基于BSA-Seq和RNA-Seq方法鉴定大豆抗豆卷叶螟候选基因

doi:10.3724/SP.J.1006.2021.04195

作物学报 ›› 2021, Vol. 47 ›› Issue (8): 1460-1471.doi: 10.3724/SP.J.1006.2021.04195

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

基于BSA-Seq和RNA-Seq方法鉴定大豆抗豆卷叶螟候选基因

曾维英(), 赖振光(), 孙祖东^*(), 杨守臻, 陈怀珠, 唐向民

广西农业科学院经济作物研究所/农业农村部西南玉米大豆间套作区农业科学观测实验站, 广西南宁 530007

收稿日期:2020-08-25 接受日期:2021-01-13 出版日期:2021-08-12 网络出版日期:2021-02-18
通讯作者: 孙祖东
作者简介:曾维英, E-mail: zengweiying_1981@163.com|赖振光, E-mail: 519229671@qq.com
基金资助:
广西自然科学基金项目(2017GXNSFDA198037);广西农业科学院科技发展基金项目(桂农科2020YM116, 2015YT58)

Identification of the candidate genes of soybean resistance to bean pyralid (Lamprosema indicata Fabricius) by BSA-Seq and RNA-Seq

ZENG Wei-Ying(), LAI Zhen-Guang(), SUN Zu-Dong^*(), YANG Shou-Zhen, CHEN Huai-Zhu, TANG Xiang-Min

Institute of Economic Crops, Guangxi Academy of Agricultural Sciences/Southwest Experimental Station of Maize-Soybean Intercrop, Ministry of Agriculture and Rural Affairs, Nanning 530007, Guangxi, China

Received:2020-08-25 Accepted:2021-01-13 Published:2021-08-12 Published online:2021-02-18
Contact: SUN Zu-Dong
Supported by:
Natural Science Foundation of Guangxi Province, China(2017GXNSFDA198037);Science and Technology Development Fund of Guangxi Academy of Agricultural Sciences (Guinongke 2020YM116, 2015YT58)

摘要/Abstract

摘要：

豆卷叶螟(Lamprosema indicata Fabricius)是重要的大豆食叶性害虫, 挖掘大豆抗豆卷叶螟相关基因对大豆抗虫品种选育和遗传改良至关重要。本研究用大豆高抗豆卷叶螟材料赶泰-2-2和高感豆卷叶螟材料皖82-178进行杂交构建F₂代分离群体, 从303个F₂代单株中挑选出高抗豆卷叶螟和高感豆卷叶螟的单株各30株, 分别构建2个极端性状的DNA混合池用于全基因组重测序以分析控制豆卷叶螟相关的候选基因。结果表明, 4个样本中共有11,963,077个单核苷酸多态性(SNPs)标记, 根据SNP-index方法关联分析, 共有329个基因位于99%置信区间外, 这些基因主要集中在7号染色体5,601,065~5,865,237 bp区间(总长为0.26 Mb)、16号染色体2,975,110~6,336,096 bp区间(总长为3.36 Mb)、18号染色体44,366,115~54,297,600 bp区间(总长为9.93 Mb)等区域内。将BSA-Seq结果与转录组测序结果进行联合分析发现, 有12个基因相关联; 最后, 结合生物信息学分析、候选基因的表达模式和基因的同源注释, 锁定CNGC4、WRKY16转录因子、AAP7、丝氨酸/苏氨酸蛋白激酶、ZPR1B等12个基因为控制豆卷叶螟性状相关的候选基因。本研究结果不仅为解析大豆抗豆卷叶螟的分子机理奠定重要的基础, 也将为大豆抗虫基因的克隆奠定坚实的理论基础。

关键词: 大豆, 豆卷叶螟, BSA-Seq, RNA-Seq, 候选基因

Abstract:

Bean pyralid is an important leaf-feeding insect in soybean. Identification of insect-tolerant genes from soybean has great significant to the crop insect-tolerant breeding and genetic improvement. In this study, an F₂ population with 303 individuals was constructed using insect-resistant line Gantai-2-2 and insect-sensitive line Wan 82-178. 30 F₂ insect-resistant individuals and 30 insect-sensitive individuals were selected respectively to construct two DNA pools which were used for the whole-genome re-sequencing. The results showed that there were a total of 11,963,077 single nucleotide polymorphism (SNPs) markers identified in two parental lines and two mixed pools. According to the association analysis of SNP-index method, a total of 329 genes were located outside the 99% confidence interval. These genes were mainly concentrated in the regions of 5,601,065-5,865,237 bp with a total of 0.26 Mb on chromosome 7, 2,975,110-6,336,096 bp with a total of 3.36 Mb on chromosome 16, and 44,366,115-54,297,600 bp with a total of 9.93 Mb on chromosome 18. Correlation analysis of BSA-Seq and transcriptome sequencing showed that 12 genes were correlated. Then, 12 candidate genes, including CNGC4, WRKY transcription factor 16, AAP7, serine/threonine protein kinase and ZPR1B were identified by bioinformatics analysis, differential expression analysis, and homologous annotation. This study laid an important foundation for the analysis of the molecular mechanism of soybean resistance to bean pyralid and the cloning of anti-insect genes.

