A SNP molecular marker related to lentinus edodes resistance trait of trichoderma atroviride and application thereof

Abstract

The present invention belongs to the field of fungal genetic breeding technology and specifically discloses a single-nucleotide polymorphism (SNP) molecular marker associated with the dark green Trichoderma resistance trait in Lentinus edodes and its application. The SNP molecular marker is located on chromosome 5 of the Lentinus edodes reference genome. At base 479,587, a C-to-T mutation occurs. Lentinus edodes with the T / T genotype exhibit high dark green Trichoderma resistance. This molecular marker can efficiently determine Trichoderma resistance during the mycelial stage of Lentinus edodes, overcoming the shortcomings of traditional breeding that relies on phenotypic identification. This accelerates the selection and breeding of new Trichoderma-resistant Lentinus edodes varieties and improves breeding efficiency.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic breeding of Lentinula edodes, and particularly relates to a molecular marker suitable for detecting Lentinula edodes Trichoderma atroviride resistance phenotype and application. The molecular marker can be used as a selection marker for breeding Lentinula edodes Trichoderma atroviride resistance, and provides a rapid screening molecular marker for breeding new Lentinula edodes Trichoderma atroviride resistance varieties with genetic stability. BACKGROUND

[0002] Lentinula edodes is the most produced 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 unique aroma and delicious taste, and is known as "Queen of Mushrooms" and "Mountain Delicacy" (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, vitamins and minerals, and has the functions of preventing tumors, increasing immunity, reducing blood lipids, resisting thrombosis, and protecting the stomach and liver (Bian Yinping. Edible Fungus Cultivation (3rd Edition). Beijing: Higher Education Press. 2017. 138-140). Due to its high nutritional and medicinal value, Lentinula edodes is deeply loved by people.

[0003] Most of the important economic traits of Lentinula edodes are quantitative traits, such as yield, number of fruiting bodies (mushroom number), single mushroom weight, early maturity, mycelial growth rate, disease resistance, etc. (Xiao et al. Association analysis and its application in fungal genetics research. Mycosystema. 2016. 35: 782-790). Unlike qualitative traits, quantitative traits mostly show continuous variation, are regulated by multiple genes, have complex genetic basis, are easily affected by environmental factors, and have no clear correspondence between phenotype and genotype, making it difficult to improve them genetically (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, commonly found in soil and decaying wood, and 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 Lentinula edodes stick rot disease. This fungus can cause both competitive and invasive diseases, causing a serious decline in the quality and yield of Lentinula edodes. Currently, there have been related reports of Trichoderma diseases in Lentinula edodes, Pleurotus ostreatus, Agaricus bisporus, Ganoderma lucidum, and Auricularia auricula-judae. So far, more than 40 species of Trichoderma can cause green mold disease in edible fungi, and the most common ones are T. atroviride, T. longibrachiatum, T. viride, T. koningii, T. pseudokoningii, and T. harzianum. Due to the many similarities in morphological characteristics and growth and development between Trichoderma fungi and Lentinula edodes, it is difficult to distinguish them and control them. Therefore, screening for Lentinula edodes strains with strong disease resistance, as well as studying the molecular genetic mechanisms of Lentinula edodes resistance to Trichoderma, and identifying key regulatory genetic loci and genes, are of great significance for the development of resistance molecular marker-assisted breeding and the promotion of sustainable development of the Lentinula edodes industry.

[0005] Genome-wide association study (GWAS) as a forward genetics method to mine trait regulatory sites was first proposed by Risch in 1996 (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 in the whole genome, genome-wide association study 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 of molecular marker-assisted selection breeding technology is the identification of DNA molecular markers closely linked to important agronomic traits. Compared with traditional phenotype selection, molecular marker-assisted breeding can be carried out at any period of biological growth, is not affected by environmental conditions, and can exclude the interference caused by allelic interaction, and has the advantages of rapidness, economy, high efficiency and accuracy.

