Ganoderma lucidum germplasm specificity identification method based on whole genome difference

Abstract

The application provides a ganoderma lucidum germplasm specificity identification method based on whole genome difference. The method adopts a differential analysis strategy for different nuclear phases: through genome sequencing, assembly and whole genome alignment, the base difference between different single nuclear hyphae is calculated; the coverage matching degree of the test strain genome and the reference genome in the database is evaluated through k-mer integrity analysis to identify the specificity of the dikaryon strain. The objective judgment is carried out by using the numerical standard, and different varieties can be effectively distinguished. The application establishes a differential whole genome analysis strategy for two nuclear phase states of ganoderma lucidum, and fills the blank of the lack of integrated identification scheme suitable for single nuclear hypha and dikaryon strain in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of bioinformatics and relates to the identification technology of edible fungi germplasm resources. More specifically, it relates to the method, system and equipment for the identification of Ganoderma lucidum germplasm specificity based on whole genome differences. Background Technology

[0002] Ganoderma lucidum is a traditional medicinal fungus in my country, and its germplasm resources are becoming increasingly abundant with the development of the industry. Specific identification of germplasm resources has become a crucial link in protecting germplasm resources, breeding new varieties, protecting intellectual property rights, and standardizing the industry's development. Two key nuclear phases exist in the life cycle of Ganoderma lucidum: monokaryotic hyphae and dikaryotic hyphae. Monokaryotic hyphae are primary hyphae formed from the germination of basidiospores, containing one nucleus within the cell, with a simple genetic background, and are the basic material for hybridization breeding. Dikaryotic hyphae are secondary hyphae (i.e., dikaryotic strains) formed by the fusion of monokaryotic hyphae of different mating types, containing two nuclei within the cell, with a complex genetic background, and are the main functional germplasm for artificial cultivation. Accurate identification of these two nuclear phases is a core requirement for Ganoderma lucidum germplasm management.

[0003] Currently, the identification of Ganoderma lucidum variety specificity mainly relies on the antagonism test (NY / T 1845-2010), which determines genetic differences by observing whether antagonistic lines are formed when the hyphae of two strains come into contact in a PDA culture dish. Specifically, this includes: 1) Strain activation: The Ganoderma lucidum strain to be identified is transferred from its storage state to a PDA slant or plate and activated in an incubator at 28-30℃ until the colonies grow to a diameter of 3-4 cm; 2) Inoculation: Using a 5 mm punch, hyphae blocks from the vigorous growth area at the edge of the colony are taken as inoculation blocks. Two points are selected on the PDA plate, 30 mm apart and 15 mm from the center of the plate, for inoculation, with the hyphae facing upwards. Three control groups were set up: the test strain paired with the control strain, the test strain paired with itself, and the control strain paired with itself, with three replicates in each group; 3) Culture: the inoculated petri dishes were placed in a constant temperature incubator at 28-30 ℃ and cultured in the dark for 15-20 days until the mycelia of the two inoculated blocks were in full contact, and then transferred to natural light conditions (light intensity > 300 ppm). Continue culturing for 5-7 days under lx); 4) Result observation and judgment: Under sufficient light, observe the morphology of hyphae at the interface between the two strains on the surface of the culture, focusing on whether it is a raised type: the hyphae at the interface are obviously raised, forming a "wall-like" type; a groove type: a depression groove appears at the interface, and hyphae growth is inhibited; an isolation type: an obvious pigment deposition zone or blank isolation zone is formed at the interface; if the tested strain and the control strain pairing group show any of the above antagonistic reaction types, it is determined that the two strains have genetic incompatibility and are different varieties; if the tested strain and the control pairing group do not have an antagonistic reaction, it cannot be determined that they are the same variety, only that their genetic background may be similar. As can be seen from the above, this method has many defects. First, it is labor-intensive and has a long cycle, which is not suitable for high-throughput identification. The antagonism experiment is a one-to-one pairing test, which requires separate preparation of culture dishes, inoculation, culture and observation for each test strain combination. The cycle of a single experiment is as long as 7-14 days. If strain activation, preparation and result analysis are included, the cycle is even longer. To conduct comprehensive identification of specific germplasm in a germplasm bank with 100 Ganoderma lucidum accessions, theoretically 4950 pairing combinations would be required, with each combination needing 3-5 replicates. This workload is unsustainable for large-scale identification in modern germplasm banks. In commercial breeding, the inefficiency of specific identification severely restricts the progress of strain screening and the promotion of new lines, making it difficult to meet the demands of the modern Ganoderma lucidum industry for rapid, large-scale, and low-cost identification. Secondly, the results are highly subjective and lack objective quantitative standards. Descriptive indicators such as the "obviousness" of antagonistic lines, the "depth" of pigment deposition, and the "degree" of hyphal elevation can vary significantly in the eyes of different experimenters. Even the same experimenter may have inconsistent judgments about the same culture dish at different times and under different conditions. Although photographic recording can partially alleviate this problem, the shooting angle, lighting conditions, and resolution can also affect the judgment.Antagonistic experiments cannot provide numerical genetic distance indicators; they can only record whether an antagonistic response is present or absent. This qualitative rather than quantitative output makes it difficult to perform statistical analysis on the results of antagonistic experiments, and it cannot meet the requirements of modern scientific research for data rigor and reproducibility.

