Agaricus bisporus AbPPO1 gene haplotype and application thereof
By screening the haplotype of AbPPO1 gene of Agaricus bisporus, the problem of unclear molecular mechanism of the white cap was solved, and efficient breeding methods were achieved. The AbPPO1 gene was identified as the control gene of the white cap, which improved the accuracy and efficiency of breeding.
Patent Information
- Application Number
- CN202510903596.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-01
- Publication Date
- 2025-08-22
AI Technical Summary
The prior art is difficult to effectively screen and analyze the molecular mechanism of the white cap of Agaricus bisporus, resulting in insufficient strain screening methods and affecting breeding efficiency.
AAAA bisporus AbPPO1 gene haplotype and its application are provided. By designing specific primers for PCR amplification and sequencing, strains with white caps were screened out.
The molecular origin of the white cap was clarified, detailed genetic information was provided for the genetic breeding of Agaricus bisporus, improved the accuracy and efficiency of breeding, and identified the AbPPO1 gene as the control gene for the white cap.
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Figure CN120519620A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of genetic breeding technology, and specifically relates to a Agaricus bisporus AbPPO1 Gene haplotypes and their applications. Background Art
[0002] Agaricus bisporus[ Agaricus bisporus (JE Lange) Imbach] is rich in polysaccharides, proteins, amino acids and other nutrients. It is the most widely cultivated and produced edible fungus in the world. The color of the Agaricus bisporus cap varies greatly. In the wild, it is mainly brown, but there are also light brown and cream colors. In 1926, it was produced. A. bisporus White strains of the species are now widely cultivated worldwide. In recent years, numerous research teams worldwide have investigated the genetic basis of cap color in Agaricus bisporus. By constructing genetic maps of segregating populations, they discovered that the major QTL controlling cap color variation is located on chromosome 8, with the white cap being controlled by a recessive allele at the PPC1 locus on chromosome 8. Furthermore, smaller QTLs have been detected on chromosomes 3, 7, 10, and 13. Although these studies used genome-wide SNP markers, the low rate of genetic recombination between markers meant that QTLs could only be mapped to the chromosomal level. Furthermore, the color and shape of edible mushrooms are related to the melanin biosynthesis pathway, but current research has primarily focused on the mechanisms of post-harvest browning in Agaricus bisporus. The polyphenol oxidase (PPO) family of enzymes has been implicated in browning, and the six PPO genes in the Agaricus bisporus genome have been shown to have distinct physiological roles. Therefore, except for the known major QTL loci for the cap color of Agaricus bisporus, the molecular mechanism of cap color variation, especially the formation of white caps in cultivated strains, is not clear, and the screening method of strains needs to be updated. Summary of the Invention
[0003] In view of the above-mentioned prior art, the present invention provides a Agaricus bisporus AbPPO1 Gene haplotype and its application can effectively screen Agaricus bisporus strains with white caps.
[0004] In order to achieve the above object, the technical solution adopted by the present invention is to provide a Agaricus bisporus AbPPO1 Gene haplotype, Agaricus bisporus AbPPO1 The nucleotide sequence of the gene haplotype is shown in SEQ ID No.1.
[0005] The present invention also includes the following technical solutions.
[0006] Further, Agaricus bisporus AbPPO1 Application of gene haplotypes in screening and breeding of white varieties of Agaricus bisporus.
[0007] Further, screening for Agaricus bisporus AbPPO1 Genetic haplotypes of Agaricus bisporus.
[0008] Furthermore, the screening method includes the step of using two pairs of primers for detection; the nucleotide sequence of primer 1 is shown as SEQ ID No.2 and SEQ ID No.3, and the nucleotide sequence of primer 2 is shown as SEQ ID No.4 and SEQ ID No.5.
[0009] Furthermore, the screening method is as follows: extracting Agaricus bisporus genomic DNA, using the Agaricus bisporus genomic DNA as a template and using two pairs of primers to prepare PCR reaction systems, performing PCR amplification, performing agarose gel electrophoresis and sequencing on the PCR amplification products, and if the amplified sequences obtained by the two pairs of primers are consistent with AbPPO1 Gene haplotype sequence alignment is consistent or contains AbPPO1 Gene haplotype sequence, indicating that the Agaricus bisporus genome contains the AbPPO1 The nucleotide sequence of the gene haplotype.
[0010] Furthermore, the annealing temperature of the PCR process was 55°C and the extension time was 10 s.
[0011] Furthermore, PCR amplification was performed using two pairs of primers, and the resulting band lengths were 221 bp and 278 bp.
