Transcriptome analysis of genetic differences between aerial and strandy hyphae of Agaricus bisporus strain As2796
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摘要:
目的 挖掘双孢蘑菌落形态变化可能涉及的重要基因,为双孢蘑菇种质资源评价、新品种选育提供科学依据。 方法 以双孢蘑菇主栽品种As2796为试验菌株,收集同一平板上生长的贴生与气生菌丝,提取样品的RNA进行转录组测序分析。 结果 通过转录组分析,A601(As2796气生组)与A602(As2796贴生组)共鉴定出965个差异表达基因(DEGs)。GO富集分析结果表明,在真菌细胞壁组成方面有疏水蛋白相关的DEGs富集,其中AGABI2DRAFT_13655、AGABI2DRAFT_136569、AGABI2DRAFT_193057属于ABH3蛋白基因家族,在气生菌丝的表达水平显著高于贴生菌丝,可能与气生菌丝的产生有关。研究还发现了疏水表面结合蛋白A(HsbA)相关基因AGABI2DRAFT_194662在气生菌丝中FPKM表达水平为0,在贴生菌丝中为10.24。通过对DEGs进一步分析,富集到8个可能与脂酶同工酶相关的DEGs,气生菌丝中这些基因的表达水平显著高于贴生菌丝。SNP/InDel分析结果表明,As2796气生组SNP数为110601,贴生组SNP数为111188,单核苷酸发生变化的主要以C:G>T: A为主,共筛选出2 374个共同InDel。 结论 成功获得双孢蘑菇不同形态菌丝的转录组数据,并从中挖掘出潜在的相关基因和SNP位点,为双孢蘑菇相关分子标记开发及辅助育种提供参考。 Abstract:Objective Excavating the important genes that may be involved in the morphological changes of Agaricus bisporus colony can provide scientific basis for the evaluation of germplasm resources and the breeding of new varieties. Methods As2796, the main cultivated variety of Agaricus bisporus, was used as the test strain. The strandy and aerial hyphae grown on the same plate were collected. RNA of the samples was extracted for transcriptome sequencing and compared with the reference genome sequence of Agaricus bisporus. Results Through transcriptome analysis, a total of 965 differentially expressed genes (DEGs) were identified in A601 (As2796 aerial group) and A602 (As2796 strandy group). GO enrichment analysis results showed that hydrophobin-related DEGs were enriched in fungal cell wall composition. Among them, AGABI2DRAFT_13655, AGABI2DRAFT_136569, and AGABI2DRAFT_193057 belong to the ABH3 protein gene family. The expression level in aerial hyphae is significantly higher thanstrandy hyphae, which may be related to the production of aerial hyphae. The study also found that the hydrophobic surface binding protein A (HsbA)-related gene AGABI2DRAFT_194662 ,the FPKM expression level of this gene is 0 in aerial hyphae and 10.24 in strandy hyphae. Through further analysis of DEGs, 8 DEGs that may be related to lipase isoenzymes were enriched. The FPKM expression levels of these genes in aerial hyphae were significantly higher than strandy hyphae. The SNP/InDel analysis results showed that the number of SNPs in the As2796 aerial group was 110601, in strandy group was 111188. The main changes in single nucleotides were C:G>T:A, and a total of 2374 common InDels were screened. Conclusion This study successfully obtained transcriptome data on mycelial morphological changes in Agaricus bisporus and unearthed potential related genes and SNP sites, which provides a reference for the evaluation of Agaricus bisporus germplasm resources and the breeding of new varieties.. -