[0004] Molecular marker technologies such as SSR (simple sequence repeat) and SNP (single nucleotide polymorphism) markers have been gradually applied to the germplasm-specific identification of Ganoderma lucidum, to some extent compensating for the shortcomings of antagonism experiments. SSR marker identification is a molecular marker technology developed based on the length polymorphism of 1-6 base tandem repeat sequences widely present in eukaryotic genomes. The copy number of repeat units varies greatly among different strains, leading to polymorphism in the length of PCR amplification products. It is the most mature molecular marker technology used in edible fungi variety identification. In Ganoderma lucidum germplasm-specific identification, researchers first need to screen for polymorphic SSR sites from the entire Ganoderma lucidum genome, design specific primers, extract genomic DNA from the test strain, perform PCR amplification and electrophoretic separation, compare the banding patterns of the test strain and the reference strain to determine genetic similarity, and achieve specific identification. SNP marker identification is based on single-base differences in the genome and is performed using techniques such as KASP, TaqMan probes, or gene chips for genotyping. In Ganoderma lucidum germplasm-specific identification, KASP (competitive allele-specific PCR) is currently the most widely used method. The experimenter first needs to screen for species-specific core SNP loci through whole-genome resequencing or simplified genome sequencing. For each locus, two competitive allele-specific forward primers and one universal reverse primer are designed. Genomic DNA is extracted from the test strain, and a 5-10 μL reaction system is prepared. The reaction is performed on a real-time PCR instrument, and the endpoint fluorescence signal is read after the reaction. The SNP genotypes of the test strain and the reference strain are compared, the allele matching degree is calculated, and a matching threshold (e.g., 98%) is set. Strains below the threshold are considered specific germplasm, while those above or equal to the threshold are considered non-specific germplasm. As can be seen, both methods have several drawbacks. First, they rely on complex experimental procedures. SSR labeling requires multiple manual steps such as PCR amplification, electrophoretic separation, and band interpretation. While SNP labeling can be partially automated, it still requires configuring the PCR reaction system, reading the fluorescence signal, and other steps, and necessitates specialized equipment such as a real-time PCR instrument and a microarray scanner. These methods require specialized molecular biology laboratory conditions and skilled experimental techniques, resulting in a high technical threshold. Secondly, they are insufficient to meet high-throughput requirements. SSR marker experiments are labor-intensive and difficult to conduct on a large scale; while SNP marker microarrays can achieve high throughput, the cost of microarray design and synthesis is high. Neither method can meet the needs of the modern Ganoderma lucidum industry for rapid, low-cost, and large-scale germplasm identification. Thirdly, they have limited coverage and insufficient resolution. Both SSR and SNP markers only detect specific sites in the genome, with a coverage of less than 0.1% of the entire genome, ignoring a large amount of variation information. If the selected site has no polymorphism between the test strain and the reference strain, it cannot be distinguished; for closely related strains with genetic similarity >95%, the polymorphism of a limited number of sites is insufficient to provide adequate resolution.Fourth, pre-screening of sites is required, which may prevent the discovery of new variations. Molecular marker methods all require pre-screening and validation of primers or probes, resulting in long development cycles and high costs. For new germplasm and unknown variation types, pre-screened sites may not be covered, limiting their application.

[0005] With the development of high-throughput sequencing technology, differential analysis at the whole-genome level has become possible. However, existing bioinformatics methods are mostly designed for single-nuclear phases and lack integration schemes applicable to both monokaryotic hyphae and binucleate strains. In particular, binucleate strains contain genetic material from two nuclei, and their genomes are combinations of two monokaryotic genomes, making direct identification using collinearity alignment methods suitable for monokaryotic genomes impossible. Therefore, there is an urgent need to develop a germplasm-specific identification method for Ganoderma lucidum that can simultaneously cover both nuclear phase states, is easy to operate, and provides objective results. Summary of the Invention

[0006] To address the problems of high workload, long cycle, strong subjectivity of results, limited resolution, and high threshold in existing technologies, this invention provides a method, system, and equipment for identifying Ganoderma lucidum germplasm specificity based on whole-genome differences.

[0007] The technical solution adopted in this invention is: a method for identifying Ganoderma lucidum germplasm specificity based on whole-genome differences, comprising:

[0008] Provide a genome database containing a specifically identified reference mononuclear genome;

[0009] For the binuclear strain to be tested: obtain whole genome sequencing data of the binuclear strain to be tested with sequencing depth that meets the analysis requirements and perform data filtering to obtain effective sequencing data; perform k-mer integrity analysis on the pairwise combinations of the effective sequencing data and the reference mononuclear genome in the genome database to obtain k-mer integrity values; compare the k-mer integrity values ​​with a preset integrity threshold to determine whether the binuclear strain to be tested is a specific germplasm;

[0010] For the monokaryotic hyphae to be tested: obtain the genome sequencing data of the monokaryotic hyphae to be tested and assemble the genome to obtain the monokaryotic genome to be tested; perform collinearity comparison analysis between the monokaryotic genome to be tested and each reference genome in the genome database, and calculate the percentage of base difference based on the Jaccard similarity coefficient; compare the percentage of base difference with a preset difference threshold to determine whether the monokaryotic hyphae to be tested is a specific germplasm.