[0012] The beneficial effects of the present invention are as follows: the present invention utilizes resequencing data from a large number of wild and cultivated strains of Agaricus bisporus from around the world to clarify the species and cultivation origins, population dynamics history, and the molecular origins of the white cap trait of this most widely cultivated edible fungus. This is the first time that the complex natural evolutionary history of large fungi represented by Agaricus bisporus has been revealed, and it provides more detailed genetic information for the genetic breeding of Agaricus bisporus. The present invention identifies a candidate domestication gene AbPPO1 , AbPPO1 A functional mutant allele of a gene, i.e. AbPPO1 The Hap_2 haplotype of the gene causes the cap to appear white and was selected during the domestication and breeding of Stropharia; Agaricus bisporus AbPPO1 The gene cap color regulation mechanism provides an important target for molecular marker-assisted breeding, and the Hap_2 haplotype can be used as a marker for the selection of white varieties of Agaricus bisporus. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 Comparison of Agaricus bisporus cap color among different groups; Figure 2Analysis of XP-CLR selective clearance; A is the comparison between France Ⅰ and Mix Ⅱ, B is the comparison between France Ⅱ and Mix Ⅱ, and C is the comparison between Greece and Mix Ⅱ; Figure 3 The melanin synthesis pathway and genes of Agaricus bisporus; the red letters indicate the genes located by GWAS, and the red arrows indicate the genes significantly upregulated in brown fruiting bodies; Figure 4 This is the Manhattan plot of the GWAS for Agaricus bisporus cap color; A and B are the analysis results of Agaricus bisporus cultivated twice respectively; Figure 5 This is the module where WGCNA is significantly correlated with cap color; Figure 6 It is the GO function enrichment of modules related to cap color in WGCNA; Figure 7 KEGG functional enrichment of modules significantly related to cap color in WGCNA; Figure 8 for AbPPO1 The haplotype block and recombination rate of the region where the gene is located; Figure 9 Agaricus bisporus AbPPO1 Gene NJ phylogenetic tree (left) and functional mutation site genotype (right); 0 / 0 represents reference, 1 / 1 represents alteration, and 0 / 1 represents heterozygosis; Figure 10 Agaricus bisporus AbPPO1 Genes contain more than 10 strains of haplotype cap color statistics; Figure 11 Agaricus bisporus AbCM Gene haplotype distribution (A) and comparison of haplotype cap color (B); Figure 12 Agaricus bisporus Ablaccase-1 Gene haplotype evolutionary network (A) and comparison of haplotype cap color (B); Figure 13 Agaricus bisporus Ablaccase-2 Gene haplotype evolutionary network (A) and comparison of haplotype cap color (B); Figure 14 NJ phylogenetic tree of GWAS candidate genes; Figure 15 for AbPPO1 NJ phylogenetic tree of the gene-constructed population (A) and the breeding history of commercial hybrid strains of Agaricus bisporus (B); the inner circle in A represents the population, and the outer circle represents the white commercial strain; Figure 16The cap color of the hybridization experiment and genetic transformation experiment between AS2796 and Bs284A; Figure 17 The statistical results of cap color of hybridization experiment and genetic transformation experiment of AS2796 and Bs284A are shown; Figure 18 These are the amino acid variation sites of the AbPPO1 gene haplotypes Hap_2, Hap_3, and Hap_41; Figure 19 Prediction of the spatial structure of AbPPO1 gene haplotypes Hap_2, Hap_3, and Hap_41; Figure 20 The results of agarose gel electrophoresis of PCR amplification products are shown. DETAILED DESCRIPTION
[0014] The specific implementation methods of the present invention are described in detail below with reference to the embodiments.
[0015] Example 1 1. Agaricus bisporus Germplasm Resequencing and Cultivation 475 Agaricus bisporus A. bisporus Core strains, 3 samples of Qilian mushroom A. qilianensis 4 servings of Agaricus tetrasporus A. sinotetrasporus and 1 serving A. subfloccosus The strain was resequenced. The strain was cultured in culture medium at 25°C for 20 days, and genomic DNA from fresh mycelium was extracted using the CTAB method. The genomic DNA was sequenced at Zhejiang Annoroad Biotechnology Co., Ltd. A 350bp paired-end library was constructed and high-throughput sequencing was performed using the MGI DNBSEQ T7 sequencing platform from BGI, with a read length of 150 pe / s and a sequencing depth of at least 100×.
[0016] Agaricus bisporus strains stored at 4°C were inoculated into a cultivation medium and activated twice. Two cultivation experiments were conducted, both under controlled indoor environmental conditions. Agaricus bisporus strains were inoculated into the cultivation medium and allowed to fully grow within 30–45 days at 25°C. Fruiting bodies emerged 15 days after covering with soil. The fruiting temperature was 18°C and the humidity was approximately 95%. Fruiting bodies were harvested when they reached 5–6 minutes of cap opening. The cap skin color was measured using a colorimeter (NR110, 3nh, Shenzhen), with a color range of 50 (dark brown) to 94 (pure white).