[0007] The present application is based on a natural population composed of 133 wild and cultivated Lentinula edodes strains, and SNP sites are screened out by GWAS analysis of resequencing data, then the population is divided into two extreme trait populations in combination with the resistance phenotype of Trichoderma atroviride in Lentinula edodes population, the genotypes of two extreme difference populations at the SNP site are obtained, a functional mutation site related to the resistance of Lentinula edodes to Trichoderma atroviride is screened out, and the effectiveness is verified in a Lentinula edodes hybrid population. SUMMARY

[0008] The present application aims to provide a molecular marker LeKX1 related to the resistance of Lentinula edodes to Trichoderma atroviride. High-throughput sequencing is used to resequence 133 wild and cultivated Lentinula edodes strains, and SNP sites are obtained by comparing the reference genome; then GWAS analysis is performed to find SNP sites associated with resistance to Trichoderma atroviride, and the SNP-Index value is calculated by combining the genotype comparison of the extreme population, the site is filtered, and it is converted into a fast and easy-to-use KASP (Kompetitive Allele Specific PCR) marker. The SNP site is located at 479587bp of chromosome 5 of the Lentinula edodes reference genome, and the base is C or T. The site is located in the exon region of the histidine phosphotransfer domain protein coding gene. The molecular marker can be used as a molecular marker for the assisted selection breeding of Lentinula edodes strains resistant to Trichoderma atroviride, and can provide a molecular marker for the cultivation of genetically stable new varieties of Lentinula edodes.

[0009] Another object of the present application is to provide a method for detecting the resistance of Lentinula edodes to Trichoderma atroviride. The marker can be used to identify Trichoderma atroviride-resistant and sensitive strains at the mycelial stage, so as to eliminate non-target strains and improve the efficiency of selection.

[0010] In order to achieve the above-mentioned purpose, the present application is realized by the following technical solutions:

[0011] The SNP molecular marker related to the resistance of Lentinula edodes to Trichoderma atroviride is located at 479587bp of 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 is mutated to T. Lentinula edodes with genotype T / T shows high resistance to Trichoderma atroviride.

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

[0013] The SNP molecular marker is applied in Lentinula edodes Trichoderma atroviride resistance breeding or Lentinula edodes Trichoderma atroviride resistance trait identification: the genotype of the SNP site is detected to determine the Lentinula edodes Trichoderma atroviride resistance, the Lentinula edodes with the T / T genotype is the Lentinula edodes Trichoderma atroviride resistance type, and the Lentinula edodes with the C / C or C / T genotype is the Lentinula edodes Trichoderma atroviride sensitive type.

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

[0015] The present application successfully obtains the SNP molecular marker for detecting the Lentinula edodes Trichoderma atroviride resistance trait, and the marker can overcome the defects of relying on phenotype selection in traditional breeding, reduce the breeding workload, shorten the breeding period, and accelerate the process of breeding new varieties of Lentinula edodes, and has the advantages of convenient and rapid detection, stable amplification, high visualization of results, etc., and can quickly screen strains with Lentinula edodes Trichoderma atroviride resistance, and is used for breeding new varieties of Lentinula edodes. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 : Comparison before and after identification of the Lentinula edodes Trichoderma atroviride infection area and the total area of Lentinula edodes mycelium. A is the original image of Lentinula edodes after being infected by Lentinula edodes Trichoderma atroviride; B is the identification area of the Lentinula edodes Trichoderma atroviride infection area; C is the identification area of the total area of Lentinula edodes.

[0017] Figure 2 : Genetic locus regulating the Lentinula edodes Trichoderma atroviride resistance trait by whole genome association analysis. The significant correlation sites detected by the association analysis are above the horizontal line. The SNP molecular marker of the present application is located on the 5th chromosome of the Lentinula edodes reference genome.

[0018] Figure 3 : Identification results of the molecular marker LeKX1. A is the identification results of 10 T / T type wild populations randomly selected (3 repeats for each sample), and B is the identification results of the hybrid population, the blue square represents the genotype T / T, the orange circle represents the genotype C / C, and the green triangle represents the genotype C / T.

[0019] Figure 4 : Violin plot of the three genotypes of Lentinula edodes strains in the Lentinula edodes population on the Lentinula edodes Trichoderma atroviride infection rate (the lower the numerical value, the higher the resistance), the Lentinula edodes Trichoderma atroviride infection rate of the T / T type Lentinula edodes is significantly lower than that of the other two genotypes (P<0.01). The T / T type data is derived from the wild population, and the C / T type and C / T type data is derived from the hybrid population.

