SNP (Single Nucleotide Polymorphism) molecular marker related to resistance character of mushroom trichoderma atroviride and application of SNP molecular marker

Abstract

The invention belongs to the technical field of fungus genetic breeding, and particularly discloses an SNP molecular marker related to resistance characters of mushroom trichoderma atroviride and application of the SNP molecular marker. The SNP molecular marker is located on a chromosome 5 of a mushroom reference genome, a base C at the 479587 bp is mutated into T, and the mushroom with the genotype of T / T shows high trichoderma atroviride resistance. The molecular marker can efficiently judge the trichoderma resistance in the mushroom hypha stage, and overcomes the defect of phenotype identification in traditional breeding, so that the breeding process of a new trichoderma-resistant mushroom variety is accelerated, and the breeding efficiency is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of shiitake mushroom genetic breeding, and specifically relates to a molecular marker suitable for detecting the dark green trichoderma resistance phenotype of shiitake mushroom and its application. The molecular marker can be used as a selection marker for dark green trichoderma resistance breeding of shiitake mushroom, providing a rapid screening molecular marker for cultivating genetically stable new varieties of shiitake mushroom dark green trichoderma resistance. Background Art

[0002] Lentinula edodes is the most productive edible mushroom in the world (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.). Lentinula edodes has a unique aroma and delicious taste, and is known as the "Queen of Mushrooms" and "Mountain Treasures" (Chang. Past and present trends in the production of Lentinula edodes in Asia. Mushroom Biology and Mushroom Products. 2002. 4: 1-8.). Lentinula edodes is rich in protein, amino acids, multiple vitamins and mineral elements, and has the effects of preventing and treating tumors, increasing immunity, lowering blood lipids, resisting thrombosis, and strengthening the stomach and protecting the liver (Bian Yinbing. Edible Fungus Cultivation (3rd Edition). Beijing: Higher Education Press. 2017. 138-140). It is deeply loved by people because of its high nutritional value 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, disease resistance, etc. (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 show 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 it difficult to carry out genetic improvement (Santoyo et al. Quantitative linkage mapping of lignin degrading enzymatic activities in Pleurotus ostreatus. Enzyme and Microbial Technology. 2008. 43 (2): 137-143.).

[0004] Trichoderma fungi belong to soil fungi, which are commonly found in soil and rotten wood. They can produce a series of enzymes to degrade polysaccharides, cell walls, etc., and secrete secondary metabolites to inhibit host growth. Trichoderma is the main source of infection for shiitake mushroom stick rot. The fungus can cause both competitive diseases and infectious diseases, causing serious declines in the quality and yield of shiitake mushrooms. At present, there are reports of Trichoderma diseases in edible fungi such as shiitake mushrooms, oyster mushrooms, Agaricus bisporus, Ganoderma lucidum, and black fungus. So far, more than 40 species of Trichoderma can cause green mold disease in edible fungi, the most common of which are mainly dark green Trichoderma (T.atroviride), long-branch Trichoderma (T.longibrachiatum), green Trichoderma (T.viride), Koning Trichoderma (T.koningii), pseudokoning Trichoderma (T.pseudokoningii) and Harzianum Trichoderma (T.harzianum). Because Trichoderma fungi have many of the same characteristics as the mycelial morphology and growth and development of shiitake mushrooms, they are difficult to distinguish and extremely difficult to prevent and control. Therefore, screening shiitake mushroom strains with strong disease resistance, studying the molecular genetic mechanism of shiitake mushroom resistance to Trichoderma, and exploring key regulatory genetic loci and genes are of great significance for developing resistance molecular marker-assisted breeding and 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 genetic 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 genome, GWAS has been widely used in various genetic studies, including human diseases 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 the blindness of selection, shorten the breeding period, and greatly improve the selection efficiency. The key to molecular marker-assisted selection breeding technology is the identification of DNA molecular markers that are closely linked to important agronomic traits. Compared with traditional phenotypic selection, molecular marker-assisted breeding can be carried out at any stage of biological growth, is not affected by environmental conditions, and can eliminate interference caused by allele interactions. It has the advantages of being fast, economical, efficient, and accurate.