[0017] Descriptive statistical analysis of the phenotypic data was performed using SPSS, including normality tests and checks for phenotypic extremes. The coefficient of variation (CV) was used to measure the phenotypic variation of Agaricus bisporus strains within the associated population. Correlations between phenotypes were calculated using the R package psych.
[0018] 2. Variant Detection and Annotation The raw sequencing reads were filtered for low quality, adapter contamination, and duplicate reads to generate high-quality clean reads. The chromosomally assembled genome of Agaricus bisporus var. bisporus H39 was used as the reference genome. The H39 genome consists of 13 chromosomes and has a genome size of 30.78 Mb. The resequencing clean reads were aligned to the H39 reference genome using BWA 0.7.17 (default parameters) to generate a sam file. The aligned sam file was converted to a bam file using SAMtools. The bam file was sorted using Picard and PCR-induced duplicates were removed. The resulting bam file was statistically analyzed using Qualimap 2.
[0019] Variant calls were performed using the joint calling strategy of GATK 4.2.0.0. Single-sample variant calls were performed using the HaplotypeCaller program with the parameter "-stand-call-conf 40.0". All sample variant files were merged using the CombineGVCFs program. Genotyping results for all samples were obtained using the GenotypeGVCFs program. Single-nucleotide polymorphisms (SNPs) were extracted using the SelectVariants program. The distribution of SNP QD, QUAL, SOR, FS, MQ, MQRankSum, and ReadPosRankSum parameters was analyzed using the VariantsToTable program. Hard filtering was performed using the VariantFiltration program with the following SNP hard filtering parameters: QUAL < 30.0 || QD < 4.0 || SOR > 3.0 || FS > 60.0 || MQ < 40.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0.
[0020] To identify high-confidence variants, SNPs were soft-filtered using VCFtools. Based on the individual sequencing depth distribution, SNPs with a minimum sequencing depth of less than 15 and a maximum sequencing depth greater than 220 were filtered out. To obtain accurate genotypes, heterozygous sites within haploids and homozygotes (heterozygosity less than 0.4%) were filtered out, as were SNPs with a genotype quality value less than 50. SNPs with SNP coverage less than 50% and a minor allele frequency (MAF) of 0 were filtered out, and only biallelic SNPs were retained. This resulted in a total of 3,724,177 high-quality SNPs.
[0021] Funannotate was used to predict and annotate genes for the reference genome H39. SnpEff was used to annotate detected variants. PLINK was used to calculate individual missingness and heterozygosity, and to calculate SNP missingness and MAF.
[0022] 3. Genetic diversity and linkage disequilibrium analysis Plink was used to calculate genetic diversity (Pi) and Tajima's D values for all SNPs in each population, using a window size of 20K and a step size of 5K. The pairwise differentiation index (Fst) was calculated for all populations using the same window and step size. LD decay within each group was calculated using PopLDdecay using a 10 kb window.
[0023] 4. Population History Inference To effectively infer the population histories of the 10 defined populations, MSMC2, PSMC2, and Beta-PSMC were used for estimation with default parameters, with a mutation rate of 1.5e-9 and an epoch time of 1 year per generation.
[0024] 5. Population differentiation and gene flow analysis To fully understand gene flow, we first calculated the f-branching test using Dsuite v.0.4r38 with the population tree as input topology. To elaborate on the proposed gene flow, we performed a qpGraph-based best model analysis using the ADMIXTOOLS package, which included population differentiation and admixture.
[0025] 6.GWAS and detection of significant association sites The heritability of phenotypic traits was calculated using GCTA, and the broad-sense heritability (H 2 ) measures the proportion of genetic variation in each trait of the strain to phenotypic variation. Single-point LMM analysis was performed using GEMMA for each trait and SNP with MAF greater than 0.05, with a significance P value set at 1×10 -4 The GWAS results were visualized using the R package CMplot for Manhattan plots and QQ-plots. Intervals with an LD less than 0.25 with significantly associated SNPs on the same chromosome were considered a SAL, and the distribution of SALs on the chromosomes was visualized using TBtool.
[0026] FastEPRR was used to calculate the recombination rate within the population, and Plink was used to calculate the haplotype blocks and homozygous segments within the population.
