the resulting fragment sizes were 125bp, 112bp, 198bp, 131bp, 211bp, 172bp, 162bp, 223bp, 176bp, 176bp, and 167bp, respectively. The fragment numbering combination is 21121111121. 2.

[0038] Mycelial culture: The mycelia of *Agaricus macrocarpa* (Xingnong No. 1 strain, Xingnong-6 strain, *Agaricus macrocarpa*-1 strain, Q1 strain, CFCC 5619 strain, CICC14086 strain, D17 strain, P122 strain) were transferred into potato dextrose agar (PDA) and cultured in the dark at 22-26℃. The mycelia were collected after 10-14 days.

[0039] Genomic DNA extraction: Genomic DNA was extracted from the hyphae using a DNA extraction kit from Jiangsu Kangwei Century Biotechnology Co., Ltd. The concentration and purity of total genomic DNA were detected by ultraviolet spectrophotometry, and the concentration of sample DNA was adjusted to 20-30 ng / uL.

[0040] Detection of InDel molecular markers: The extracted DNA was subjected to whole-genome InDel marker PCR amplification using the detection primers in Table 1;

[0041] The PCR amplification system consisted of a total volume of 20 μL, including: 1 μL of sample DNA template, 1 μL each of InDel forward and reverse primers, 8.5 μL of 2×San Taq PCR Mix, and 8.5 μL of ddH2O.

[0042] PCR reaction conditions: The first stage was pre-denaturation at 94℃ for 5 min, the second stage was denaturation at 94℃ for 40 s, annealing at 47-56℃ for 45 s, extension at 72℃ for 1 min, for a total of 36 cycles, and the third stage was final extension at 72℃ for 7 min; stored at 4℃.

[0043] Electrophoresis detection: 3 μL of the PCR amplification product was loaded onto an agarose gel containing nucleic acid dye for electrophoresis. The agarose gel concentration was 3% (v / v), the electrophoresis buffer was 1×TAE, the voltage was 130 V, the current was 280 mA, the power was 100 W, and the electrophoresis time was 2 h. Images were then taken and the results analyzed (see appendix). Figure 1 -Appendix Figure 13 );

[0044] Eleven pairs of InDel-labeled primers were used for PCR amplification of the test strain. The number and relative molecular weight of allelic fragments amplified by each InDel-labeled primer were determined by using the DNA molecular weight control D2000 bp DNA ladder. The strain that matched the number combination 21121111121 was identified as Pleurotus ostreatus CFCC5619.

[0045] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0046] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A primer set for detecting *Agaricus macrocarpa* strain CFCC5619, characterized in that, The primer set consists of 11 primer pairs, including SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, and SEQ ID NO.21 and SEQ ID NO.

22.

2. A detection kit for *Agaricus macrocarpa* strain CFCC5619, characterized in that, The detection kit includes the detection primer set as described in claim 1.

3. The application of the detection primer set of claim 1 and the detection kit of claim 2 in the identification of Pleurotus ostreatus CFCC5619 strain.

4. A method for identifying the germplasm of *Agaricus macrocarpa* strain CFCC5619, characterized in that, Includes the following steps: S1: Perform PCR amplification of the genome of the mushroom to be tested using the detection primer set described in claim 1 or the detection kit described in claim 2; S2: Perform electrophoresis on the PCR amplification products. If the resulting fragment sizes are 125bp, 112bp, 198bp, 131bp, 211bp, 172bp, 162bp, 223bp, 176bp, 176bp, and 167bp respectively, then the tested *Stropharia macrocarpa* is *Stropharia macrocarpa* strain CFCC5619.

Images