[0020] Figure 5: The situation of three genotypes of strains after being infected by Trichoderma atroviride. A is the image of T / T type Lentinula edodes strain after being infected by Trichoderma atroviride; B is the image of C / T type Lentinula edodes strain after being infected by Trichoderma atroviride; C is the image of C / C type Lentinula edodes strain after being infected by Trichoderma atroviride. DETAILED DESCRIPTION

[0021] Example 1: Development of molecular markers associated with Lentinula edodes Trichoderma atroviride resistance traits

[0022] Re-sequencing of Lentinula edodes strains: DNA samples were extracted from mycelium of 133 Lentinula edodes strains, including 99 wild strains and 34 cultivated strains. The DNA samples were sent to Berry & Co. (Beijing, China) for library preparation and sequencing. According to the manufacturer's instructions, a total of 1.5 μg of genomic DNA was used for each sample to construct a double-end sequencing library (Illumina) with an insert size of approximately 500 bp. These libraries were sequenced on the Illumina HiSeq2500 platform with a double-end read length of 125 bp. Trimmomatic 0.33 software was used to remove adapter sequences and low-quality reads with default parameters.

[0023] 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 software GATK (Genome Analysis Toolkit) to obtain VCF files. To obtain high-quality variant data, VariantFiltration was used for hard filtering of the results, and quality control was performed by the 'QUAL<30.0||QD<13.0||MQ<20.0||FS>20.0||MQRankSum<-3.0||ReadPosRankSum<-3.0||BaseQRankSum<-3.0' command, resulting in 1096394 high-quality SNP sites for GWAS analysis.

[0024] Resistance phenotype data determination: Trichoderma atroviride was plate center confrontation cultured with 133 Lentinula edodes strains. The Petri dish was 90 mm in diameter, about 20 mL of PDA medium was poured into each plate, and a 8 mm diameter circular Lentinula edodes mycelium block was inoculated at the center position of the Petri dish. After 10 days of culture at 25°C, the Lentinula edodes mycelium block at the center position was removed, and a 5-day-old Trichoderma atroviride mycelium block of the same size was inoculated at the same position. Re-culture at 25°C, take photos to record the phenotype when Trichoderma atroviride is inoculated for 7 days. Observe and count the interaction between Trichoderma atroviride mycelium and Lentinula edodes mycelium before and after confrontation. Measure the center lesion area by ImageJ software, and calculate the Trichoderma atroviride infection rate (TIR) according to the following formula: TIR = (Trichoderma atroviride infection area - center mycelium block area) / (total Lentinula edodes mycelium area - center mycelium block area). Quantify the resistance of Lentinula edodes strains to Trichoderma atroviride, the higher the Trichoderma atroviride infection rate, the lower the resistance value of the strain Figure 1 ).

[0025] Genome-wide association analysis: Linear mixed model in FaST-LMM was used for GWAS analysis (Lippert, Listgarten, Liu, et al. FaST linear mixed models for genome-wide association studies. Nature Methods, 2011, 8, 833-835.), with Trichoderma atroviride infection rate as the phenotype, to obtain the sites significantly associated with Lentinula edodes resistance to Trichoderma atroviride Figure 2 ).

[0026] The higher the Trichoderma atroviride infection rate, the lower the resistance value of the strain. Two populations with extreme trait differences were selected, i.e. 5 strains with the highest and lowest Trichoderma atroviride infection rates as high-resistance and high-susceptibility populations, respectively. The SNP site information of these 10 strains obtained from GWAS analysis was extracted, and the allele distribution frequency (SNP-Index) in the two populations was calculated according to the Trichoderma atroviride infection rate. Secondary SNP site screening was performed to screen sites with large differences in SNP-Index between the two populations, and 20 sites significantly associated with Trichoderma atroviride resistance and with large differences in SNP typing frequency in the extreme trait populations were obtained. Finally, by combining the test Lentinula edodes population, sites with different parental genotypes and capable of forming genotype segregation were selected as candidate sites for verification to ensure that the test Lentinula edodes population has two or more genotypes at the site. The candidate site is located at position 479587 bp of chromosome 5 of the Lentinula edodes wpm-1 reference genome, and the base is C or T. Sequence analysis of the site found that it is located in the exon region of the histidine phosphotransfer domain protein coding gene, and the SNP molecular marker is named LeKX1.