[0027] 7. Haplotype analysis Since most mutations in genes are synonymous, their amino acid sequence and function remain unchanged. Only a few variants have the potential to alter the amino acid sequence, including nonsynonymous SNPs, frameshift indels, and variants in splice sites, promoter regions, start codons, and stop codons. These functional mutations in combination generate distinct haplotypes, thus allowing for the examination of relationships between haplotypes and traits. If these haplotypes are significantly correlated, the gene is likely to be associated with trait variation. Haplotypes were identified using DnaSP v5, and haplotype networks were constructed using POPART. Analysis of variance (ANOVA) was used to test for significant differences between major haplotypes (involving at least 10 samples).
[0028] FastEPRR was used to calculate the recombination rate within the population, and Plink software was used to calculate the haplotype blocks and runs of homozygosity (ROH) within the population. Alphafold2 ( https: / / colab.research.google.com / github / sokrypton / ColabFold / blob / main / AlphaFold2.ipynb?pli=1#scrollTo=kOblAo-xetgx ) predicted the protein structures of haplotypes, and compared the protein structures pairwise using PyMOL v.3.0.3.
[0029] 8. Transcriptome Analysis Transcriptome sequencing was performed on fruiting bodies with significant color variation in the cap. RNA from the cap tissue was extracted using the Quick RNA Isolation Kit (Huayueyang biotech, Beijing, China), with three biological replicates per strain. RNA degradation and contamination were assessed using agarose gel electrophoresis, and RNA quality was assessed using a Unano-1000 Micro-spectrophotometer (UMI Instrument, Hangzhou, China) and an Agilent 2100 bioanalyser (Agilent Technologies). Qualified RNA was sent to Annoroad for sequencing using the Illumina HiSeq sequencing platform.
[0030] The transcriptome sequencing raw data was assessed for sequencing quality using FastQC (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ), and Trimmomatic was used for data filtering to obtain clean data. The clean data was aligned to the reference genome using Hisat2. A. bisporusH39, gene-level quantification was performed for all samples using featureCounts, and normalized gene expression levels were obtained using FPKM conversion. Significantly differentially expressed genes were calculated using DESeq2, and gene co-expression network analysis was performed using the R package WGCNA, and co-expression networks were visualized using Cytoscape.
[0031] 9. AbPPO1 Gene haplotype functional verification Agaricus bisporus strain As2796 (white cap, AbPPO1 Hap_2 / Hap_3 ) and Bs284A (brown cap, AbPPO1 Hap_41 ) homokaryon offspring. Then hybridization and fruiting were performed, and the statistical genotypes were measured. AbPPO1 Hap _2 / Hap_41 and AbPPO1 Hap_3 / Hap_41 The color of the fruiting body cap.
[0032] In the gene function verification, primers Abgpd-F and Abgpd-R (nucleotide sequences are shown in SEQ ID No.8 and SEQ ID No.9) were used to amplify the promoter gpd II of Agaricus bisporus, and primers AbPPO1-F and AbPPO1-R (nucleotide sequences are shown in SEQ ID No.10 and SEQ ID No.11) were used to amplify the promoter gpd II of strain Bs284A (brown cap). AbPPO1 Hap_41 Gene. Then use the one-step cloning kit to clone the promoter and AbPPO1 Hap_41 The gene was ligated into the plasmid pEHg-gdp-hsp20 digested with BamHI and BstEII to generate the expression vector pHg-LeAbgpd-PPO1. This plasmid contains a Lentinula edodes gpd promoter expressing the hygromycin resistance gene and a Agaricus bisporus gpd promoter expressing the AbPPO1 Hap_41 The plasmid was transformed into strain As2796 (white cap) via Agrobacterium-mediated transformation. Primary screening was performed using PDA medium containing 10 μg / mL hygromycin and 400 μg / mL cefotaxime. Resistant bacterial blocks were then transferred to PDA medium containing 20 μg / mL hygromycin for secondary screening. PCR verification was performed using OEPPO1-F and OEPPO1-R (nucleotide sequences shown in SEQ ID No. 12 and SEQ ID No. 13), and positive transformants amplified a 2720 bp band, which contained 2228 bp. AbPPO1 Hap_41The positive transformants were subjected to fruiting experiments, and the cap color was measured and counted.
[0033] Experimental results and analysis: Commercial strains of Agaricus bisporus are derived only from the original variant and are mainly distributed in the Mix II population of the original variant. Figure 1 The statistical analysis of the differences in the cap colors of the three populations (Greece, France Ⅱ, France Ⅰ, Mix Ⅲ, Mix Ⅱ, Mix Ⅰ, America and Highland) showed that the cap color of the Mix II population was significantly whiter than that of the other populations except Highland and America ( Figure 1 ).