[0007] The present invention is based on a natural population consisting of 133 wild and cultivated shiitake mushroom strains, and uses resequencing data to perform GWAS analysis to initially screen out SNP sites. Combined with the resistance phenotype of dark green Trichoderma in the shiitake mushroom population, the population is divided into two extreme trait groups, and the genotypes of the two extreme difference groups at the SNP site are obtained. Functional mutation sites related to shiitake mushroom resistance to dark green Trichoderma are screened, and the effectiveness is verified in a shiitake hybrid population. Summary of the invention

[0008] The purpose of the present invention is to provide a molecular marker LeKX1 related to dark green Trichoderma resistance of shiitake mushrooms. Using high-throughput sequencing, 133 wild and cultivated shiitake mushroom strains were resequenced, and SNP sites were obtained by comparing the reference genome; then GWAS analysis was performed to find SNP sites associated with dark green Trichoderma resistance, and combined with the genotype comparison of extreme groups, the SNP-Index value was calculated, the site was filtered, and it was converted into a fast and easy-to-use KASP (Kompetitive AlleleSpecific PCR) marker. The SNP site is located at the 479587bp of chromosome 5 of the shiitake mushroom reference genome, and its base is C or T. The site is located in the exon region of the histidine phosphotransfer domain protein encoding gene. The molecular marker can be used as a molecular marker for auxiliary selection breeding of dark green Trichoderma resistant 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 dark green Trichoderma resistance trait of Lentinus edodes. The dark green Trichoderma resistance and sensitive strains can be identified at the hyphae stage using the marker, thereby eliminating non-target strains and improving the selection efficiency.

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

[0011] The SNP molecular marker associated with dark green Trichoderma resistance of Lentinula edodes, the molecular marker is located on chromosome 5 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 C mutates to T at the 479587bp, and the Lentinula edodes with the genotype T / T show high resistance to dark green Trichoderma.

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

[0013] The application of the SNP molecular marker in the breeding of dark green Trichoderma resistance of shiitake mushrooms or the identification of dark green Trichoderma resistance traits: the dark green Trichoderma resistance of shiitake mushrooms is determined by detecting the genotype of the SNP site, the shiitake mushrooms with the T / T genotype are the dark green Trichoderma resistance type, and the shiitake mushrooms with the C / C or C / T genotype are the dark green Trichoderma sensitive type. The shiitake mushrooms to be tested are amplified by the KASP primers, and the genotype of the SNP site is detected according to the fluorescence signal.

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

[0015] The present invention successfully obtains a SNP molecular marker for detecting the dark green Trichoderma resistance trait of shiitake mushrooms. The use of this marker for auxiliary selection of shiitake mushrooms with Trichoderma resistance 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. The marker has the advantages of convenient and rapid detection, stable amplification, and highly visualized results. It can quickly screen strains with dark green Trichoderma resistance and is used for the breeding of new shiitake mushroom varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Comparison of the dark green Trichoderma infection area and the total area of ​​shiitake mushroom hyphae before and after identification. A is the original image of shiitake mushrooms after being infected by dark green Trichoderma; B is the identification area of ​​the dark green Trichoderma infection area; C is the identification area of ​​the total area of ​​shiitake mushrooms.

[0017] Figure 2 : Genetic loci regulating dark green Trichoderma resistance traits by genome-wide association analysis. The horizontal line is the significant association loci detected by association analysis. The SNP molecular marker of the present invention is located on chromosome 5 of the reference genome of Lentinus edodes.

[0018] Figure 3 : The results of molecular marker LeKX1 typing. A is the typing results of 10 randomly selected T / T wild populations (three replicates for each sample), B is the typing results of the hybrid population, blue squares represent genotypes of T / T, orange circles represent genotypes of C / C, and green triangles represent genotypes of C / T.

[0019] Figure 4 : Violin plot of the dark green Trichoderma infection rate of three genotypes in the shiitake mushroom population (the lower the value, the higher the resistance). The dark green Trichoderma infection rate of T / T shiitake was significantly lower than that of the other two genotypes (P<0.01). The data of T / T type came from the wild population, and the data of C / T type and C / T type came from the hybrid population.

[0020] Figure 5:The three genotypes of strains were infected by dark green Trichoderma. A is the image of T / T type shiitake strains infected by dark green Trichoderma; B is the image of C / T type shiitake strains infected by dark green Trichoderma; C is the image of C / C type shiitake strains infected by dark green Trichoderma. DETAILED DESCRIPTION

[0021] Example 1: Development of molecular markers related to the resistance trait of Lentinus edodes to Trichoderma viride

[0022] Resequencing of Lentinus edodes strains: DNA samples were extracted from the mycelium of 133 Lentinus edodes strains, including 99 wild strains and 34 cultivated strains. The DNA samples were sent to Berry Genomics (Beijing, China) for library preparation and sequencing. A total of 1.5 μg of genomic DNA was used per sample to construct paired-end sequencing libraries (Illumina) with an insert size of approximately 500 bp according to the manufacturer's instructions. The libraries were sequenced on the Illumina HiSeq2500 platform with a paired-end read length of 125 bp. Trimmomatic0.33 software was used to remove adapter sequences and low-quality reads with default parameters.

