SNP (Single Nucleotide Polymorphism) molecular marker related to mushroom number character and application of SNP molecular marker

Abstract

The invention belongs to the technical field of genetic breeding of lentinus edodes, and particularly discloses an SNP molecular marker related to the mushroom number character of lentinus edodes and application of the SNP molecular marker. The SNP molecular marker is located on a chromosome 1 of a mushroom reference genome, a base A at the 53001166bp is mutated into G, and mushrooms with genotypes of A / G and G / G show the number of more mushrooms. The molecular marker can efficiently judge the number of shiitake mushrooms in the shiitake mushroom hypha stage, and overcomes the defect of phenotype identification in traditional breeding, so that the breeding process of a new high-yield shiitake mushroom variety is accelerated, and the breeding efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to the field of shiitake mushroom genetic breeding technology, and specifically relates to a molecular marker suitable for detecting the number phenotype of shiitake mushrooms and its application. The molecular marker can be used as a selection marker for breeding high-number shiitake mushrooms, providing a rapid screening molecular marker for cultivating new genetically stable high-number shiitake mushroom varieties. Background Art

[0002] Shiitake mushrooms are the world's most produced edible fungi (Royse et al. Current Overview of Mushroom Production in the World. In: Diego CZ, Pardo-Giménez A eds., Edible and Medicinal Mushrooms: Technology and Applications. Hoboken: Wiley, 2017.5-13.), playing an extremely important role in my country's edible fungi industry. China is also the world's largest producer, exporter, and consumer of shiitake mushrooms (Li Yuemei. The shiitake mushroom industry has good development prospects. Food Science. 2005.07:261-266.). Lentinula edodes have a unique aroma and delicious flavor, earning them the reputation of "Queen of Mushrooms" and "Mountain Treasure" (Chang. Past and present trends in the production of Lentinula edodes in Asia. Mushroom Biology and Mushroom Products. 2002. 4: 1-8.). They are also rich in protein, amino acids, multiple vitamins, and minerals, and have been shown to prevent and treat tumors, boost immunity, lower blood lipids, resist thrombosis, and strengthen the stomach and liver (Bian Yinbing. Cultivation of Edible Fungi (3rd ed.). Beijing: Higher Education Press. 2017. 138-140). They are deeply loved by people for their high nutritional and medicinal value.

[0003] Most of the important economic traits of shiitake mushrooms are quantitative traits, such as yield, number of fruiting bodies (number of mushrooms), single mushroom weight, early maturity, mycelial growth rate, and disease resistance (Xiao Yang et al. Association analysis and its application in fungal genetics research. Journal of Mycology. 2016. 35: 782-790). Unlike quality traits, quantitative traits mostly exhibit continuous variation, are regulated by multiple genes, have a complex genetic basis, and are easily affected by environmental factors. There is no clear correspondence between phenotype and genotype, making genetic improvement difficult (Santoyo et al. Quantitative linkage mapping of lignin degrading enzymatic activities in Pleurotus ostreatus. Enzyme and Microbial Technology. 2008. 43(2): 137-143.).

[0004] Lentinula edodes yield is a complex trait that can be divided into two interrelated component traits: number of fruiting bodies (NF) and weight of single fruiting body (WF). The inventor's research group used multivariate statistical methods to analyze the correlations between 10 important agronomic traits of fruiting bodies in two hybrid populations and one natural population, classifying them as three main factors: single mushroom characteristics, yield, and precocity. They found that NF and WF were negatively correlated, making them difficult to improve simultaneously. Furthermore, NF and WF showed a more pronounced positive correlation than WF (Gong WB et al. Phenotypic evaluation and analysis of important agronomic traits in the hybrid and natural populations of Lentinulaedodes. Scientia Horticulturae, 2014, 179: 271-276). Therefore, screening for high-number-of-fruiting-bodies strains and developing molecular marker-assisted breeding techniques for high-number-of-fruiting-bodies are of great significance for promoting the sustainable development of the shiitake mushroom industry.

[0005] Genome-wide association study (GWAS) was first proposed by Risch in 1996 as a forward genetics method for discovering trait regulatory sites (Risch and Merikangas. The future of genetic studies of complex human diseases. Science, 1996, 273(3): 350-354.). By identifying genetic markers associated with target phenotypes across the entire genome, GWAS has been widely used in various genetic studies, including human and plant diseases (Visscher, Brown, McCarthy, et al. Five years of GWAS discovery. The American Journal of Human Genetics, 2012, 90(1): 7-24.).