[0034] To identify the molecular signals associated with cap color, selective sweep analysis was performed between different groups, and the results were as follows Figure 2 As shown in the figure, 577 selected genes were detected when France Ⅰ was compared with Mix Ⅱ; 577 selected genes were detected when France Ⅱ was compared with Mix Ⅱ; and 405 selected genes were detected when Greece was compared with Mix Ⅱ. The selected genes that existed in all three pairwise comparisons included the chromosome 3 transcription factor PPR1, the chromosome 10 gene Stearoyl-CoA desaturase (SCD) and chromosome 13 genes β-hexosaminidase 1 (β-Hex). PPR1 is essential for oxidative stress and spore formation in yeast division bodies. SCD catalyzes desaturation reactions by inserting double bonds in fatty acyl chains to produce unsaturated fatty acids, which play a vital role in the synthesis of polyunsaturated fatty acids. β-Hex belongs to the glycosyl hydrolase family and is involved in the utilization of chitosan, cell growth, and pathogen invasion. The selected genes also include multiple melanin synthesis pathway genes: polyketide synthase (PKS), polyphenol oxidase (PPO1), laccase (LAC), 4-aminobenzoate hydroxylase (4ABH), such as Figure 3 As shown, these selected genes were also located in the cap color GWAS. Figure 2 middle.
[0035] Based on the cap color data of two cultivations, 415 samples were analyzed by using the linear mixed model (LMM) implemented in the tool GEMMA. A. bisporus A genome-wide association study (GWAS) was conducted to locate A. bisporusThe loci that determine corolla color during the domestication and breeding selection of the hybrid were identified using both genotypic and phenotypic datasets. A total of 205 significant trait-associated SNPs (-Log10(p)>4.0) were identified, such as Figure 4 As shown, 38 significantly associated loci (SALs) related to cap color were subsequently defined based on LD in the significant association region. They were distributed on 13 chromosomes, and co-localized intervals existed on chromosomes 7, 8, 10, 12, and 13 ( Figure 4 GWAS located 12 genes (CM, PKS, PPO, PDH, LAC, 4ABH) on chromosomes 7, 8, 10, 11, and 12, which are known genes in the melanin synthesis pathway of Agaricus bisporus ( Figure 1 and Figure 3 GWAS gene mapping results showed that the major effect QTL quantitative trait locus (QTL) for Agaricus bisporus cap color variation was located on chromosome 8, and the results of small effect QTLs on chromosomes 3, 7, 10, and 13 were consistent.
[0036] To further explore A. bisporus The whole genome gene expression profile of the strain was analyzed, and weighted gene co-expression network analysis (WGCNA) was performed in this strain, and 18 different co-expression modules were found, which may be related to the cap color (correlation > 0.50, p <0.01), including 108 genes mapped by GWAS, including the melanin synthesis pathway AbCM 、 Ab4ABH 、 AbPKS Gene( Figure 5 ). We focused on the co-expression modules (including candidate genes) related to cap color and the pathways involved. The results showed the mechanism of action of the related pathways. Post-translational cell differentiation, reproduction, ATP hydrolysis, amino acid metabolism and transport, protein modification, redox, osmotic pressure response, ion transport, abiotic stress response, etc. ( Figure 6 and Figure 7 ), indicating that the physiological processes related to cap color mainly involve growth and development and environmental adaptation.
[0037] On chromosome 8, where the main QTL for Agaricus bisporus cap color variation is located, the GWAS co-localization interval was located. AbPPO1 Gene, encoding a key enzyme in melanin synthesis, a polyphenol oxidase (PPO), involved in the melanin synthesis pathway of GHB, L-DOPA, and catechol ( Figure 3 ). Calculate the recombination rate and haplotype block of the first 100kb of chromosome 8 and find AbPPO1 The recombination rate of the gene and the surrounding area is 0, and there are many haplotype blocks and long homozygous segments (such as Figure 8 ), indicating AbPPO1 Gene variation is caused by mutation, not genetic recombination. AbPPO1 There are 24 functional mutation sites in the gene, and 113 haplotypes have been identified (e.g. Figure 9 and Table 1), and 98% of wild strains AbPPO1 The gene is a homozygous genotype of haplotype Hap_4 - Hap_113, and the cultivated strain AbPPO1 The genotype is homozygous or heterozygous for the three unique haplotypes (Hap_1, Hap_2, Hap_3). AbPPO1 The correlation between gene haplotype and color was analyzed. The cap color of haplotypes containing more than 10 strains was statistically analyzed. It was found that the strain with haplotype Hap_2 had the whitest cap, followed by the haplotypes Hap_5 and Hap_13, which were most closely related to Hap_2. The cap color of the haplotypes from France I-II and Greece was the darkest ( Figure 10 Agaricus bisporus PPO The six genes in the family have distinct expression patterns. PPO Gene function is conserved within a population. AbPPO3 and AbPPO4 The expression level is extremely high (mean FPKM>12,000), which is consistent with the reported studies; on chromosome 8 AbPPO1 and AbPPO6 There are multiple functional mutation sites. AbPPO1 The expression level meanFPKM>200 indicates constitutive expression. AbPPO6 The expression level was extremely low (mean FPKM < 2.5), suggesting that cap color variation should be mainly due to gene function variation rather than changes in gene expression.