[0023] Variant analysis: Based on the reference genome of Lentinula edodes wpm-1 (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.), the high-throughput sequencing data was analyzed using the software GATK (Genome Analysis Toolkit) to obtain VCF files. In order to obtain high-quality variant data, VariantFiltration was used to perform hard filtering on the results, and quality control was performed using the command 'QUAL<30.0||QD<13.0||MQ<20.0||FS>20.0||MQRankSum<-3.0||ReadPosRankSum<-3.0||BaseQRankSum<-3.0', and 1,096,394 high-quality SNP sites were obtained for GWAS analysis.

[0024] Resistance phenotype data determination: Dark green Trichoderma and 133 Lentinus edodes strains were cultured in the center of the plate. The diameter of the culture dish was 90 mm, and about 20 mL of PDA culture medium was poured into each plate. A circular Lentinus edodes mycelium block with a diameter of 8 mm was inoculated in the center of the culture dish. After culturing at 25°C for 10 days, the Lentinus edodes mycelium block in the center was removed, and a dark green Trichoderma mycelium block of the same size that had grown for 5 days was inoculated in the same position. The strains were re-cultured at 25°C. The phenotypes were recorded after 7 days of inoculation with dark green Trichoderma. The interaction between dark green Trichoderma hyphae and Lentinus edodes hyphae before and after confrontation was observed and counted. The area of ​​the central lesion was determined by ImageJ software. The dark green Trichoderma infection rate was calculated according to the following formula: dark green Trichoderma infection rate (TIR) ​​= (dark green Trichoderma infection area - central hyphae block area) / (total growth area of ​​Lentinus edodes hyphae - central hyphae block area). The resistance of Lentinus edodes strains to dark green Trichoderma was digitally quantified. The higher the dark green Trichoderma infection rate, the lower the resistance value of the strain ( Figure 1 ).

[0025] 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.), and the dark green Trichoderma infection rate was used as the phenotype to obtain loci significantly associated with the dark green Trichoderma resistance trait of Lentinula edodes ( Figure 2 ).

[0026] The higher the infection rate of Trichoderma, the lower the resistance value of the strain. Two groups with extreme trait differences, namely 5 strains with the highest and lowest infection rates of Trichoderma, were selected as high-resistant and high-susceptible groups, and the SNP site information significantly associated with the dark green Trichoderma infection rate obtained in GWAS of these 10 strains was extracted. The distribution frequency (SNP-Index) of alleles in the two populations was calculated according to the dark green Trichoderma infection rate, and a secondary SNP point screening was performed to screen sites with large differences in SNP-Index between the two populations, and 20 sites that were significantly associated with dark green Trichoderma resistance and had large differences in SNP typing frequencies in extreme trait groups were obtained. Finally, combined with the test shiitake mushroom population, sites with different parental genotypes and able to form genotype separation were selected as candidate sites for verification to ensure that the test shiitake mushroom population had more than two genotypes at this site. The candidate site is located at the 479587bp of chromosome 5 of the Lentinus edodes wpm-1 reference genome, and its base is C or T. Sequence analysis revealed that the site is located in the exon region of the histidine phosphotransfer domain protein encoding gene, and the SNP molecular marker was named LeKX1.

[0027] Table 1 LeKX1 marker genotypes and dark green Trichoderma infection rates in extreme trait populations

[0028]

[0029] Example 2: Verification of the molecular marker LeKX1 associated with the dark green Trichoderma resistance trait of Lentinus edodes

[0030] KASP primers were designed for the obtained molecular marker LeKX1, the forward primer sequences were shown in SEQ ID NO.1 and 2, and the reverse universal primer sequence was shown in SEQ ID NO.3, wherein the forward primer shown in SEQ ID NO.1 carried a FAM fluorescent marker, and the forward primer shown in SEQ ID NO.2 carried a HEX fluorescent marker, and the primers were synthesized by Wuhan Tianyi Huiyuan Biological Co., Ltd.

[0031] A hybrid population of Lentinula edodes was constructed with YS44 and Qihe No. 2 (L2) as parents, with a total of 68 hybrid strains. Since the LeKX1 marker genotypes of the parents were C / T and C / C, respectively, there was a lack of T / T individuals in the hybrid population. Therefore, T / T individuals in the wild population were used to compare the differences in dark green Trichoderma resistance among the three genotypes. The dark green Trichoderma infection rates of 68 hybrid strains and 71 T / T wild strains were measured in the same way.

