Application of SNP (Single Nucleotide Polymorphism) molecular marker associated with shiitake mushroom sterility pleat abnormal character in shiitake mushroom breeding
By applying SNP molecular markers (NG_snp) associated with sterile fold abnormal traits in shiitake mushroom breeding and early screening using AS-PCR technology, the problems of long breeding cycle and high elimination rate of shiitake mushrooms are solved, which improves breeding efficiency and reduces resource waste.
Patent Information
- Application Number
- CN202411714047.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-27
- Publication Date
- 2025-04-04
AI Technical Summary
The mushroom breeding cycle is long and the elimination rate is high, especially during the mushroom production stage, which leads to ineffective work and waste of resources, affecting breeding efficiency.
A SNP molecular marker (NG_snp) associated with sterile fold abnormal traits of shiitake mushrooms was developed, and early screening was carried out through AS-PCR technology to identify strains of sterile fold abnormal traits, thereby eliminating abnormal traits in advance and improving breeding efficiency.
Through early screening, the elimination rate in the mushroom production stage is significantly reduced, the efficiency of mushroom breeding is improved, and the consumption of manpower and material resources is reduced.
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Figure CN119753196A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of molecular genetic breeding, and particularly relates to an application of a SNP molecular marker associated with an abnormal trait of aseptic fringe of shiitake mushrooms in shiitake mushroom breeding. Background Art
[0002] Shiitake mushrooms are widely popular among consumers due to their high nutritional and medicinal value. They are the most widely cultivated and highest-yielding mushroom variety in my country.
[0003] In the breeding of edible fungi such as shiitake mushrooms, monokaryons are usually prepared by protoplast monokaryon preparation or spore monokaryon separation to obtain monokaryons, and then traditional breeding methods such as single-single hybridization or single-double hybridization are used to aggregate the ideal traits of the parents into new varieties through hybridization combinations. Generally, there are four stages: 1) The compatible monokaryon strains are paired to form new binuclear strains, and these binuclear strains are purified to form mother strains (primary strains), which is usually called the pairing stage; 2) The mother strain is inoculated into the culture medium to grow into the original seed stage of the cultivated species (secondary species); 3) The original seed is inoculated into the mushroom stick and the culture stage of the spawn is completed; 4) The mushroom stick enters the mushroom shed for the phenotypic screening stage of mushroom production. The entire breeding cycle usually takes 8-10 months, and strains are eliminated in the constructed population in the four stages, especially in the mushroom production stage, some strains are eliminated due to the formation of abnormal fruiting bodies without sterile folds.
[0004] Due to the long breeding cycle of shiitake mushrooms and the high elimination rate, especially the eliminated strains in the final fruiting stage, a lot of ineffective work has been caused, a lot of manpower, material resources and time costs have been wasted, the difficulty of shiitake mushroom breeding has been increased, and the breeding efficiency has been restricted. If the abnormal characteristics of sterile folds are located and molecular markers are developed for early screening in the mother stage, the strains with abnormal characteristics of sterile folds that can only be found in the fruiting stage can be identified, which will significantly improve the breeding efficiency of shiitake mushrooms. The method of using molecular markers to assist in directional screening of parents with specific traits and breeding offspring has been widely used in crop breeding, but this method has not been used in edible fungi at present. With the maturity of molecular marker technology and the completion of shiitake mushroom genome sequencing, the application of molecular markers for directional breeding in edible fungi such as shiitake mushrooms is inevitable. AS-PCR molecular markers are an application of high-throughput sequencing technology developed based on SNP markers, which have the advantages of high accuracy, good stability, and fast and easy typing. Summary of the invention
[0005] In order to solve the problem of early screening of abnormal sterile gills traits in shiitake mushrooms, the present invention provides an application of SNP molecular markers associated with abnormal sterile gills traits in shiitake mushrooms in shiitake mushroom breeding. The SNP molecular markers associated with abnormal sterile gills traits in shiitake mushrooms can assist in screening shiitake mushroom strains with abnormal sterile gills traits, and can be used for early screening of abnormal sterile gills traits in shiitake mushrooms, thereby improving the efficiency of shiitake mushroom breeding.