[0011] Preferably, the method for determining whether the binuclear strain to be tested is a specific germplasm includes: when the k-mer integrity value is ≥98%, the binuclear strain to be tested is determined to be highly similar to the pairwise combinations of the current reference mononuclear genome and is a non-specific germplasm; when the k-mer integrity value is <98%, the binuclear strain to be tested contains a new mononuclear component and is a specific germplasm. More specifically, when the germplasm to be tested is a binuclear strain, the binuclear identification process is performed: the whole genome of the binuclear strain is resequencing using a next-generation sequencing platform to obtain sequencing reads with a sequencing depth of not less than 30×; k-mer counting is performed on the sequencing reads using Meryl software, and the k-mer integrity percentage between the sequencing reads and the reference genome is calculated using Merqury software, where the k-mer length k=21, and the reference genome is a pairwise combination of mononuclear genomes already included in the genome database; the combination with the highest k-mer integrity is selected as the identification result. When the k-mer integrity is ≥98%, the binuclear strain is determined to be non-specific germplasm, and when the k-mer integrity is <98%, it is determined to be specific germplasm. The 98% k-mer integrity threshold is determined based on statistical data of k-mer integrity from a large number of known binuclear strains of the same variety. Testing shows that the k-mer integrity of different binuclear strains within the same variety with their corresponding mononuclear combinations is usually above 98%; the k-mer integrity between different varieties is usually significantly lower than 98%. Choosing 98% as the threshold for distinguishing between specific and non-specific germplasm effectively balances identification sensitivity and specificity. For fine differentiation of mutants or continuously bred lines within the same variety, further identification can be achieved by increasing the threshold.

[0012] Preferably, the method for determining whether the monokaryotic hyphae to be tested is a specific germplasm includes: when the percentage of base difference is ≤1%, the monokaryotic hyphae to be tested is determined to be highly similar to the current reference genome and is a non-specific monokaryotic hyphae; when the percentage of base difference is >1%, the monokaryotic hyphae to be tested has a significant genetic difference from the current reference genome and is a specific monokaryotic hyphae. More specifically, when the germplasm to be tested is monokaryotic hyphae, the monokaryotic identification process is performed: long-read sequencing is performed on the monokaryotic hyphae using a third-generation sequencing platform, and simultaneously sequencing is performed using a second-generation sequencing platform to obtain sequencing reads. After data quality control and preprocessing, the genome is initially assembled using the third-generation sequencing data, and error correction and polishing are performed using the second-generation sequencing data to assemble the monokaryotic genome to be tested. The nucmer tool of the MUMmer software is used to perform collinearity alignment between the monokaryotic genome to be tested and the reference genome in the genome database. The alignment parameter—mincluster—is set to 500, and the base difference value is calculated based on the Jaccard similarity coefficient. The result with the smallest base difference value is selected as the identification result. When the base difference value is ≤1%, the monokaryotic hyphae are determined to be non-specific germplasm; when the base difference value is >1%, it is determined to be specific germplasm. The 1% base difference threshold is determined statistically based on a large amount of collinearity alignment data of known monokaryotic hyphae of the same variety. Tests showed that the base difference between different monokaryotic hyphae within the same variety is usually less than 1%; the base difference between different varieties is usually significantly higher than 1%. 1% was chosen as the threshold for distinguishing between specific and non-specific germplasm. For fine differentiation of mutants or continuously bred lines within the same variety, further identification can be achieved by lowering the threshold.

[0013] As a preferred method, obtaining whole-genome sequencing data of the target binuclear strain with a sequencing depth sufficient for analysis includes: performing whole-genome paired-end sequencing of the target binuclear strain using a next-generation sequencing platform. The next-generation sequencing platform is preferably an Illumina HiSeq series sequencer, with the sequencing mode being PE150 paired-end sequencing.

[0014] Preferably, the data filtering process includes at least one of the following: removing reads containing sequencing adapter sequences; cutting and removing non-AGCT bases at the 5' end; filtering sequences with N base content greater than 10%; filtering sequences with low-quality bases (quality value less than or equal to 5) content greater than 50%; and removing PCR repeats.

[0015] Preferably, the method for obtaining genome sequencing data of the monokaryotic hyphae to be tested and assembling the genome includes: performing long-read sequencing of the monokaryotic hyphae to be tested using a third-generation sequencing platform, and simultaneously performing paired-end sequencing using a second-generation sequencing platform; after data preprocessing (removing mitochondrial sequences), the second-generation and third-generation sequencing data are uploaded to an analysis platform, which automatically performs the following steps: using the third-generation sequencing data for preliminary genome assembly, and using the second-generation sequencing data for error correction and refinement of the assembly results to obtain the monokaryotic genome to be tested. The third-generation sequencing platform is preferably a PacBio Sequel sequencer.

[0016] Preferably, the percentage of base differences is calculated based on the Jaccard similarity coefficient, and the calculation formula is: percentage difference = [1 - (size of common part of the genome to be tested + size of common part of the reference genome) / (total size of the genome to be tested + total size of the reference genome)] × 100%; wherein, the size of the common part is determined by the collinearity alignment analysis results.

[0017] Preferably, the collinearity alignment analysis is performed with parameters of a minimum matching cluster length of not less than 500 base pairs and a thread number of not less than 20 for whole genome alignment. The alignment results are analyzed to obtain collinearity blocks, and the percentage of base differences is calculated based on the collinearity blocks.

[0018] Preferably, the genome database is constructed in the following manner: collecting Ganoderma lucidum germplasm resources, extracting mononuclear mycelia through protoplast isolation technology, and culturing them to obtain mononuclear hyphae; extracting genomic DNA from the mononuclear hyphae and performing third-generation sequencing and second-generation sequencing; after genome assembly, quality assessment and specificity identification, the mononuclear genomes that meet the quality standards are included in the genome database; the genome database is regularly updated and expanded.