[0032] DNA of hybrid strains and wild strains was extracted using the CTAB method: appropriate amount of mycelium (about 0.1 g) was placed in a ceramic mortar, and after adding liquid nitrogen, it was quickly ground into powder, and 500 μL of extraction buffer (475 μL of CTAB buffer: 100 mmol / LTris-HCl (pH=7.8), 20 mmol / L EDTA, 1.4 mol / L NaAc, 2% (w / v) CTAB; 25 μL of 5% SDS) was added, and the mixture was thoroughly mixed for 30 s, and the mixture was placed in a 65°C water bath for 20 min. 500 μL of PCI (phenol: chloroform: isoamyl alcohol = 25:24:1) was added, and the mixture was thoroughly mixed for 30 s, and centrifuged at 13000 r / min for 2-4 min. The supernatant was transferred to a new 1.5 mL centrifuge tube, and an equal volume of isopropanol was added, and the mixture was allowed to stand at room temperature for 5-10 min. Centrifuged at 13000 r / min for 2-4 min, and the precipitate was washed twice with 70% alcohol. The alcohol was dried on a clean bench, 40 μL ddH2O was added to dissolve it, and the concentration was measured using an ultra-micro UV spectrophotometer.

[0033] The DNA of wild and hybrid strains was typed by KASP markers. 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, 0.6°C reduction per cycle, 10 cycles; 95°C denaturation for 15 s, 55°C annealing extension for 1 min, a total of 28 cycles. The reading conditions of the fluorescence signal value were 30°C for 30 s. If only the FAM fluorescence signal was detected, the LeKX1 marker genotype of the strain to be tested was C / C type; if only the HEX fluorescence signal was detected, the LeKX1 marker genotype of the strain to be tested was T / T type; if both fluorescence signals were detected at the same time, the LeKX1 marker genotype of the strain to be tested was C / T ( Figure 3 ).

[0034] The results of population genotyping and dark green Trichoderma resistance phenotype were statistically analyzed (Wilcoxon test). The results showed that for individuals with genotype T / T, the dark green Trichoderma infection rate was significantly lower than that of individuals with C / C and C / T types (P<0.01), indicating that the LeKX1 marker is significantly associated with the dark green Trichoderma resistance of Lentinus edodes, and when the marker mutates to T, Lentinus edodes has a higher dark green Trichoderma resistance ( Figure 4 and Figure 5 ).

[0035] The infection rate of Trichoderma viride less than 0.05 was defined as resistant strains, and that greater than 0.07 was defined as sensitive strains. The infection rates of Trichoderma viride of the three genotypes were statistically analyzed, and it was found that 55 of the 71 T / T strains were resistant strains, 23 of the 40 C / T strains were sensitive strains, and 14 of the 28 C / C strains were sensitive strains. When only resistant strains were identified, the accuracy of the LeKX1 marker could reach 77.5%. When identifying sensitive strains while identifying resistant strains, the comprehensive accuracy of the LeKX1 marker was 66.18%.

[0036] The above identification results show that the molecular marker LeKX1 and the detection method provided by the present invention can accurately and efficiently judge the dark green Trichoderma resistance of Lentinus edodes at the mycelial stage, effectively reduce the cultivation scale of breeding materials, reduce the workload of later field phenotypic identification, accelerate the breeding process, and improve breeding efficiency. Therefore, the LeKX1 molecular marker described in the present invention can effectively identify the dark green Trichoderma resistance trait of Lentinus edodes, and can be used for molecular marker-assisted breeding of dark green Trichoderma resistant Lentinus edodes strains.

Claims

1. A SNP molecular marker associated with resistance to dark green Trichoderma edodes, characterized in that: The molecular marker is located on chromosome 5 of the edodes reference genome, and the base C mutates to T at 479587 bp. The edodes with the genotype T / T show high resistance to dark green Trichoderma.

2. The KASP primer used to amplify 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 resistance of Lentinus edodes to dark green Trichoderma, characterized in that: The kit contains a reagent for detecting the 479587 bp base of chromosome 5 of Lentinus edodes.

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

3.

5. Application of the SNP molecular marker according to claim 1 in breeding of Lentinus edodes resistance to Trichoderma viride.

6. Use of the SNP molecular marker according to claim 1 in the identification of resistance traits of Lentinus edodes to Trichoderma viride.

7. Use of the KASP primer according to claim 2 in breeding of Lentinus edodes resistance to Trichoderma viride.

8. Use of the KASP primers according to claim 2 in identifying the resistance trait of Lentinus edodes to Trichoderma viride.

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