家蚕茧层率相关基因筛选及其功能分析

任晓晓; 孙运朋; 卿卓; 杨万军; 罗朝斌

doi:10.19303/j.issn.1008-0384.2022.007.004

家蚕茧层率相关基因筛选及其功能分析

doi: 10.19303/j.issn.1008-0384.2022.007.004

贵州省农业科学院蚕业研究所，贵州　贵阳　550006

基金项目: 贵州省科技计划项目（黔科合基础［2020］1Y133、黔科合成果［2019］4214）；贵州省农业科学院青年科技基金项目（黔农科院青年科技基金［2020］09）

详细信息

作者简介:
任晓晓（1991−），女，硕士，助理研究员，研究方向：家蚕遗传育种（E-mail：1016375902@qq.com）

通讯作者:
罗朝斌（1964−），男，硕士，研究员，研究方向：家蚕遗传育种（E-mail：1839916377@qq.com）

中图分类号: S 881.2
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出版历程
- 收稿日期: 2021-09-15
- 录用日期: 2021-09-15
- 修回日期: 2021-12-20
- 网络出版日期: 2022-08-07
- 刊出日期: 2022-07-28

Identification and Functional Analysis of Genes Related to Cocoon Shell Ratio in Bombyx mori

Sericultural Institute, Guizhou Academy of Agricultural sciences, Guiyang, Guizhou　550006, China

摘要

摘要: 目的挖掘家蚕茧层率相关基因，以期为家蚕茧层率性状分子遗传改良提供参考依据。方法以多丝量家蚕品种菁松和中丝量家蚕品种芙蓉为亲本构建BC₁代分离群体。在BC₁代雄性群体中挑选极端高/低茧层率个体构建子代DNA混池，运用BSA-seq方法对茧层率关联区域进行定位，并运用BLAST软件对关联区域的编码基因进行GO和KEGG等数据库注释及功能预测。结果重测序数据与家蚕参考基因组平均比对率为98.86%，平均基因组覆盖度为95.79%（1×）和88.63%（5×）；变异检测共获得26 557 646个SNPs；∆(SNP-index)定位到3个与茧层率显著相关的区域，分别为Chr.2:4430～4930 kb、Chr.4:12350～12920 kb和Chr.13:3230～3730 kb，共包含70个编码基因。通过GO与KEGG注释，有58个基因注释到GO数据库，涉及生物过程、分子功能和细胞组分三大类；有19个基因注释到KEGG通路，分布于34个代谢通路中。通过KEGG代谢通路分析，筛选出10个可能对家蚕茧层率有重要调控作用的基因，推测其参与了家蚕丝腺细胞运动、能量代谢和蛋白质合成加工。结论运用BSA-seq方法在家蚕第2、4和13号染色体上定位到与茧层率关联的区域，筛选到10个可能与茧层率密切相关的候选基因，为茧层率关键调控基因精细定位及克隆奠定基础。
- 家蚕 /
- 茧层率 /
- BSA-seq /
- 基因注释
Abstract: Objective Genes related to the cocoon shell ratio of Bombyx mori were investigated for improvement on silkworm productivity. Method Segregated BC₁ populations of a highly productive silkworm, Jingsong, and a moderately productive Furong were established. DNA pools were constructed by mixing 30 high and low cocoon shell ratio individuals from the BC₁male populations. BSA-seq was applied to identify the regions relevant to the target trait. The coding genes at the candidate regions were annotated with BLAST software in GO and KEGG databases. Result The percentage of clean reads that matched the reference genome of P50 was 98.86%, and those of the average genome coverage 95.79% for 1× and 88.63% for 5×. Variant calling resulted in 26 557 646 SNPs. Three QTLs for the cocoon shell ratio detected by ∆(SNP-index) were in the intervals of 4 430–4930 kb on Chr.2, 12350–12920 kb on Chr.4, and 3230–3730 kb on Chr.13. There were 70 coding genes found in the associated regions. According to the GO database annotation, 58 genes were categorized in the groups of biological process, molecular function, or cellular component. The 19 genes annotated in the KEGG database distributed in 34 metabolic pathways. Of which, 10 might play important roles in regulating the cocoon shell ratio and the functions involving the silk gland cell movement, energy metabolism, and/or protein synthesis. Conclusion The genes related to the silkworm cocoon shell ratio were identified by BSA-seq method. Ten genes were speculated to possibly associate with the silk formation. The present study only managed to locate the regions on chromosomes 2, 4, and 13 of the genes in B. mori that might related to the cocoon shell ratio. Further study will be needed to finely map and clone the key regulatory genes in the highly complex regulatory mechanism.
- Bombyx mori /
- silkworm cocoon shell ratio /
- BSA-seq /
- gene annotation