[0019] This invention also provides a Ganoderma lucidum germplasm-specific identification system based on genome-wide differences, comprising:

[0020] A genome database module for providing a genome database containing a specifically identified reference mononuclear genome;

[0021] The data upload module is used to acquire whole genome sequencing data of binucleate strains to be tested with sequencing depths that meet the analysis requirements and to perform data filtering to obtain effective sequencing data; it also acquires genome sequencing data of monokaryotic hyphae to be tested and performs genome assembly to obtain the monokaryotic genome to be tested.

[0022] The analysis mode selection module is used to select between dual-core and single-core analysis modes based on the whole-genome sequencing data obtained from the data upload module.

[0023] The analysis execution module automatically invokes the corresponding analysis process based on the analysis mode selected by the analysis mode selection module. For the binuclear strain to be tested: the whole genome sequencing data of the binuclear strain to be tested with a sequencing depth that meets the analysis requirements is obtained and the data is filtered to obtain effective sequencing data; the effective sequencing data is paired with a reference monokaryotic genome in the genome database to perform k-mer integrity analysis and obtain k-mer integrity values; the k-mer integrity values ​​are compared with a preset integrity threshold to determine whether the binuclear strain to be tested is a specific germplasm. For the monokaryotic hyphae to be tested: the genome sequencing data of the monokaryotic hyphae to be tested is obtained and the genome is assembled to obtain the monokaryotic genome to be tested; the monokaryotic genome to be tested is compared with each reference genome in the genome database to perform collinearity alignment analysis and calculate the percentage of base difference based on the Jaccard similarity coefficient; the percentage of base difference is compared with a preset difference threshold to determine whether the monokaryotic hyphae to be tested is a specific germplasm.

[0024] The results display module is used to output the judgment results of the analysis and execution module.

[0025] This invention also provides a device for identifying Ganoderma lucidum germplasm specificity based on whole-genome differences, including a processor and a memory; the processor and the memory are connected via a communication bus; wherein, the processor is used to call and execute a Ganoderma lucidum germplasm specificity identification program stored in the memory; the memory is used to store the program, which is at least used to execute the Ganoderma lucidum germplasm specificity identification method based on whole-genome differences, specifically including: performing k-mer integrity analysis on binucleate strains, or performing genome assembly and whole-genome alignment on monokaryotic hyphae to determine the Ganoderma lucidum germplasm specificity.

[0026] The beneficial effects of this invention are:

[0027] (1) This invention establishes a differentiated whole-genome analysis strategy for the two nuclear phases of Ganoderma lucidum, filling the gap in the existing technology for the lack of an integrated identification scheme applicable to both monokaryotic hyphae and binucleate strains. For binucleate strains, since they contain two sets of monokaryotic genomes, direct collinearity alignment cannot be performed. This invention creatively adopts the k-mer integrity analysis method to identify the strain by evaluating the coverage of sequencing reads to the reference genome combination. For monokaryotic hyphae, collinearity alignment and base difference calculation methods are used to achieve accurate base level difference assessment.

[0028] (2) This invention establishes numerical judgment criteria: for binuclear strains, the judgment threshold is ≥98% k-mer integrity as non-specific germplasm and <98% as specific germplasm; for monokaryotic hyphae, the judgment threshold is ≤1% base difference as non-specific germplasm and >1% as specific germplasm. This numerical judgment system eliminates the bias of subjective interpretation of traditional antagonistic experimental results and improves the objectivity and accuracy of the identification results.

[0029] (3) This invention analyzes differences at the whole genome level, without relying on preset sites, and improves the resolution to the base level. Its coverage and resolution are significantly better than molecular marker technologies (SSR, SNP, etc.), solving the problems of limited coverage and insufficient resolution of molecular marker technologies.

[0030] (4) The web-based automated analysis platform developed based on the identification system of this invention enables non-bioinformatics professionals to complete high-throughput identification, solving the problem of high technical barriers in traditional methods. The identification process is simplified to DNA extraction-sequencing-data analysis, which greatly shortens the cycle and realizes high-throughput and rapid identification of Ganoderma lucidum germplasm. Attached Figure Description

[0031] Figure 1 A flowchart of a method for identifying Ganoderma lucidum germplasm specificity based on whole-genome differences, provided in an embodiment of the present invention. Detailed Implementation

[0032] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0033] To facilitate understanding of this invention, the key terms involved in this invention are explained below.

[0034] Ganoderma lucidum germplasm: the genetic material of Ganoderma lucidum, including monokaryotic hyphae, binucleate strains, basidiospores, and other materials that can be used for propagation or research.

[0035] Monokaryotic hyphae: Primary hyphae formed from the germination of basidiospores, containing a single nucleus within the cell, with a single genetic background, serving as the basic material for hybridization breeding.

[0036] Binary mycelial strains: secondary mycelia formed by the fusion of monokaryotic mycelia of different mating types. The cells contain two nuclei and have a complex genetic background. They are the main functional germplasm for artificial cultivation.

[0037] Mononuclear genome: refers to the genome obtained by extracting two mononuclear cells from Ganoderma lucidum using protoplast isolation technology, culturing mononuclear hyphae, extracting DNA, sequencing using third-generation sequencing technology, and assembling the genome.

[0038] Specificity identification: The process of determining the degree of genetic difference between the germplasm to be tested and known germplasms through whole-genome differential analysis, and determining whether the germplasm to be tested has specificity.