[0006] The present invention is achieved through the following technical solutions:
[0007] The present invention provides an application of a SNP molecular marker associated with an abnormal trait of sterile fringe of shiitake mushrooms in shiitake mushroom breeding, wherein the SNP molecular marker associated with an abnormal trait of sterile fringe of shiitake mushrooms is named NG_snp, and the NG_snp corresponds to the physical position 3472150 of chromosome 3 in the shiitake mushroom reference genome L808-1, and is located at the gene
[0008] In maker-chr3-snap-gene-34.175, the base here is G or C.
[0009] Based on the same inventive concept, the present invention provides an AS-PCR primer set for detecting SNP molecular markers associated with the abnormal trait of sterile gills of Lentinus edodes, wherein the AS-PCR primer set comprises a forward primer and a reverse common primer;
[0010] The forward primer includes wz_snp_f 4.1 and wz_snp_f 4.2, the nucleotide sequence of wz_snp_f 4.1 is shown in SEQ ID NO.1, and the nucleotide sequence of wz_snp_f 4.2 is shown in SEQ ID NO.2;
[0011] The reverse common primer includes wz_snp_r r4, and the nucleotide sequence of wz_snp_r r4 is shown in SEQ ID NO.3.
[0012] Based on the same inventive concept, the present invention provides an application of an AS-PCR primer set for detecting SNP molecular markers associated with the abnormal trait of sterile fringe of shiitake mushrooms in shiitake mushroom breeding.
[0013] Based on the same inventive concept, the present invention provides an application of an AS-PCR primer set for detecting SNP molecular markers associated with abnormal sterile gills traits of shiitake mushrooms in early screening of abnormal sterile gills traits of shiitake mushrooms.
[0014] Based on the same inventive concept, the present invention provides an early screening method for abnormal trait of sterile fringe of shiitake mushrooms, the early screening method comprising: early screening of abnormal trait of sterile fringe of shiitake mushrooms based on the genotype of SNP molecular marker NG_snp;
[0015] The NG_snp corresponds to the physical position 3472150 of chromosome 3 in the Lentinus edodes reference genome L808-1, located in the gene maker-chr3-snap-gene-34.175, where the base is G or C.
[0016] Furthermore, the early screening method specifically includes:
[0017] Detect the genotype of NG_snp in the genome of the tested mushroom;
[0018] Perform early screening for abnormal sterile folds of the tested shiitake mushroom based on the genotype of the NG_snp;
[0019] Among them, the GG genotype strain of the NG_snp exhibits abnormal characteristics of no sterile gills, and the GC or CC genotype strain of the NG_snp exhibits normal fruiting body characteristics with sterile gills.
[0020] Furthermore, the detection of the genotype of NG_snp in the genome of the tested shiitake mushroom specifically includes:
[0021] Divide the tested mushrooms into two groups, one of which uses wz_snp_f 4.1 and wz_snp_r r4 as primers to perform PCR amplification on the genomic DNA of the tested mushrooms, and perform electrophoresis detection on the PCR amplification products to obtain electrophoresis pattern A;
[0022] Another group uses wz_snp_f 4.2 and wz_snp_r r4 as primers to perform PCR amplification on the genomic DNA of the tested mushroom, and performs electrophoresis detection on the PCR amplification product to obtain electrophoresis pattern B;
[0023] Compare the electrophoresis pattern A and the electrophoresis pattern B to obtain the genotype of NG_snp in the genome of the tested shiitake mushroom;
[0024] Among them, the nucleotide sequence of wz_snp_f 4.1 is shown as SEQ ID NO.1, the nucleotide sequence of wz_snp_f 4.2 is shown as SEQ ID NO.2, and the nucleotide sequence of wz_snp_r r4 is shown as SEQ ID NO.3.
