双孢蘑菇As2796菌株气生与贴生菌丝遗传差异的转录组分析

戴建清; 陈文智; 曾志恒; 蔡志欣; 陈美元

双孢蘑菇As2796菌株气生与贴生菌丝遗传差异的转录组分析

福建省农业科学院食用菌研究所/特色食用菌繁育与栽培国家地方联合工程研究中心，福建　福州　350013

基金项目: 福建省科技计划公益类专项（2023R1033002）；国家现代农业产业技术体系专项（CARS-20）；福建省种业创新与产业化工程项目（zycxny2021011）；福建省农业高质量发展超越“5511”协同创新工程项目（XTCXGC2021007）；福建省农业科学院科技创新团队建设项目（CXTD2021007-1）

详细信息

作者简介:
戴建清（1972 —），男，高级农艺师，主要从事食用菌良种繁育研究，E-mail：317152396@qq.com

通讯作者:
陈美元（1972 — ），男，教授级高级工程师，主要从事双孢蘑菇遗传育种研究，E-mail: 401061326@qq.com

中图分类号: S646
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出版历程
- 收稿日期: 2024-08-09
- 修回日期: 2024-09-01
- 网络出版日期: 2024-10-31

Transcriptome analysis of genetic differences between aerial and strandy hyphae of Agaricus bisporus strain As2796

The Edible mushroom Institute of Fujian Academy of Agricultural Sciences, National and Local Joint Engineering Research Center for Breeding & Cultivation of Featured Edible mushroom, Fuzhou　350013, Fujian, China

摘要

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。
- 双孢蘑菇 /
- As2796 /
- 贴生菌丝 /
- 气生菌丝 /
- 转录组测序 /
- 差异表达基因
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..
- Agaricus bisporus /
- As2796 /
- Strandy hypha /
- Aerial hypha /
- Transcriptome sequencing /
- Differentially expressed genes (DEGs)

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图 1 双孢蘑菇As2796菌株（A）、贴生菌丝（B）和气生菌丝（C）

红色箭头为气生菌落，黑色箭头为贴生菌落。

Figure 1. Plate of Agaricus bisporus As2796 strain (A)、strandy mycelium (B) and aerial hypha (C)

Red arrows are aerial hypha and black arrows are strandy mycelium.

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图 2 差异表达基因火山图和数目统计图

Figure 2. Volcano plot and number statistics chart of DEGs

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图 3 双孢蘑菇贴生菌丝与气生菌丝共同差异表达基因的COG分类

Figure 3. COG annotation of common DEGs between strandy mycelium and aerial hypha of Agaricus bisporus

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图 4 差异表达基因GO二级节点注释统计图

Figure 4. GO secondary node annotation chart of DEGs

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图 5 双孢蘑菇贴生菌丝与气生菌丝共同差异表达基因的GO富集分析

Figure 5. GO enrichment analysis of common DEGs between strandy mycelium and aerial hypha of Agaricus bisporus

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图 6 差异表达基因KEGG通路富集分析

Figure 6. KEGG pathway analysis of differentially expressed genes

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图 7 吲哚二萜生物碱生物合成通路（A）和星形孢菌素生物合成通路（B）相关基因表达量分析

Figure 7. Gene expression related to indole diterpene alkaloid biosynthesis (A) and Staurosporine biosynthesis (B)

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图 8 SNP位点统计（A）和突变分布图（B）

Figure 8. SNP site statistics (A) and mutation distribution map (B)

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图 9 InDel Veen图（A）和位点统计图（B）

Figure 9. InDel Veen diagram (A) and site statistical diagram (B)

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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

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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

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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 FPKM		Log₂FC	Pfam蛋白注释 Pfam protein annotation	基因注释 Gene annotation
编号 NO	基因ID Gene ID	A601	A602	Log₂FC	Pfam蛋白注释 Pfam protein annotation	基因注释 Gene annotation
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]

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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

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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 Genome	11	2	0	0	1	2	0	6	106	1235	1559	121	1	0	0	2	0	0	0	0
A601	外显子 Exonic	7	1	0	0	1	0	0	3	2	99	221	14	1	0	0	2	0	0	0	0
A602	基因组 Genome	14	3	0	0	1	2	0	11	100	1080	1434	121	3	0	0	3	0	0	0	0
A602	外显子 Exonic	9	1	0	0	1	0	0	7	2	100	229	15	3	0	0	3	0	0	0	0

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