[0039] This invention provides a method for identifying Ganoderma lucidum germplasm specificity based on whole-genome differences. A differential analysis strategy is employed for different nuclear phases: genome sequencing, assembly, and whole-genome alignment are performed on monokaryotic hyphae to calculate base differences; k-mer integrity analysis is performed on binkaryotic strains to assess the degree of matching with reference genome assemblies. Objective judgment is made using numerical standards (1% base difference threshold, 98% integrity threshold), which can effectively distinguish different varieties (k-mer integrity among binkaryotic strains of the same variety is typically ≥98%, while the difference among monokaryotic hyphae of the same variety is typically ≤1%). The specific steps are explained below.

[0040] 1. Construction of a genome database

[0041] This embodiment details the method for constructing a genome database. The genome database contains a set of reference mononuclear genomes that have been specifically identified. The database construction process is as follows: Ganoderma lucidum germplasm resources are collected, and single cell nuclei are extracted from the cells using protoplast isolation technology. The isolated mononuclear nuclei are then cultured on a suitable culture medium to obtain mononuclear hyphae. Genomic DNA is extracted from the mononuclear hyphae and subjected to third-generation sequencing and second-generation sequencing. After genome assembly, assembly quality assessment, and specific identification, mononuclear genomes that meet the quality standards are included in the database. Each mononuclear genome in the database has a unique identifier and is regularly updated and expanded. In one specific embodiment, the genome database used contains specifically identified Ganoderma lucidum mononuclear genomes, currently including 81 mononuclear genomes, and is updated regularly. The mononuclear genome was obtained as follows: mononuclear hyphae were isolated from Ganoderma lucidum binuclear strains using protoplast isolation technology, genomic DNA was extracted, and sequenced using PacBio HiFi third-generation sequencing technology and Illumina second-generation sequencing technology. After data quality control and removal of mitochondrial sequences, preliminary assembly was performed using Canu software, redundant sequences were removed using Purge_dups software, gaps were filled using third-generation sequencing data, and polishing was performed using second-generation sequencing data to obtain a high-quality mononuclear genome sequence, which was then included in the database after specific identification.

[0042] 2. Specific identification of binucleate strains

[0043] This embodiment details the complete process for specific identification of binuclear strains, using binuclear strain YSWL-5 as an example.

[0044] Step 1: Genomic DNA Extraction

[0045] Take 300-500 mg of mycelium from the binuclear strain YSWL-5, freeze it at -50℃, dry it in a freeze dryer for 18 hours, break it up with steel balls, and extract genomic DNA using the CTAB method. The specific procedure is as follows: Add 2 mL of CTAB extraction buffer (CTAB 4g, NaCl 10g, 1M Tris-HCl (pH=8) 20ml, 0.5M EDTA 8ml, bring the volume to 200ml), transfer to a new centrifuge tube, and incubate at 65℃ for at least 1 hour, inverting and mixing every 15 minutes during this period; centrifuge at 12000 rpm for 20 minutes at 4℃; take 1 mL of supernatant, add an equal volume of phenol:chloroform:isopropanol (25:24:1) mixture, gently mix for 10 minutes, and centrifuge at 12000 rpm for 10 minutes at 4℃; take 0.8 mL of supernatant, transfer to a new centrifuge tube, add an equal volume of chloroform:isopropanol (24:1) mixture, gently mix for 10 minutes, and centrifuge at 12000 rpm for 10 minutes at 4℃; take 0.6 mL of supernatant, transfer to a new centrifuge tube, and add 0.4 mL of... Pre-chilled isopropanol at -20°C was gently shaken for 5 min, placed in a -20°C freezer for 20 min, and centrifuged at 8000 rpm for 10 min at 4°C. The supernatant was discarded, and the precipitate was washed with 0.6 mL of 70% ethanol. The precipitate was centrifuged at 12000 rpm for 3 min at room temperature, and the supernatant was discarded. The precipitate was centrifuged at 12000 rpm for 1 min at room temperature, air-dried / ultra-cleaned, and 0.1 mL of TE buffer was added. The precipitate was gently shaken to dissolve and incubated overnight at 4°C. 0.5 μL of 10 mg / mL RNase A was added, and the mixture was incubated in a 37°C metal bath for 1 h to obtain the DNA extract. The DNA concentration and purity were determined using NanoDrop, and the A260 / A280 ratio should be between 1.8 and 2.0.

[0046] Step 2: Whole genome resequencing

[0047] The extracted genomic DNA was sent to a sequencing company for whole-genome resequencing using the Illumina HiSeq X Ten sequencing platform. The sequencing mode was PE150 paired-end sequencing, with a sequencing depth of at least 30×. Library construction was performed using the Illumina DNA PCR-Free Prep kit, following the manufacturer's standard procedures. After sequencing, two raw data files in FASTQ format, R1 and R2, were obtained. The sequencing data volume needed to meet the coverage requirements of subsequent k-mer analysis, typically at least 1 Gb.