[0025] Furthermore, the electrophoresis pattern A and the electrophoresis pattern B are compared to obtain the genotype of NG_snp in the tested shiitake mushroom genome, specifically including:
[0026] Compare the electrophoretic pattern A and the electrophoretic pattern B:
[0027] If a band appears at the 170 bp position of the electrophoresis pattern A and no band appears at the 170 bp position of the electrophoresis pattern B, the genotype of the tested Lentinus edodes NG_snp is GG;
[0028] If bands appear at the 170 bp position in both the electrophoresis pattern A and the electrophoresis pattern B, the genotype of the tested Lentinus edodes NG_snp is GC;
[0029] If no band appears at the 170 bp position of the electrophoresis pattern A and a band appears at the 170 bp position of the electrophoresis pattern B, the genotype of the tested Lentinus edodes NG_snp is CC.
[0030] Preferably, the early screening method is carried out at any one of the pairing stage, original seed stage and culture stage of shiitake mushroom breeding.
[0031] Based on the same inventive concept, the present invention also provides a kit for early screening of abnormal sterile gills of shiitake mushrooms, the kit comprising the above-mentioned AS-PCR primer set for detecting SNP molecular markers associated with abnormal sterile gills of shiitake mushrooms.
[0032] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
[0033] 1. The present invention discloses an application of a SNP molecular marker associated with the abnormal sterile gills trait of shiitake mushrooms in shiitake mushroom breeding. The SNP molecular marker associated with the abnormal sterile gills trait of shiitake mushrooms includes NG_snp. The GG genotype strain of NG_snp exhibits the abnormal sterile gills trait, and the GC and CC genotype strains exhibit normal fruiting body traits. Therefore, the molecular marker NG_snp can be used for the early screening of the abnormal sterile gills trait of shiitake mushrooms, and the strains with the abnormal sterile gills trait that can only be found in the mushroom fruiting stage can be identified in advance in the mother strain stage, thereby significantly improving the shiitake mushroom breeding efficiency and reducing the consumption of manpower and material resources.
[0034] 2. The present invention is used to detect the AS-PCR primer set of SNP molecular markers associated with the abnormal sterile gills trait of shiitake mushrooms. The AS-PCR primer set can identify the SNP molecular marker NG_snp associated with the abnormal sterile gills trait of shiitake mushrooms through AS-PCR technology, and can be used for early screening of abnormal sterile gills traits of shiitake mushrooms or shiitake mushroom breeding, thereby effectively improving the efficiency of shiitake mushroom breeding. BRIEF DESCRIPTION OF THE DRAWINGS
[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.
[0036] Figure 1 This is a comparison chart of normal fruiting bodies of Lentinus edodes and fruiting bodies with abnormal sterile gills: A is the morphology of fruiting bodies with abnormal sterile gills at different developmental stages; B is the morphology of normal fruiting bodies at different developmental stages.
[0037] Figure 2 This is the primary screening and identification map of the AS-PCR primers based on the SNP site of maker-chr3-sanp-gene-34.175 of the present invention. DETAILED DESCRIPTION
[0038] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.
[0039] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.
[0040] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.
[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0042] The following will describe in detail the application of a SNP molecular marker associated with the abnormal trait of aseptic frills of Lentinus edodes in Lentinus edodes breeding in combination with the embodiments and experimental data.
[0043] Example 1
[0044] This example uses the AS-PCR primer set to detect the abnormal trait of sterile folds of shiitake mushrooms in the early stage of breeding, including the following steps:
[0045] 1) Extracting DNA from the mycelial stage of the strain to be tested;
[0046] 2) According to the SNP at position 3472150 on gene maker-chr3-snap-gene-34.175, AS-PCR specific primers were designed, including two forward primers wz_snp_f 4.1 and wz_snp_f 4.2, and one reverse primer wz_snp_r r4. The mycelial DNA of the mushroom strain to be tested was used as a template, and two pairs of primers were used for PCR amplification respectively;
[0047] 3) Determine the genotype of the strain to be tested based on the DNA electrophoresis results after PCR amplification and judge the mushroom phenotype.