HTML全文

图 1 分子标记及关联信号在染色体上的分布

从外到内依次为参考基因组染色体坐标、染色体上基因分布（颜色越深表示基因密度越大）、SNP密度分布（圆点越密集表示SNP密度越大）、InDel密度分布（三角形越密集表示InDel密度越大）、Index值在染色体上的分布。

Figure 1. Distribution of SNPs, InDels, and associated signals on chromosome

Shown from outside inward: chromosome coordinates of reference genome, genes distribution on chromosome (darker color indicates greater gene density), SNP density distribution (density of dots corresponds to that of SNP), InDel density distribution (density of triangles reflects that of InDel), and distribution of indices on chromosome.

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图 2 H池和L池SNP-index、∆（SNP-index）分布情况

图中不同颜色表示不同的染色体，横坐标为1～28号染色体上每个window的具体物理位置，纵坐标为位置所对应的Index值。

Figure 2. SNP-index and ∆(SNP-index) distribution of H-pool and L-pool.

Different colors represent different chromosomes; x-axis is for physical location of each window on chromosomes 1 to 28; y-axis is for Index corresponding to respective locations.

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图 3 候选基因的GO注释

Figure 3. GO annotation of candidate genes

下载: 全尺寸图片幻灯片

表 1 测序数据质量

Table 1. Statistics on quality of sequencing data

样品编号 Sample ID	原始数据量 Raw data/bp	过滤后数据量 Clean base/bp	原测序reads 数 Raw reads	过滤后reads 数 Clean reads	Q₂₀/%	Q₃₀/%	GC含量 GC content/%
菁松（JS）	380002170	6295123605	42251670	41738170	98.10	94.04	38.83
芙蓉（FR）	5853501108	5771738817	38764908	38269934	97.91	93.56	38.96
H-pool	16080222540	15911340150	106491540	105500096	98.13	94.11	38.89
L-pool	16401318000	16232070628	108618000	107624014	98.13	94.11	38.66
Q₂₀：高质量测序数据中质量值≥20的碱基所占百分比；Q₃₀：高质量测序数据中质量值≥30的碱基所占百分比。 Q₂₀：The percentage of the bases whose Phred value are more than 20; Q₃₀：The percentage of the bases whose Phred value are more than 30.

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表 2 质控数据与参考基因组比对情况

Table 2. Matching between quality control data and reference genome

样品编号 Sample ID	比对率 Mapped ratio/%	比对到基因组上的reads比例 Properly ratio/%	平均测序深度 Average depth	基因组覆盖度（1×） Genome coverage(1×) /%	基因组覆盖度（5×） Genome coverage(5×) /%
菁松（JS）	98.84	82.72	10.34	94.70	84.71
芙蓉（FR）	98.88	82.28	9.44	94.41	82.61
H-pool	98.90	81.29	26.02	97.04	93.59
L-pool	98.82	82.38	27.59	97.02	93.59
1×覆盖度：1 个碱基覆盖的位点占基因组的百分比；5×覆盖度：5个碱基覆盖的位点占基因组的百分比。 Coverage 1×: the percentage of at least 1 base-covered site in reference genome; Coverage 5×: the percentage of at least 5 base-covered sites in reference genome.

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表 3 关联区域信息统计

Table 3. Statistics of the related genes

染色体编号 Chromosome ID	关联区域起点 Start of associated regions/bp	关联区域终点 End of associated regions/bp	关联区域长度 Associated region size/Mb	SNP数量 SNP number	关联区域内基因个数 Gene number in the associated regions
第13染色体 Chr 13	3230000	3730000	0.50	11	13
第4染色体 Chr 4	12350000	12920000	0.50	39	48
第2染色体 Chr 2	4430000	4930000	0.57	17	9
合计 Total			1.57	67	70