[0048] Step 3: Raw Data Processing

[0049] The raw image data output from the sequencing instrument was converted into sequence data through base identification and stored in FASTQ format. Subsequently, the raw sequencing data was processed for quality control using FASTP software. Specific filtering criteria included: (1) removing reads containing sequencing adapter sequences; (2) removing non-AGCT bases at the 5' end; (3) filtering sequences with N base content >10%; (4) filtering sequences with low-quality bases (quality value ≤5) content >50%; and (5) removing PCR repeats. After processing, the number of reads before and after filtering, the Q30 ratio, and the GC content were statistically analyzed to obtain effective sequencing data (clean reads). For example, in this embodiment, the raw data volume was 1.53 Gb (0.77 Gb at the R1 end and 0.77 Gb at the R2 end), and the effective data volume after filtering was approximately 1.48 Gb, with Q30 >85%.

[0050] Step 4: k-mer integrity analysis

[0051] Eighty-one mononuclear genomes already included in the genome database were paired to form C(81,2)=3240 reference genome combinations. For each combination, k-mer counts (k=21) were performed on clean reads using Meryl software (v1.4), and the k-mer integrity percentage between clean reads and reference genome combinations was calculated using Merqury software (v1.3). The analysis command line is as follows:

[0052] meryl k=21 count output read1.meryl reads1.fastq.gz

[0053] meryl k=21 count output read2.meryl reads2.fastq.gz

[0054] meryl union-sum output example.meryl read*.meryl

[0055] merqury.sh example.meryl reference.fa example

[0056] The reference.fa file is a merged FASTA file containing two mononuclear genomes. After analysis, the combination with the highest k-mer integrity was selected as the identification result.

[0057] Step 5: Specificity determination

[0058] The integrity percentage of the combination with the highest k-mer integrity is compared with a 98% threshold. If the integrity is ≥98%, it is determined to be non-specific germplasm; if the integrity is <98%, it is determined to be specific germplasm. In this example, the k-mer integrity of the combination with G07801+G07802 is 99.57%, which is the highest value. Since 99.57% ≥ 98%, YSWL-5 is determined to be non-specific germplasm.

[0059] 3. Specific identification of monokaryotic hyphae

[0060] This embodiment details the complete process for the specific identification of monokaryotic hyphae, taking monokaryotic hyphae G00301 as an example.

[0061] Step 1: Monokaryotic mycelial culture and DNA extraction

[0062] The monokaryotic mycelium G00301 was inoculated onto PDA plates (potato dextrose agar) and incubated at 28°C for 7-10 days until the colony diameter was approximately 5 cm. 300-500 mg of fresh mycelium was collected, ground in liquid nitrogen, and genomic DNA was extracted using the CTAB method. The concentration and purity were determined using NanoDrop.

[0063] Step 2: Third-generation sequencing and second-generation sequencing

[0064] The extracted genomic DNA was sent to a sequencing company for both third-generation (NG) and second-generation (NG) sequencing. NNG sequencing was performed using the PacBio Sequel IIe platform in HiFi sequencing mode to obtain high-fidelity long read data. NNG sequencing was performed using the Illumina NovaSeq 6000 platform in PE150 mode for error correction and polishing. In this embodiment, the HiFi sequencing data volume was 0.84 Gb, and the Illumina sequencing data volume was 1.38 Gb (0.69 Gb at the R1 end and 0.69 Gb at the R2 end).

[0065] Step 3: Genome Assembly

[0066] Mitochondrial sequences were removed from the third-generation sequencing data using BLAST. Initial assembly was performed using Canu software (v2.2) with the following parameters: genomeSize=50m, minReadLength=1000. After assembly, redundant sequences caused by heterozygous regions were removed using Purge_dups software. Gap filling was performed using third-generation HiFi data, and the assembly results were polished using second-generation Illumina data (using Pilon software, iterated for 3 rounds) to obtain high-quality single-nucleus genome sequences. For example, in this embodiment, the final assembled genome size was approximately 48.2 Mb, and the contig N50 was approximately 3.6 Mb.

[0067] Step 4: Collinearity Analysis and Base Difference Calculation

[0068] The assembled single-nucleus genome to be tested was collinearly aligned with 81 reference genomes in the genome database one by one. The alignment was performed using the nucmer tool in MUMmer software (v4.0), with the following parameter settings:

[0069] nucmer --mincluster 500 -t 20 -p t1 genome1.fa genome2.fa

[0070] show-coords -c -r t1.delta > t1.coords

[0071] Here, `--mincluster 500` indicates a minimum alignment cluster length of 500 bp, and `-t 20` indicates the use of 20 threads. After alignment, a self-written program is used to parse the collinearity analysis results and extract the following fields: reference genome common portion size, comparison genome common portion size, reference genome size, and comparison genome size.

[0072] Based on the extracted data, the base difference value is calculated with reference to the Jaccard similarity coefficient, using the following formula:

[0073] Base difference (%) = [1 - (size of common portion of reference genome + size of common portion of comparison genome) / (size of reference genome + size of comparison genome)] × 100%

[0074] After the calculation is completed, the result with the smallest base difference value is selected as the identification result.

[0075] Step 5: Specificity determination

[0076] The result with the smallest base difference value is compared with a 1% threshold. If the difference value is ≤1%, it is determined to be non-specific germplasm; if the difference value is >1%, it is determined to be specific germplasm. In this example, the base difference value with the reference genome G00301 is 0.68%, which is the minimum value. Since 0.68% ≤ 1%, G00301 is determined to be a non-specific mononuclear genome.

[0077] Example 1: Identification of specific binucleate strains (unknown sample)

[0078] Materials: The binary strain YSYS-1 to be tested.