[0006] Molecular marker-assisted breeding can reduce blind selection, shorten breeding cycles, and significantly improve selection efficiency. The key to molecular marker-assisted selection is the identification of DNA markers closely linked to important agronomic traits. Compared to traditional phenotypic selection, molecular marker-assisted breeding can be used at any stage of biological growth, regardless of environmental conditions, and can eliminate interference caused by allelic interactions. It offers the advantages of being rapid, cost-effective, efficient, and accurate.

[0007] This study is based on a natural population consisting of 133 wild and cultivated shiitake mushroom strains. GWAS analysis was performed using resequencing data to initially screen out SNP sites. Combined with the mushroom number phenotype in the shiitake mushroom population, the population was divided into two extreme trait groups, and the genotypes of the two extreme difference groups at the SNP site were obtained. Functional mutation sites related to the mushroom number trait of shiitake mushrooms were screened and their effectiveness was verified in a shiitake mushroom hybrid population. Summary of the Invention

[0008] The purpose of the present invention is to provide a molecular marker LeGS1 related to the number of shiitake mushrooms. Utilizing high-throughput sequencing, 133 wild and cultivated shiitake mushroom strains were resequenced, and SNP sites were obtained by comparing with the reference genome; GWAS analysis was then performed to find SNP sites associated with the number of shiitake mushrooms, and combined with genotype comparisons of extreme populations, their SNP-Index values were calculated, site filtering was performed, and the sites were converted into fast and easy-to-use KASP (Kompetitive Allele Specific PCR) markers. The SNP site is located at 5300116bp on chromosome 1 of the shiitake mushroom reference genome, and its base is A or G. The molecular marker can be used as a molecular marker for assisted selection breeding of high-number shiitake mushroom strains, and can provide a molecular marker for cultivating new genetically stable shiitake mushroom varieties.

[0009] Another object of the present invention is to provide a method for detecting the mushroom count trait of Lentinus edodes. Utilizing the marker, strains with high and low mushroom counts can be identified at the hyphae stage, thereby eliminating non-target strains and improving selection efficiency.

[0010] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0011] A SNP molecular marker associated with the mushroom number trait of Lentinula edodes, wherein the molecular marker is located on chromosome 1 of the Lentinula edodes reference genome (Shen, Xie, Liu, et al. Near-gapless genome and transcriptome analyses provide insights into fruiting body development in Lentinula edodes. International Journal of Biological Macromolecules, 2024, 263: 130610.), and the base A mutates to G at the 5300116bp position. Lentinula edodes with genotypes A / G and G / G show a large number of mushrooms.

[0012] KASP primers were designed for the SNP molecular markers, and the sequences of the primers were shown in SEQ ID NO. 1-3.

[0013] The SNP molecular marker is used in breeding or identifying mushroom abundance in Lentinus edodes. The genotype of the SNP locus is detected to determine the number of Lentinus edodes mushrooms. Lentinus edodes with the A / A genotype are classified as having a small number of mushrooms, while those with the A / G or G / G genotypes are classified as having a large number of mushrooms. The KASP primers are used to amplify the mushrooms to be tested, and the genotype of the SNP locus is detected based on the fluorescence signal.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0015] The present invention develops a SNP molecular marker that can be used to detect the mushroom number trait of Shiitake mushrooms. Using this marker to assist in the selection of Shiitake mushroom mushroom number traits can overcome the shortcomings of traditional breeding that relies on phenotype for selection, and can reduce the breeding workload, shorten the breeding years, and accelerate the process of breeding new Shiitake mushroom varieties. This marker has the advantages of convenient and rapid detection, stable amplification, and highly visualized results. It can quickly screen out strains with high mushroom numbers for the selection of new Shiitake mushroom varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Genome-wide association analysis of genetic loci regulating mushroom number traits. In Figure A, the areas above the horizontal line indicate significant association loci from the association analysis. The red arrows indicate SNP markers identified by the present invention. Figure B shows the corresponding QQ plot from the genome-wide association analysis.

[0017] Figure 2 Molecular marker LeGS1 typing results. Blue squares represent the G / G genotype, orange circles represent the A / A genotype, and green triangles represent the A / G genotype. Figures A, B, and C show the typing results for some of the 71 strains, respectively.

[0018] Figure 3 : Box plot of mushroom number of three genotypes in the hybrid population of Lentinus edodes. The number of Lentinus edodes A / A was significantly lower than that of the other two genotypes (P<0.01).