[0027] Table 1 LeKX1 marker genotypes and Trichoderma atroviride infection rates in the extreme trait population

[0028]

[0029] Example 2: Verification of the molecular marker LeKX1 associated with the Trichoderma atroviride resistance trait of Lentinula edodes

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

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

[0032] CTAB method was used to extract DNA from the hybrid strains and wild strains: an appropriate amount of mycelium (about 0.1 g) was placed in a ceramic mortar, and after adding liquid nitrogen, it was quickly 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, mixed thoroughly for 30 s, and incubated at 65°C for 20 min. 500 μL of PCI (phenol: chloroform: isopropyl alcohol = 25:24:1) was added, mixed thoroughly 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. It 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 with 70% alcohol twice. The alcohol was blown dry on a clean bench, 40 μL of ddH2O was added for dissolution, and the concentration was determined using an ultramicro UV spectrophotometer.

[0033] The KASP marker typing is performed on the DNA of the wild population and hybrid population strains, and the KASP reaction system is as follows: 250 ng of DNA (4.5 μL), 2 μL of 2x KASP Master mixture, and 0.5 μL of primer system mixture (the concentration of the forward primer is 12 μmol / L, and the concentration of the reverse universal primer is 30 μmol / L). The reaction conditions are as follows: 95 °C pre-denaturation for 10 min; 95 °C denaturation for 15 s, gradient annealing at 61-55 °C for 1 min, 10 cycles of decreasing 0.6 °C each time; 95 °C denaturation for 15 s, annealing at 55 °C for 1 min, a total of 28 cycles. The reading condition of the fluorescence signal value is 30 °C for 30 s. If only the FAM fluorescence signal is detected, the LeKX1 marker genotype of the strain to be detected is C / C type; if only the HEX fluorescence signal is detected, the LeKX1 marker genotype of the strain to be detected is T / T type; if both fluorescence signals are detected, the LeKX1 marker genotype of the strain to be detected is C / T( Figure 3 ).

[0034] The population genotype typing results and the deep green Trichoderma resistance phenotype are statistically analyzed (Wilcoxon test). The results show that the deep green Trichoderma infection rate of the individual with the genotype T / T is significantly lower than that of the individuals with the genotypes C / C and C / T (P < 0.01), indicating that the LeKX1 marker is significantly related to the resistance of Lentinula edodes to deep green Trichoderma, and when the marker is mutated to T, the Lentinula edodes has higher resistance to deep green Trichoderma Figure 4 and Figure 5 ).

[0035] The deep green Trichoderma infection rate of the individual with the genotype T / T is significantly lower than that of the individuals with the genotypes C / C and C / T (P < 0.01), indicating that the LeKX1 marker is significantly related to the resistance of Lentinula edodes to deep green Trichoderma, and when the marker is mutated to T, the Lentinula edodes has higher resistance to deep green Trichoderma

[0036] The above identification results show that the molecular marker LeKX1 and the detection method provided by the present application can accurately and efficiently determine the deep green Trichoderma resistance of Lentinula edodes at the mycelial stage, effectively reduce the cultivation scale of breeding materials, reduce the workload of late-stage phenotype identification, accelerate the breeding process, and improve the breeding efficiency. Therefore, the LeKX1 molecular marker described in the present application can effectively identify the deep green Trichoderma resistance of Lentinula edodes, and can be used for the molecular marker assisted selection of deep green Trichoderma resistant Lentinula edodes strains.

Claims

1. Application of SNP molecular markers in identification of resistance to Trichoderma viride in Lentinus edodes, characterized in that: The molecular marker is located on chromosome 5 of the Lentinus edodes wpm-1 reference genome, and the base C mutates to T at 479587 bp. Lentinus edodes with the T / T genotype show high resistance to Trichoderma aureum. The Lentinus edodes wpm-1 reference genome has an Accession number of GWHERGK00000000 in NGDC.

2. Application of KASP primers in identification of resistance to Trichoderma viride in Lentinus edodes, characterized by The primer sequences are shown in SEQ ID NO. 1-3.

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