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表 4 候选基因的KEGG通路分析

Table 4. KEGG pathway of genes in candidate regions

一级代谢 Primary metabolism	二级代谢 Secondary metabolism	三级代谢 Tertiary metabolism	通路编号 Ko ID	基因编号 Gene ID
新陈代谢 Metabolism	聚糖生物合成与代谢 Glycan biosynthesis and metabolism	糖胺聚糖降解 Glycosaminoglycan degradation	ko00531	KWMTBOMO07657
	聚糖生物合成与代谢 Glycan biosynthesis and metabolism	O-聚糖生物合成 Other types of O-glycan biosynthesis	ko00514	KWMTBOMO02149
	脂质代谢 Lipid metabolism	初级胆汁酸生物合成 Primary bile acid biosynthesis	ko00120	KWMTBOMO02138
有机体系统 Organismal systems	免疫系统 Immune system	RIG-I样受体信号通路 RIG-I-like receptor signaling pathway	Ko04622	KWMTBOMO02145； KWMTBOMO07659； KWMTBOMO02143
		Toll样受体信号通路 Toll-like receptor signaling pathway	ko04620	KWMTBOMO07659
		NOD样受体信号通路 NOD-like receptor signaling pathway	ko04621	KWMTBOMO07659
	内分泌系统 Endocrine system	胰高血糖素信号通路 Glucagon signaling pathway	Ko04922	KWMTBOMO07653； KWMTBOMO07652； KWMTBOMO02146
		甲状腺激素信号通路 Thyroid hormone signaling pathway	ko04919	KWMTBOMO07653； KWMTBOMO07652
		胰岛素信号通路 Insulin signaling pathway	ko04910	KWMTBOMO02146
	神经系统 Nervous system	长时程增强效应 Long-term potentiation	ko04720	KWMTBOMO07653； KWMTBOMO07652
	神经系统 Nervous system	神经营养因子信号通路 Neurotrophin signaling pathway	ko04722	KWMTBOMO07659
	环境适应 Environmental adaptation	生理节律 Circadian rhythm	ko04710	KWMTBOMO00853
环境信息加工 Environment information processing	信号转导 Signal transduction	Ras信号通路 Ras signaling pathway	ko04014	KWMTBOMO02152
		丝裂原活化蛋白激酶信号通路 MAPK signaling pathway	Ko04013	KWMTBOMO02145； KWMTBOMO07659； KWMTBOMO02143
		钙离子信号通路 Calcium signaling pathway	ko04020	KWMTBOMO02146
		低氧诱导因子-1信号通路 HIF-1 signaling pathway	ko04066	KWMTBOMO07653； KWMTBOMO07652
		Wnt信号通路 Wnt signaling pathway	ko04310	KWMTBOMO07653； KWMTBOMO07652； KWMTBOMO00853
		环磷酸腺苷信号通路 cAMP signaling pathway	ko04024	KWMTBOMO07653； KWMTBOMO07652
		Notch信号通路 Notch signaling pathway	ko04330	KWMTBOMO07653； KWMTBOMO07652
		Jak-STAT信号通路 Jak-STAT signaling pathway	ko04630	KWMTBOMO07653； KWMTBOMO07652
		TGF-β信号通路 TGF-beta signaling pathway	ko04350	KWMTBOMO07653； KWMTBOMO00853； KWMTBOMO07652
		刺猬信号通路 Hedgehog signaling pathway	ko04341	KWMTBOMO00853
细胞进程 Cellular processes	细胞生长和死亡 Cell growth and death	细胞周期 Cell cycle	Ko04110	KWMTBOMO07653； KWMTBOMO07652； KWMTBOMO00853
		细胞凋亡 Apoptosis	ko04214	KWMTBOMO07659
		卵母细胞减数分裂 Oocyte meiosis	ko04114	KWMTBOMO00853
	细胞通讯 Cell communication	黏着连接 Adherens junction	ko04520	KWMTBOMO07653； KWMTBOMO07652
	运输与分解代谢 Transport and catabolism	内吞作用 Endocytosis	ko04144	KWMTBOMO07659
	运输与分解代谢 Transport and catabolism	溶酶体 Lysosome	ko04142	KWMTBOMO07657
遗传信息加工 Genetic information processing	折叠、组装和降解 Folding, sorting and degradation	泛素介导的蛋白质水解 Ubiquitin mediated proteolysis	ko04120	KWMTBOMO07659； KWMTBOMO02114； KWMTBOMO00853
		蛋白质在内质网上的加工 Protein processing in endoplasmic reticulum	ko04141	KWMTBOMO02147； KWMTBOMO00853
		蛋白酶体 Proteasome	ko03050	KWMTBOMO02148
	转录 Transcription	转录因子 Basal transcription factors	ko03022	KWMTBOMO02120
	转录 Transcription	剪接体 Spliceosome	ko03040	KWMTBOMO02133
	翻译 Translation	核糖体 Ribosome	ko03010	KWMTBOMO02140

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