[0019] Figure 4 :Phenotypic conditions of three genotype strains. DETAILED DESCRIPTION

[0020] Example 1: Development of molecular markers related to the number of shiitake mushrooms

[0021] Resequencing of Lentinus edodes Strains: DNA samples were extracted from the mycelia of 133 Lentinus edodes strains, including 99 wild strains and 34 cultivated strains. DNA samples were sent to Berry Genomics (Beijing, China) for library preparation and sequencing. A total of 1.5 μg of genomic DNA per sample was used to construct paired-end sequencing libraries (Illumina) with an insert size of approximately 500 bp according to the manufacturer's instructions. These libraries were sequenced on an Illumina HiSeq2500 platform with paired-end reads of 125 bp. Trimmomatic 0.33 software was used to remove adapter sequences and low-quality reads with default parameters.

[0022] Variant analysis: Based on the Lentinula edodes wpm-1 reference genome (Shen, Xie, Liu, et al. Near-gapless genome and transcriptome analyses provide insights into fruiting body development in Lentinula edodes. International Journal of Biological Macromolecules, 2024, 263:130610.), high-throughput sequencing data were analyzed using the GATK (Genome Analysis Toolkit) software to generate VCF files. To obtain high-quality variant data, the results were hard filtered using VariantFiltration. Quality control was performed using the 'QUAL<30.0||QD<13.0||MQ<20.0||FS>20.0||MQRankSum<-3.0||ReadPosRankSum<-3.0||BaseQRankSum<-3.0' command. 1,096,394 high-quality SNP loci were obtained for GWAS analysis.

[0023] Determination of mushroom number phenotypic data: 133 Lentinus edodes strains were cultivated in 2013 and 2017, and the total number of Lentinus edodes harvested in a single bag during the fruiting period was counted.

[0024] Genome-wide association analysis: GWAS analysis was performed using the linear mixed model in Fast-LMM (Lippert, Listgarten, Liu, et al. Fast linear mixed models for genome-wide association studies. Nature Methods, 2011, 8, 833-835.). The number of shiitake mushrooms in 2013 and 2017 was used as the phenotype, and loci significantly associated with the number of shiitake mushrooms were obtained ( Figure 1 ).

[0025] Eleven strains consistently ranked in the top 20 for mushroom number across both the 2013 and 2017 growth cycles were selected as a high-mushroom population. Eleven strains consistently ranked in the bottom for this trait were simultaneously selected as a low-mushroom population, forming a comparative population of extreme phenotypes (n=22). For these 22 strains, SNP loci significantly associated with mushroom number, as determined by GWAS analysis, were extracted. The allele distribution frequencies (SNP-Index) in the two populations were calculated based on the phenotypic values. A secondary SNP screening was performed, selecting sites with a ΔSNP-Index greater than 0.5 in both populations. Finally, based on the test populations, sites were selected where both parental genotypes were heterozygous, ensuring that the hybrid population contained all genotypes. The candidate locus is located at bp 5300116 on chromosome 1 of the Lentinus edodes wpm-1 reference genome, where the bases are either A or G. This SNP marker was named LeGS1.

[0026] Table 1 Genotype and phenotypic values of LeGS1 marker in extreme trait populations

[0027]

[0028] Example 2: Validation of the molecular marker LeGS1 associated with the number of Lentinus edodes mushrooms

[0029] KASP primers were designed for the obtained molecular marker LeGS1. The forward primer sequences are shown in SEQ ID NOs. 1 and 2, and the reverse universal primer sequence is shown in SEQ ID NO. 3. The forward primer shown in SEQ ID NO. 1 carries a FAM fluorescent marker, and the forward primer shown in SEQ ID NO. 2 carries a HEX fluorescent marker. The primers were synthesized by Wuhan Tianyi Huiyuan Biotechnology Co., Ltd.

[0030] A hybrid population of 71 Lentinus edodes strains was constructed using YL246 and Qihe No. 2 (L2) as parents. All alleles for the molecular marker LeGS1 were heterozygous in both parents. The hybrid population was cultivated, and the total number of Lentinus edodes harvested in a single bag during fruiting was counted.