[0048] Step 1) Specific method:
[0049] 1. Inoculate the mycelium to be tested into PDA medium, culture at 23-25℃ in the dark, and collect the mycelium after 10-15 days;
[0050] 2. CTAB method to extract genomic DNA from mycelium: ① Place freeze-dried Lentinus edodes mycelium in a 2 mL centrifuge tube and add 2-3 steel balls; ② Place the centrifuge tube in a multi-sample tissue grinder, set the frequency to 60 Hz, and grind for 30 seconds until it becomes a uniform powder; ③ Add CTAB extract (CTAB 2 g, 1 mol / L Tris-HCl 10 mL, 0.2 mol / L ETA 10 mL, 5 mol / L NaCl) preheated at 65 °C for 1 h. ④ Centrifuge at 12000r / min and 4°C for 20min, and dispense the supernatant into two tubes, 800μL each; ⑤ Add a mixture of phenol, chloroform and isoamyl alcohol in a volume ratio of 25:24:1 to the above supernatant, gently mix for 10min, centrifuge at 12000r / min and 4°C for 10min, and transfer the supernatant to a new centrifuge tube; ⑥ Add a mixture of chloroform and isoamyl alcohol in a volume ratio of 24:1 to the above centrifuge tube, and gently Mix for 10 minutes, centrifuge at 12000r / min and 4℃ for 10 minutes, take the supernatant (record the volume) and transfer it to another centrifuge tube; ⑦ Add 2 / 3 volume of -20℃ pre-cooled isopropanol relative to the supernatant in step ⑥ to the above centrifuge tube, mix, let it stand at -20℃ for 1h, then centrifuge at 8000r / min and 4℃ for 10min, and discard the supernatant; ⑧ Add 1mL of 70% ethanol to the obtained precipitate and wash it once, centrifuge at 8000r / min at room temperature for 5min; ⑨ Discard the ethanol and blow dry in the clean bench; add 100μL 10×TE buffer, tap gently to dissolve the precipitate, add 1μL 10mg / mL RNaseA and bathe at 37℃ for 1h to remove RNA; ⑩ Store the obtained DNA extract in a -20℃ refrigerator for later use.
[0051] Step 2) Specific method:
[0052] 1. The total volume of the PCR amplification reaction system is 20 μL, consisting of 2×PCR Premix Taq TM 10 μL of (1.25U / 25μL Taq DNA enzyme, 0.4mM / L dNTP, 0.3mM / L PCR buffer), 10 μmol / L AC-PCR marker forward primers wz_snp_f4.1, wz_snp_f4.2 and reverse primer wz_snp_r r4 (sequences in Table 2), 1 μL each, concentration 20-30ng / μL template DNA extracted in step 1 2 μL, ddH 2 O 6 μL;
[0053] 2. PCR reaction conditions: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 1 min, annealing at 56°C for 45 s, extension at 72°C for 1 min, 35 cycles; maintain at 72°C for 10 min, and store at 4°C.
[0054] Step 3) Specific method:
[0055] 5 μL of the product obtained by PCR amplification was spotted on agarose gel containing nucleic acid dye, the volume percentage concentration of agarose gel was 2.5%, the electrophoresis buffer was 1×TAE, the voltage was 90V for 5 hours, and the photo was taken. The bands were statistically analyzed: the genotype of the strain that could only amplify the bands with primers wz_snp_f 4.1 and wz_snp_r r4 was recessive homozygous f NG f NG (GG), the mushrooms showed NG (sterile fold) characteristics; the genotype of the strain that could amplify the bands with both the wz_snp_f 4.1, wz_snp_r r4 and wz_snp_f4.2, wz_snp_r r4 pairs of primers was dominant heterozygous Ff NG (GC), the mushroom production showed the Norm trait; the genotype of the strain that could only amplify the bands with primers wz_snp_f 4.2 and wz_snp_r r4 was dominant homozygous FF (CC), and the mushroom production showed the Norm trait (such as Figure 1 B).