表 1 样品测序数据及与参考基因组的序列比对结果
Table 1. Statistics of experimental data and alignnment with reference on genome sequence
编号
ID样品
Sample总Read数
Total clean reads比对到参考基因组的Reads数目
Mapped reads比对到参考基因组的Reads百分比
Mapped reads/%GC含量
GC content/%≥Q30的百分比
≥Q30/%A601 As2796气生 42 155 674 25 410 008 60.28 49.12 92.00 A602 As2796贴生 45 225 288 28 324 702 62.63 49.28 92.37 表 2 新基因功能注释结果统计
Table 2. Number of new genes with functional annotation
用于功能注释的数据库
Annotated Database获得注释的新基因数目
Annotated Gene Number长度在≥300~ 1000 的新基因数目
Gene Number (300≤gene length<1000 )长度≥ 1000 的新基因数目
Gene Number (Gene length≥1000 )COG 15 12 3 GO 21 13 7 KEGG 38 21 14 KOG 20 12 8 Pfam 34 20 12 Swissprot 24 15 8 nr 132 70 57 All 139 76 57 表 3 部分差异表达基因
Table 3. Some significantly differentially expressed genes
编号
NO基因ID
Gene ID相对表达量FPKM
Relative expression FPKMLog2FC Pfam蛋白注释
Pfam protein annotation基因注释
Gene annotationA601 A602 1 AGABI2DRAFT_194497 15.6653 0.0000 −6.78 — — 2 AGABI2DRAFT_177536 20.5777 0.0000 −4.47 — 假设蛋白OH76DRAFT_1359727 3 AGABI2DRAFT_120985 1.0452 0.0000 −3.64 — 假设蛋白AN958_00580 4 AGABI2DRAFT_203489 2.3380 0.0001 −3.85 — 假设蛋白 AN958_02967 5 AGABI2DRAFT_44066 6.2585 0 −3.75 HhH-GPD超家族碱基
切除DNA修复蛋白假设蛋白 AN958_02739 6 AGABI2DRAFT_113583 0.0000 4.2489 4.73 — 假设蛋白 AN958_04089 7 AGABI2DRAFT_117617 0.0008 6.9437 3.30 — 假设蛋白 AN958_03520 8 AGABI2DRAFT_179131 0.0010 2.5569 4.28 — 假设蛋白 AN958_00324 9 AGABI2DRAFT_194662 0 10.2374 4.66 疏水表面结合蛋白A 假设蛋白AN958_09554 10 AGABI2DRAFT_136569 196.301 32.0293 −2.58 真菌疏水蛋白 假定的疏水蛋白 [双孢蘑菇] 11 AGABI2DRAFT_193057 999.26 189.163 −2.38 真菌疏水蛋白 假定的疏水蛋白 [双孢蘑菇] 12 AGABI2DRAFT_136557 19733 3086.17 −2.67 真菌疏水蛋白 假定的疏水蛋白 [双孢蘑菇] 13 AGABI2DRAFT_143464 100.031 896.356 3.21 真菌疏水蛋白 假定的疏水蛋白 [双孢蘑菇] 14 AGABI2DRAFT_178707 31.7555 666.848 4.35 真菌疏水蛋白 假定的疏水蛋白 [双孢蘑菇] 表 5 SNP突变统计表
Table 5. SNP mutation statistics table
编号 ID T:A>G:C C:G>G:C T:A>A:T C:G>T:A C:G>A:T T:A>C:G 转化 Ts 颠换 Tv 转换/颠换比 ts/tv A601 6312 5221 7641 42864 6405 42158 85022 25579 3.32 A602 6276 5239 7586 43273 6338 42476 85749 25439 3.37 表 6 全基因组和编码区InDel长度分布表
Table 6. Genome-wide and coding region InDel sites length distribution
编号
ID区域
Region−10 −9 −8 −7 −6 −5 −4 −3 −2 −1 1 2 3 4 5 6 7 8 9 10 A601 基因组
Genome11 2 0 0 1 2 0 6 106 1235 1559 121 1 0 0 2 0 0 0 0 A601 外显子
Exonic7 1 0 0 1 0 0 3 2 99 221 14 1 0 0 2 0 0 0 0 A602 基因组
Genome14 3 0 0 1 2 0 11 100 1080 1434 121 3 0 0 3 0 0 0 0 A602 外显子
Exonic9 1 0 0 1 0 0 7 2 100 229 15 3 0 0 3 0 0 0 0 -
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