[0079] Methods: The procedures for identifying binuclear strains described in Example 1 were followed in detail. Genomic DNA was extracted from the hyphae of the binuclear strain and resequencing was performed using an Illumina HiSeq PE150 platform (paired-end sequencing, 1.57 Gb data, 0.79 Gb R1 end and 0.79 Gb R2 end). The sequencing data was uploaded via a web-based platform, selecting the "dual-core" mode. The system automatically performed k-mer integrity analysis on each pairwise combination with 81 genomes in the database.

[0080] Results: k-mer integrity was <98% for all 3240 combinations, with the highest value being 93.07% (G00101+G00503 combination).

[0081] Judgment: The highest value of k-mer integrity is 93.07% < 98% threshold, so YSYS-1 is determined to be a specific germplasm (containing a new mononuclear component), and it is recommended to conduct mononuclearization experiments for further verification.

[0082] Example 2: Identification of nonspecific binuclear strains (unknown sample)

[0083] Materials: The binary strain YSWL-5 to be tested.

[0084] Methods: The procedures for identifying binuclear strains described in Example 1 were followed in detail. Genomic DNA was extracted from the hyphae of the binuclear strain and resequencing was performed using an Illumina HiSeq PE150 platform (paired-end sequencing, 1.53 Gb data, 0.77 Gb R1 end and 0.77 Gb R2 end). The sequencing data was uploaded via a web-based platform, selecting the "dual-core" mode. The system automatically performed k-mer integrity analysis on each pairwise combination with 81 genomes in the database.

[0085] Result: The k-mer integrity of the combination with G07801+G07802 is 99.57%, which is the highest value.

[0086] Judgment: With k-mer integrity ≥ 99.57% threshold, YSWL-5 is determined to be a non-specific germplasm.

[0087] This invention also provides a Ganoderma lucidum germplasm-specific identification system based on whole-genome differences, including a genome database module, a data upload module, an analysis mode selection module, an analysis execution module, and a result display module. The genome database module provides a genome database containing a reference mononuclear genome that has been specifically identified. The data upload module acquires the whole-genome sequencing data of the target dual-kine strain with a sequencing depth sufficient for analysis and performs data filtering to obtain valid sequencing data; it also acquires the genome sequencing data of the target mononuclear hyphae and assembles the genome to obtain the target mononuclear genome. The analysis mode selection module selects either a dual-kine analysis mode or a mononuclear analysis mode based on the whole-genome sequencing data acquired by the data upload module. The analysis execution module automatically invokes the corresponding analysis process according to the analysis mode selected by the analysis mode selection module. For the binuclear strain to be tested: the whole genome sequencing data of the binuclear strain to be tested with a sequencing depth that meets the analysis requirements is obtained and the data is filtered to obtain effective sequencing data; the effective sequencing data is paired with a reference monokaryotic genome in the genome database to perform k-mer integrity analysis and obtain k-mer integrity values; the k-mer integrity values ​​are compared with a preset integrity threshold to determine whether the binuclear strain to be tested is a specific germplasm. For the monokaryotic hyphae to be tested: the genome sequencing data of the monokaryotic hyphae to be tested is obtained and the genome is assembled to obtain the monokaryotic genome to be tested; the monokaryotic genome to be tested is collinearly aligned with each reference genome in the genome database, and the percentage of base difference is calculated based on the Jaccard similarity coefficient; the percentage of base difference is compared with a preset difference threshold to determine whether the monokaryotic hyphae to be tested is a specific germplasm. The result display module is used to output the determination results of the analysis execution module. In a specific embodiment, a web-based automated analysis platform was developed based on the above identification system. The platform operation process is as follows: Users log in to the platform, enter the data upload page, upload sequencing data files. For dual-core analysis, two FASTQ format files (R1 and R2) need to be uploaded; for single-core analysis, a third-generation sequencing data FASTQ file and a second-generation sequencing data FASTQ file need to be uploaded. Select the dual-core or single-core analysis mode, fill in the sample name and strain number; submit the task, click "Start" in the task center, and perform the analysis based on the genome database module and analysis execution module. Wait for the analysis to complete; view the specificity determination results, click "Export" to download the complete identification report in Excel format, which includes the comparison results.

[0088] This invention also provides a device for identifying Ganoderma lucidum germplasm specificity based on whole-genome differences, including a processor and a memory; the processor and the memory are connected via a communication bus; wherein, the processor is used to call and execute a Ganoderma lucidum germplasm specificity identification program stored in the memory; the memory is used to store the program, which is at least used to execute the Ganoderma lucidum germplasm specificity identification method based on whole-genome differences, specifically including the following steps: performing k-mer integrity analysis on binucleate strains, or performing genome assembly and whole-genome alignment on monokaryotic hyphae, to determine the Ganoderma lucidum germplasm specificity.

[0089] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A method for identifying Ganoderma lucidum germplasm-specific characteristics based on genome-wide differences, characterized in that: include: Provide a genome database containing a specifically identified reference mononuclear genome; For the binuclear strain to be tested: obtain whole genome sequencing data of the binuclear strain to be tested with sequencing depth that meets the analysis requirements and perform data filtering to obtain effective sequencing data; perform k-mer integrity analysis on the pairwise combinations of the effective sequencing data and the reference mononuclear genome in the genome database to obtain k-mer integrity values; compare the k-mer integrity values ​​with a preset integrity threshold to determine whether the binuclear strain to be tested is a specific germplasm; For the monokaryotic hyphae to be tested: obtain the genome sequencing data of the monokaryotic hyphae to be tested and assemble the genome to obtain the monokaryotic genome to be tested; perform collinearity comparison analysis between the monokaryotic genome to be tested and each reference genome in the genome database, and calculate the percentage of base difference based on the Jaccard similarity coefficient; compare the percentage of base difference with a preset difference threshold to determine whether the monokaryotic hyphae to be tested is a specific germplasm.