[0056] Example 2
[0057] This example verifies the association between the abnormal trait of sterile fringe of Lentinus edodes and the SNP molecular marker NG_snp.
[0058] The fruiting bodies of the F2 generation of the strain "Huxiang F2" will have the abnormal trait of no gilling (NG) during the fruiting stage, which is manifested as the formation of normal primordia, and the differentiation of cap and stipe in the primordia. However, as the fruiting bodies develop, the cap cannot expand outwards. Even if there is a small amount of expansion, no gilling is formed on the inner surface of the cap. This is an abnormal trait of fruiting body formation ( Figure 1 A). Therefore, when the inventors used the shiitake mushroom strain "Huxiang F2" as a parent for breeding operations, they developed an AS-PCR molecular marker that is easy to genotype for NG appearing in the fruiting stage, which can be used for early screening of NG in the breeding population.
[0059] In the previous study, the inventors counted the percentage of strains eliminated in the fruiting stage among the 1028 offspring of the self-pollination population of the constructed "Huxiang F2" strain. The strains of the self-pollination offspring of "Huxiang F2" that could enter the fruiting stage accounted for 34.82% of the total population, and the fruiting bodies showing NG accounted for 9.50% of the number of strains entering the fruiting stage. Subsequently, NG abnormal fruiting strains and normal fruiting (Normal fruiting, Norm) control strains were randomly selected to conduct phenotypic stability fruiting tests under three cultivation modes: single-film mushroom sticks, water-retaining film mushroom sticks, and factory-based breathable bags. It was found that the Norm strain and the NG abnormal trait strain showed consistent performance under the three cultivation modes, and were genetically stable traits that were not affected by the environment. In order to further determine the genetic laws of NG, four strains with greatly different traits, namely "L808", "Zunhua 168", "Qihe 2" and "Shiitake Mushroom 60", were selected as parents, and 4 self-pollinated and 6 hybrid populations were constructed, with a total of 7350 strains. The proportion of strains eliminated due to abnormal NG traits during the fruiting stage was statistically analyzed, confirming that the abnormal NG trait was a recessive trait controlled by a single locus.
[0060] In order to locate a single site regulating the NG abnormal trait, the present invention uses a self-pollinated offspring population of the "Huxiang F2" strain and a mixed population of multiple hybrid offspring as the positioning population, and selects NG abnormal trait strains and Norm strains from the self-pollinated and multiple hybrid offspring, respectively, and locates the site to the gene maker-chr3-snap-gene-34.175 on chromosome 3 of the reference genome through DNA mixed pool sequencing, BSA positioning analysis (the reference genome is "L808-1), transcriptome differential gene analysis, and SNP marker analysis. The specific physical location and SNP site variation details are shown in Table 1. Ensure that the mutation site of the candidate gene maker-chr3-snap-gene-34.175 is co-segregated with the phenotype, and design AS-PCR typing primers at the SNP site of the deleterious mutation. The primer sequences are shown in Table 2. The SNP site of the deleterious mutation is placed at the 3' end of the forward primer. wz_snp_f 4.1 is the forward primer for the NG abnormal trait, and wz_snp_f 4.2 is the forward primer of Norm. Select a site without mutation to design a common reverse primer.
[0061] Table 1. Location of genes regulating the abnormal trait of sterile folds and SNP variation sites
[0062]
[0063] In Table 1 , C664G represents the 664th base of the CDS sequence of the maker-chr3-snap-gene-34.175 gene, where cytosine mutated to guanine.