[0038] Table 1 Variation information of melanin synthesis genes mapped by GWAS
[0039] The mutations of melanin synthesis-related genes on chromosomes 7, 10, 11, and 12, which are involved in the color variation of Agaricus bisporus cap located by GWAS, were counted (Table 1). AbCM Gene( Figure 3), there is a functional mutation site, and 78.80% of the strains are homozygous genotypes. The homozygous genotype Reference: C / C exists in eight populations, and only exists in the domesticated population Mix Ⅱ. Another homozygous genotype (Alt: T / T) exists only in the America, Mix Ⅰ, and Greece populations. The heterozygous genotype (Het: C / T) has a dark cap color and exists in the other seven populations except Mix Ⅱ ( Figure 11 ). Participates in the DHN melanin synthesis pathway AbPKS Gene( Figure 3 ), there are 222 functional mutation sites, only 7.71% of the strains are homozygous genotypes, and all commercial strains are heterozygous genotypes. Transcriptome analysis found that its expression level was significantly upregulated in brown fruiting bodies. AbPDH There are 25 functional mutation sites in the gene, 12.53% of the strains are homozygous genotypes, 33 haplotypes were identified, and commercial strains are homozygous or heterozygous for the haplotypes Hap_1, Hap_2, Hap_3, and Hap_4. AbLaccase Gene, AbLaccase-1 There are 15 functional mutation sites in the gene, 77% of wild strains are homozygous genotypes, and 34 haplotypes have been identified ( Figure 12 ), the commercial strains are homozygous or heterozygous for the Hap_1, Hap_2, and Hap_3 haplotypes; AbLaccase-2 There are 12 functional mutation sites in the gene, 69% of wild strains are homozygous genotypes, and 41 haplotypes have been identified ( Figure 13 ), the commercial strains are homozygous or heterozygous for the haplotypes Hap_1, Hap_2, Hap_3, and Hap_4; AbLaccase-3 There were 15 functional mutation sites in the gene, 51.81% of the strains were homozygous genotypes, and 14 haplotypes were identified. The commercial strains were homozygous or heterozygous for the Hap_1, Hap_2, and Hap_3 haplotypes. AbLaccase-1 and AbLaccase-2 The haplotype color statistics of the gene showed that there was no significant difference between different haplotypes ( Figure 12 and Figure 13 ), transcriptome analysis found that their expression levels were significantly upregulated in brown fruiting bodies. Ab4ABH Participate in the GHB melanin synthesis pathway ( Figure 3 ), the proportion of homozygous genotypes is between 20% and 30%.
[0040] To gain some preliminary insights into the selection process for these 11 genes, we used a panel of genes that included those detected by selective sweep analysis and GWAS. AbPKS 、 AbPPO1 、 AbPDH 、 AbLaccase-1 、 AbLaccase-2 、 AbLaccase-3 and 5 Ab4ABH Genes were subjected to phylogenetic analysis, except AbPPO1 In the phylogenetic relationships of genes other than the β-catenin gene, cultivated strains and wild strains cannot be distinguished, and gene evolution is inconsistent with population division ( Figure 14 ), so the cap color variation may not be significantly related to their functional mutation evolution. AbPPO1 In the phylogenetic relationship, commercial strains from Mix Ⅰ / Ⅱ / Ⅲ clustered together, the Highland population clustered into a separate group, and most of America, France Ⅰ, France Ⅱ, and Greece formed clusters separately. This is consistent with the mixed origin of the original variant of Agaricus bisporus, and is basically consistent with the phylogenetic relationship of the population and the haplotype evolution network ( Figure 8 and Figure 15 A). The above research results show that AbPPO1 The genes not only directly reflect the evolutionary relationships among Agaricus bisporus populations, but are also directly related to domestication.