2. The method as described in claim 1, characterized in that, The method for determining whether the binuclear strain to be tested is a specific germplasm includes: when the k-mer integrity value is ≥98%, the binuclear strain to be tested is determined to be highly similar to the pairwise combinations of the current reference mononuclear genome and is a non-specific germplasm; when the k-mer integrity value is <98%, the binuclear strain to be tested contains a new mononuclear component and is a specific germplasm.

3. The method as described in claim 1, characterized in that, The method for determining whether the monokaryotic hyphae to be tested is a specific germplasm includes: when the percentage of base difference is ≤1%, the monokaryotic hyphae to be tested is determined to be highly similar to the current reference genome and is a non-specific monokaryotic hyphae; when the percentage of base difference is >1%, the monokaryotic hyphae to be tested has significant genetic differences from the current reference genome and is a specific monokaryotic hyphae.

4. The method as described in claim 1, characterized in that, Obtain whole-genome sequencing data of the target binuclear strain with a sequencing depth sufficient for analysis, including: performing whole-genome paired-end sequencing of the target binuclear strain using a next-generation sequencing platform; filtering the sequencing data, including at least one of the following: removing reads containing sequencing adapter sequences; cleaving and removing non-AGCT bases at the 5' end; filtering sequences with N base content greater than 10%; filtering sequences with low-quality base content greater than 50%; and removing PCR repeats.

5. The method as described in claim 1, characterized in that, The method for obtaining genome sequencing data of monokaryotic hyphae to be tested and assembling the genome includes: using a third-generation sequencing platform to perform long-read sequencing of the monokaryotic hyphae to be tested, and simultaneously using a second-generation sequencing platform to perform paired-end sequencing; after data preprocessing, the third-generation sequencing data is used for preliminary genome assembly, and the second-generation sequencing data is used for error correction and refinement of the assembly results to obtain the monokaryotic genome to be tested.

6. The method as described in claim 1, characterized in that, The percentage of base differences is calculated based on the Jaccard similarity coefficient, and the formula is: Percentage of differences = [1 - (size of common part of the genome to be tested + size of common part of the reference genome) / (total size of the genome to be tested + total size of the reference genome)] × 100%; wherein, the size of the common part is determined by the collinearity alignment analysis results.

7. The method as described in claim 1, characterized in that, The collinearity alignment analysis uses parameters such as a minimum matching cluster length of not less than 500 base pairs and a thread number of not less than 20 to perform whole-genome alignment. The alignment results are analyzed to obtain collinearity blocks, and the percentage of base differences is calculated based on the collinearity blocks.

8. The method as described in claim 1, characterized in that, The genome database is constructed as follows: Ganoderma lucidum germplasm resources are collected, single nuclei are extracted using protoplast isolation technology, and single-nucleate hyphae are obtained by culturing; genomic DNA of the single-nucleate hyphae is extracted and subjected to third-generation sequencing and second-generation sequencing; after genome assembly, quality assessment and specificity identification, single-nucleate genomes that meet the quality standards are included in the genome database; the genome database is regularly updated and expanded.

9. A Ganoderma lucidum germplasm-specific identification system based on genome-wide differences, characterized in that, include: A genome database module for providing a genome database containing a specifically identified reference mononuclear genome; The data upload module is used to acquire the whole genome sequencing data of the binucleate strains to be tested with sequencing depths that meet the analysis requirements, and to perform data filtering to obtain effective sequencing data; The genome sequencing data of the monokaryotic hyphae to be tested were obtained and the genome was assembled to obtain the monokaryotic genome to be tested; The analysis mode selection module is used to select between dual-core and single-core analysis modes based on the whole-genome sequencing data obtained from the data upload module. The analysis execution module is used to automatically invoke the corresponding analysis process based on the analysis mode selected by the analysis mode selection module. For the binuclear strain to be tested: obtain whole genome sequencing data of the binuclear strain to be tested with sequencing depth that meets the analysis requirements and perform data filtering to obtain effective sequencing data; perform k-mer integrity analysis on the pairwise combinations of the effective sequencing data and the reference mononuclear genome in the genome database to obtain k-mer integrity values; compare the k-mer integrity values ​​with a preset integrity threshold to determine whether the binuclear strain to be tested is a specific germplasm; For the monokaryotic hyphae to be tested: obtain the genome sequencing data of the monokaryotic hyphae to be tested and assemble the genome to obtain the monokaryotic genome to be tested; perform collinearity comparison analysis between the monokaryotic genome to be tested and each reference genome in the genome database, and calculate the percentage of base difference based on the Jaccard similarity coefficient; compare the percentage of base difference with a preset difference threshold to determine whether the monokaryotic hyphae to be tested is a specific germplasm; The results display module is used to output the judgment results of the analysis and execution module.

10. A Ganoderma lucidum germplasm-specific identification device based on whole-genome differences, characterized in that, It includes a processor and a memory; the processor and the memory are connected via a communication bus; wherein the processor is used to call and execute a program stored in the memory; the memory is used to store the program, which is at least used to execute the Ganoderma lucidum germplasm specificity identification method based on whole-genome differences as described in any one of claims 1-8.

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