[0064] Table 2. AS-PCR typing primers for the co-analyzed SNP sites for the abnormal trait of sterile folds
[0065]
[0066]
[0067] 1. Using four strains with relatively different traits, namely, "L808", "Zunhua 168", "Qihe 2" and "Shiitake 60", as parents, 24 samples were selected from the BF hybrid populations of the four self-pollination and six hybrid populations constructed, and the method of Example 1 was used for preliminary screening and verification (the results are shown in Figure 2 )(12 NG abnormal trait strains, green font; 12 Norm strains, red font). According to the above description, the NG abnormal trait is a recessive trait controlled by a single locus, so the genotype of the NG abnormal trait strain is a recessive homozygous f NG f NG The genotype of the Norm strain may be dominant homozygous FF or dominant heterozygous Ff NG. Use wz_snp_f 4.1 and wz_snp_r r4 as primers to detect the recessive gene f NG , wz_snp_f 4.2 and wz_snp_rr4 were used as primers to detect the dominant gene F. Figure 2 As shown in the figure, the NG trait strains tested can only amplify bands using primers wz_snp_f 4.1 and wz_snp_r r4, while primers wz_snp_f 4.2 and wz_snp_r r4 cannot amplify bands (green numbers), indicating that the NG trait strains have only homozygous f NG f NG Genotype, while strains 28 and 46 of the Norm trait were homozygous FF genotypes, and strains 4, 5, 10, 12, 25, 36, 50, and 56 were heterozygous genotypes Ff NG In addition, 11 and 49 were Norm strains with normal fruiting phenotypes, but AS-PCR detection showed that they were homozygous genotypes f NG f NG , which was considered as a false positive due to misidentification. Only two of the 24 strains were misidentified, and the accuracy of the initial screening was 91.7%, which can be used for population expansion identification applications.
[0068] 2. Population expansion verification experiment was conducted on 7 populations with mushroom photos and traceable and accurate phenotype identification from the hybrid populations of previous years. According to the fruiting period fruiting body images and phenotype evaluation data tables, 25 strains of NG and Norm trait samples were selected from each population. The expanded population verification involved 11 parents in total, and the AS-PCR molecular marker primers of the present invention were used for verification. The population information and verification accuracy are shown in Table 3. The 11 parents of the expanded population include 7 hybrid populations constructed by A, B, D, E, F, G, K, N, R, U and T. The identification accuracy of NG traits in each population ranged from 91% to 100%, with an average accuracy of 98%, and the identification accuracy of Norm traits ranged from 91.2% to 100%, with an average accuracy of 97.1%. The false positive and false negative rates were both lower than 3%, proving that the AS-PCR molecular markers of the present invention can be used for population identification of NG traits.
[0069] Table 3. AS-PCR validation results for NG traits in expanded population
[0070]
[0071]
[0072] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.
[0073] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.
[0074] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.
Claims
1. Application of a SNP molecular marker associated with the abnormal trait of sterile folds of shiitake mushrooms in shiitake mushroom breeding, characterized in that: The SNP molecular marker associated with the abnormal trait of sterile folds of Lentinus edodes is named NG_snp, and the NG_snp corresponds to the physical position 3472150 of chromosome 3 in the Lentinus edodes reference genome L808-1, located at the gene In maker-chr3-snap-gene-34.175, the base here is G or C.
2. An AS-PCR primer set for detecting SNP molecular markers associated with the abnormal trait of sterile fringe of Lentinus edodes, characterized in that: The AS-PCR primer set includes a forward primer and a reverse common primer; The forward primer includes wz_snp_f 4.1 and wz_snp_f 4.2, the nucleotide sequence of wz_snp_f 4.1 is shown in SEQ ID NO.1, and the nucleotide sequence of wz_snp_f 4.2 is shown in SEQ ID NO.2; The reverse common primer includes wz_snp_r r4, and the nucleotide sequence of wz_snp_r r4 is shown in SEQ ID NO.