[0041] Currently, the two most widely used white cap commercial strains in the world are both from the Mix II group. They are represented by strains U1 and U3, including the U hybrid series of strains A15, 901 and other strains they systematically bred; and the Chinese AS2796 hybrid series represented by AS2796, including strains W192, W2000 and other strains. AbPPO1 The genotypes are both Hap_1 / Hap_2, one of its hybrid parents has the pure white mutant haplotype Hap_2, and the other parent has the cream haplotype Hap_1 ( Figure 15 B). For hybrid strains of the Chinese AS2796 series, AbPPO1 The gene haplotype is haplotype Hap_2 / Hap_3, and Hap_3 is most closely related to Highland, which is consistent with the literature report that one parent is a U series strain (with haplotype Hap_2) and the other is a Chinese wild strain (with haplotype Hap_3). Figure 15 B). Therefore, AbPPO1 Gene mutations are the primary genetic basis for the diverse cap color phenotypes of Agaricus bisporus. During domestication, a cream-capped strain mutated to produce the pure white cap haplotype Hap_2. This mutant was then hybridized with cream-capped strains with haplotypes Hap_1 and Hap_3 in breeding, resulting in a pure white cap strain that is now widely cultivated worldwide. This coincides with the hybridization history of commercial white-capped Agaricus bisporus strains.
[0042] It is worth noting that the commercial strain A15, which is now widely cultivated, was bred using the U1 system. AbPPO1 The gene has only Hap_1 haplotype, but A15 and 901 have exactly the same genetic background as the U series, and Ablaccase-1, Ablaccase-2 、 Ablaccase-3 、 Ab4ABH The haplotype of the gene is consistent with the U1 haplotype, so we believe that continuous systematic breeding has caused the AbPPO1 The loss of the gene Hap_2 haplotype. In short, AbPPO1 As the main effect gene of cap color variation, the gene has rich haplotypes, and the evolution of the gene is basically consistent with the population evolution. Some haplotypes are easily lost in the continuous breeding of cultivated strains, so AbPPO1 The gene is not only the main effect gene that controls the color of the Agaricus bisporus cap, but also prone to mutation, which can easily lead to various changes in the color of the Agaricus bisporus cap.
[0043] Pass AS2796 (white, AbPPO1 Hap_2 / Hap_3 ) and Bs284A (brown, AbPPO1 Hap_41 ) Homokaryon hybridization experiment, measurement and statistics of hybrid offspring ( AbPPO1 Hap_2 / Hap_41 or AbPPO1 Hap_3 / Hap_41 ) of the cap color, the cap color of the white strain AS2796 was 91.51 ± 0.68, the brown strain Bs284A was 64.72 ± 5.07, and the hybrid offspring was 77.96 ± 5.56, which was close to the average cap color of the parents (76.44). Figure 16 and Figure 17 As shown in the figure, CK represents AS2796 (white, AbPPO1 Hap_2 / Hap_3 ), WT_Hap_41 expressed Bs284A (brown, AbPPO1 Hap_41 ), Hybrid indicates the hybrid offspring of AS2796 and Bs284A, and TG_Hap_41 indicates the haplotype expressed in AS2796 by Agrobacterium-mediated genetic transformation AbPPO1 Hap _41 strains.
[0044] Haplotypes were expressed in the white strain AS2796 by Agrobacterium-mediated genetic transformation AbPPO1 Hap_41 , AbPPO1 Hap_41 The transgenic strain had a cream-colored cap and was 86.63 ± 1.42 ( Figure 16and Figure 17 ),show AbPPO1 Functional mutations cause color variation in the Agaricus bisporus cap. 3 haplotypes AbPPO1 Hap_2 、 AbPPO1 Hap_3 and AbPPO1 Hap_41 There are 18 amino acid variation sites ( Figure 18 ), protein structure prediction reveals haplotype AbPPO1 Hap_2 and AbPPO1 Hap_3 The spatial structure of the α-D-type ... AbPPO1 Hap_2 and AbPPO1 Hap_41 、 AbPPO1 Hap_3 and AbPPO1 Hap_41 The spatial structure of the two groups cannot achieve significant three-dimensional overlapping effects, and the RMSDs are 0.327 and 0.343 respectively ( Figure 19 ),show AbPPO1 Amino acid variations cause changes in protein structure and therefore alter protein function.
[0045] This study mapped the cap color-related genes to chromosome 8 AbPPO1 The gene is a key enzyme in melanin synthesis. The results of haplotype analysis showed that AbPPO1 The haplotype Hap_2 of the gene is crucial for selecting the white cap phenotype. AbPPO1 The haplotype evolution of the genus was positively correlated with population evolution and changes in cap color.