[0031] DNA from hybrid strains was extracted using the CTAB method: an appropriate amount of mycelium (approximately 0.1 g) was placed in a ceramic mortar and pestle. After adding liquid nitrogen, the mixture was rapidly ground into a powder. 500 μL of extraction buffer (475 μL of CTAB buffer: 100 mmol / L Tris-HCl (pH 7.8), 20 mmol / L EDTA, 1.4 mol / L NaAc, 2% (w / v) CTAB; 25 μL of 5% SDS) was added, the mixture was thoroughly mixed for 30 seconds, and the mixture was incubated in a 65°C water bath for 20 minutes. 500 μL of PCI (phenol:chloroform:isoamyl alcohol = 25:24:1) was added, the mixture was thoroughly mixed for 30 seconds, and the mixture was centrifuged at 13,000 rpm for 2-4 minutes. The supernatant was transferred to a new 1.5 mL centrifuge tube, and an equal volume of isopropanol was added. The mixture was allowed to stand at room temperature for 5-10 minutes. The mixture was then centrifuged at 13,000 rpm for 2-4 minutes, and the pellet was washed twice with 70% ethanol. The alcohol was dried in a clean bench, 40 μL of ddH 2 O was added to dissolve it, and the concentration was measured using an ultra-micro UV spectrophotometer.

[0032] The DNA of the hybrid population strain was subjected to KASP labeling typing. The KASP reaction system was as follows: 250 ng DNA (4.5 μL), 2×KASP Master mixture 2 μL, 0.5 μL primer system mixture (forward primer concentration was 12 μmol / L, reverse universal primer concentration was 30 μmol / L). The reaction conditions were: 95°C pre-denaturation for 10 min; 95°C denaturation for 15 s, 61-55°C gradient annealing for 1 min, decreasing 0.6°C each cycle, 10 cycles; 95°C denaturation for 15 s, 55°C annealing and extension for 1 min, a total of 28 cycles. The reading conditions for the fluorescence signal value were 30°C for 30 s. If only the FAM fluorescence signal was detected, the LeGS1 marker genotype of the strain to be tested was A / A type; if only the HEX fluorescence signal was detected, the LeGS1 marker genotype of the strain to be tested was G / G type; if both fluorescence signals were detected, the LeGS1 marker genotype of the strain to be tested was A / G ( Figure 2 ).

[0033] The results of population genotyping and the number of mushrooms in the shiitake mushrooms were statistically analyzed (Wilcoxon test). The results showed that there were 23 strains with the A / A genotype, with an average number of mushrooms per bag of 14.64; there were 39 strains with the A / G genotype, with an average number of mushrooms per bag of 25.8; and there were 9 strains with the G / G genotype, with an average number of mushrooms per bag of 29.26. For individuals with the A / A genotype, the number of mushrooms in the shiitake mushrooms was significantly lower than that of individuals with the A / G and G / G types (P<0.01), indicating that the LeGS1 marker is significantly correlated with the number of mushrooms in the shiitake mushrooms, and when the marker mutates to G, the shiitake mushrooms have a higher number of mushrooms ( Figure 3 and Figure 4 ).

[0034] The above identification results demonstrate that the molecular marker LeGS1 and detection method provided by the present invention can accurately and efficiently determine the number of mushrooms in Lentinus edodes at the mycelial stage, effectively reducing the cultivation scale of breeding materials, alleviating the workload of later field phenotypic identification, accelerating the breeding process, and improving breeding efficiency. Therefore, the LeGS1 molecular marker described in the present invention can effectively identify the number of mushrooms in Lentinus edodes and can be used for molecular marker-assisted breeding of high-number-of-mushroom strains.

Claims

1. A SNP molecular marker associated with the number of shiitake mushrooms, characterized by: The molecular marker is located on chromosome 1 of the Lentinus edodes reference genome, and the base A mutates to G at 5300116 bp. Lentinus edodes with genotypes of A / G and G / G show multiple mushrooms.

2. The KASP primer for amplifying the SNP molecular marker according to claim 1, characterized in that: The primer sequences are shown in SEQ ID NO. 1-3.

3. A kit for detecting the number of shiitake mushrooms, characterized in that: The kit contains a reagent for detecting the 5300116 bp base of chromosome 1 of the Lentinus edodes reference genome.

4. The kit according to claim 3, wherein The kit includes two forward primers and one reverse universal primer. The forward primer sequences are shown in SEQ ID NOs. 1 and 2, and the reverse universal primer sequence is shown in SEQ ID NO.

3.

5. Use of the SNP molecular marker according to claim 1 in breeding of high-number mushrooms of Lentinus edodes.

6. Use of the SNP molecular marker according to claim 1 in the identification of mushroom number traits of Lentinus edodes.

7. Use of the KASP primer according to claim 2 in breeding of high mushroom numbers of Lentinus edodes.

8. Use of the KASP primers according to claim 2 in identifying the number of mushrooms in Lentinus edodes.

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