3.
3. Use of the AS-PCR primer set for detecting SNP molecular markers associated with the abnormal trait of sterile gills of Lentinus edodes as claimed in claim 2 in Lentinus edodes breeding.
4. Use of the AS-PCR primer set for detecting SNP molecular markers associated with the abnormal sterile gill trait of Lentinus edodes as claimed in claim 2 in early screening of the abnormal sterile gill trait of Lentinus edodes.
5. An early screening method for abnormal characteristics of aseptic folds of shiitake mushrooms, characterized in that: The early screening method comprises: performing early screening on the abnormal trait of sterile folds of shiitake mushrooms based on the genotype of the SNP molecular marker NG_snp; The NG_snp corresponds to the physical position 3472150 of chromosome 3 in the Lentinus edodes reference genome L808-1, located in the gene maker-chr3-snap-gene-34.175, where the base is G or C.
6. The early screening method for abnormal shiitake mushroom sterile folds according to claim 5, characterized in that: The early screening method specifically includes: Detect the genotype of NG_snp in the genome of the tested mushroom; Perform early screening for abnormal sterile folds of the tested shiitake mushroom based on the genotype of the NG_snp; Among them, the GG genotype strain of the NG_snp exhibits abnormal characteristics of no sterile gills, and the GC or CC genotype strain of the NG_snp exhibits normal fruiting body characteristics with sterile gills.
7. The early screening method for abnormal shiitake mushroom sterile folds according to claim 6, characterized in that: The detecting of the genotype of NG_snp in the genome of the tested mushroom specifically comprises: Divide the tested mushrooms into two groups, one of which uses wz_snp_f 4.1 and wz_snp_r r4 as primers to perform PCR amplification on the genomic DNA of the tested mushrooms, and perform electrophoresis detection on the PCR amplification products to obtain electrophoresis pattern A; Another group uses wz_snp_f 4.2 and wz_snp_r r4 as primers to perform PCR amplification on the genomic DNA of the tested mushroom, and performs electrophoresis detection on the PCR amplification product to obtain electrophoresis pattern B; Compare the electrophoresis pattern A and the electrophoresis pattern B to obtain the genotype of NG_snp in the genome of the tested shiitake mushroom; Among them, the nucleotide sequence of wz_snp_f 4.1 is shown as SEQ ID NO.1, the nucleotide sequence of wz_snp_f 4.2 is shown as SEQ ID NO.2, and the nucleotide sequence of wz_snp_r r4 is shown as SEQ ID NO.
3.
8. The early screening method for abnormal shiitake mushroom sterile folds according to claim 7, characterized in that: The comparing the electrophoresis pattern A and the electrophoresis pattern B to obtain the genotype of NG_snp in the tested shiitake mushroom genome specifically includes: Compare the electrophoretic pattern A and the electrophoretic pattern B: If a band appears at the 170 bp position of the electrophoresis pattern A and no band appears at the 170 bp position of the electrophoresis pattern B, the genotype of the tested Lentinus edodes NG_snp is GG; If bands appear at the 170 bp position of both the electrophoresis pattern A and the electrophoresis pattern B, the genotype of the tested Lentinus edodes NG_snp is GC; If no band appears at the 170 bp position of the electrophoresis pattern A and a band appears at the 170 bp position of the electrophoresis pattern B, the genotype of the tested Lentinus edodes NG_snp is CC.
9. The early screening method for abnormal sterile fringe characteristics of shiitake mushrooms according to claims 5 to 8, characterized in that: The early screening method is carried out at any stage of the pairing stage, the original seed stage and the cultivation stage of shiitake mushroom breeding.
10. A kit for early screening of abnormal characteristics of aseptic folds of shiitake mushrooms, characterized in that: The kit comprises the AS-PCR primer set for detecting the SNP molecular marker associated with the abnormal trait of sterile gills of Lentinus edodes as described in claim 2.