[0046] In summary, by resequencing the whole genome of 475 Agaricus bisporus accessions worldwide and combining it with population structure analysis, we systematically revealed the previously undescribed population history and complex network evolution process of Agaricus bisporus, and provided insights into the origin of the current population. In addition, through GWAS, haplotype analysis, and transcriptome analysis, we found AbPPO1 、 AbCM 、 AbPKS 、 AbLaccase-1 、 AbLaccase-2 、 Ab4ABH Genes that affect melanin formation at different levels lead to color variation in the Agaricus bisporus cap. A candidate domestication gene has been identified. AbPPO1 , AbPPO1A functional mutant allele of the gene causes the cap to appear white and was selected during the domestication and breeding of Stropharia bisporus. This study, based on the evolution and domestication history of Agaricus bisporus, reveals the genetic characteristics that lead to the white trait and provides gene targets for the utilization and improvement of Agaricus bisporus germplasm resources.
[0047] Identified AbPPO1 Hap_2 The nucleotide sequence of the gene is shown in SEQ ID No.1.
[0048] Example 2 AbPPO1 The Hap_2 haplotype of the gene can be used as a marker for the selection of white varieties of Agaricus bisporus according to AbPPO1 Combination of amino acid variations in genes, designed for specific recognition AbPPO1 Hap_2 Two pairs of haplotype MNP primers: Primer 1 (PF1 and PR1), PCR amplification fragment length 221 bp; the sequence of PF1 is shown in SEQ ID No. 2; the sequence of PR1 is shown in SEQ ID No. 3; Primer 2 (PF2 and PR2), PCR amplification fragment length 278 bp; the sequence of PF2 is shown in SEQ ID No. 4; the sequence of PR2 is shown in SEQ ID No. 5.
[0049] DNA from Agaricus bisporus strains was extracted using a plant DNA extraction kit (Bomaide). PCR reactions were prepared using 2× Es Taq MasterMix (Congwei Century). The PCR protocol was set according to the reagent instructions, with an annealing temperature of 55°C and an extension time of 20 seconds. The PCR products were subjected to 2% agarose gel electrophoresis and sent to BGI Sequencing for Sanger sequencing.
[0050] Figure The figure shows the 2% agarose gel electrophoresis obtained by the above method. Lanes 1-3 in the figure were detected with primer 1, which is marked as PF1 in the figure. The DNA samples detected were from Agaricus bisporus strains of Hap_1 / Hap_2, Hap_41 and Hap_2 / Hap_3 haplotypes respectively; lanes 4-6 in the figure were detected with primer 2, which is marked as PF2 in the figure. The DNA samples detected were from Agaricus bisporus strains of Hap_1 / Hap_2, Hap_41 and Hap_2 / Hap_3 haplotypes respectively; the band results showed that primers 1 and 2 can amplify multiple haplotypes. Gene sequence; Sequencing sequence and Hap_2 Haplotype sequence alignment is consistent or contains Hap_2Haplotype, indicating that the genome of Agaricus bisporus strains has Hap_2 Haplotype, with white quality screening value.
[0051] Hap_2 The sequence amplified by gene primer 1 (221 bp) is shown as SEQ ID No. 6; Hap_2 The sequence amplified by gene primer 2 (278 bp) is shown as SEQ ID No. 7.
[0052] Although the specific embodiments of the present invention have been described in detail in conjunction with the embodiments, this should not be construed as limiting the scope of protection of this patent. Within the scope described by the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.
Claims
1. A type of Agaricus bisporus AbPPO1 Gene haplotype, characterized by Agaricus bisporus AbPPO1 The nucleotide sequence of the gene haplotype is shown in SEQ ID No.
1.
2. The Agaricus bisporus according to claim 1 AbPPO1 Application of gene haplotypes in screening and breeding of white varieties of Agaricus bisporus.
3. The use according to claim 2, characterized in that: Agaricus bisporus AbPPO1 Genetic haplotypes of Agaricus bisporus.
4. The use according to claim 3, characterized in that: The screening method includes the steps of using two pairs of primers for detection; the nucleotide sequence of primer 1 is shown as SEQ ID No. 2 and SEQ ID No. 3, and the nucleotide sequence of primer 2 is shown as SEQ ID No. 4 and SEQ ID No.
5.
5. The use according to claim 4, characterized in that The screening method is as follows: extracting Agaricus bisporus genomic DNA, using the Agaricus bisporus genomic DNA as a template and using two pairs of primers to prepare PCR reaction systems, performing PCR amplification, performing agarose gel electrophoresis and sequencing on the PCR amplification products, and if the amplified sequences obtained by the two pairs of primers are consistent with AbPPO1 Gene haplotype sequence alignment is consistent or contains AbPPO1 Gene haplotype sequence, indicating that the Agaricus bisporus genome contains the AbPPO1 The nucleotide sequence of the gene haplotype.
6. The use according to claim 5, characterized in that: The annealing temperature of the PCR process was 55°C and the extension time was 10 s.
7. The use according to claim 5, characterized in that: PCR amplification was performed with two pairs of primers, and the resulting band lengths were 221 bp and 278 bp.