Key words: soybean, bean pyralid, BSA-Seq, RNA-Seq, candidate genes

曾维英, 赖振光, 孙祖东, 杨守臻, 陈怀珠, 唐向民. 基于BSA-Seq和RNA-Seq方法鉴定大豆抗豆卷叶螟候选基因[J]. 作物学报, 2021, 47(8): 1460-1471.

ZENG Wei-Ying, LAI Zhen-Guang, SUN Zu-Dong, YANG Shou-Zhen, CHEN Huai-Zhu, TANG Xiang-Min. Identification of the candidate genes of soybean resistance to bean pyralid (Lamprosema indicata Fabricius) by BSA-Seq and RNA-Seq[J]. Acta Agronomica Sinica, 2021, 47(8): 1460-1471.

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链接本文: https://zwxb.chinacrops.org/CN/10.3724/SP.J.1006.2021.04195

https://zwxb.chinacrops.org/CN/Y2021/V47/I8/1460

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样本 Sample	原始序列数 Raw reads	原始碱基数 Raw bases	过滤后序列数 Clean reads	过滤后碱基数 Clean bases	有效率 Clean_data/ raw_data (%)	GC含量 Clean_GC_rate (%)	Q20含量 Q20 (%)	Q30含量Q30 (%)
皖82-178 Wan 82-178	438,210,500	65,731,575,000	432,474,442	64,871,166,300	98.69	35.57	96.50	92.13
赶泰-2-2 Gantai-2-2	441,716,184	66,257,427,600	433,990,902	65,098,635,300	98.25	35.60	96.34	91.79
高感子代池 Highly susceptible pool	206,898,280	31,034,742,000	202,406,834	30,361,025,100	97.83	35.54	96.44	92.01
高抗子代池 Highly resistant pool	210,122,884	31,518,432,600	206,805,618	31,020,842,700	98.42	35.46	96.29	91.70

样本 Sample	过滤后序列数 Clean reads	比对上数据 Mapping reads	比对率 Mapping rate (%)	优质比对率 Properly paired ratio (%)	平均测序深度 Mean depth	1×覆盖度Coverage ≥ 1× (%)	5×覆盖度 Coverage ≥ 5× (%)	10×覆盖度 Coverage ≥ 10× (%)	20×覆盖度 Coverage ≥ 20× (%)
皖82-178 Wan 82-178	43,4152,986	433,232,376	99.79	99.73	65.78	96.67	96.12	95.67	94.62
赶泰-2-2 Gantai-2-2	435,703,158	434,715,984	99.77	99.72	65.98	96.68	96.13	95.68	94.60
高感子代池 Highly susceptible pool	203,202,650	202,820,220	99.81	99.76	30.78	95.75	94.55	93.29	83.88
高抗子代池 Highly resistant pool	207,33,600	207,143,542	99.76	99.71	31.44	95.76	94.56	93.32	84.44

变异位点信息 Variation sites information	皖82-178 Wan 82-178	赶泰-2-2 Gantai-2-2	高感子代池 Highly susceptible pool	高抗子代池 Highly resistant pool
内含子Intron	324,202	324,333	230,606	231,332
基因区间Intergenic region	2,589,457	2,588,540	1,851,943	1,867,897
可变剪切位点Splicing	612	620	449	444
基因上游Upstream	192,755	192,753	133,797	136,203
基因下游Downstream	162,024	161,574	113,186	114,671
基因上游/基因下游Upstream/downstream	12,038	12,088	8296	8733
5°非翻译区UTR5°	23,658	23,746	68,071	16,818
3°非翻译区UTR3°	29,843	29,595	20,949	21,579
终止子提前Stop gain	1652	1649	1177	1225
终止子丢失Stop loss	270	276	197	204
同义突变Synonymous	48,462	48,433	35,671	35,492
非同义突变Non-synonymous	67,206	67,040	49,333	49,653
SNP总数SNP numbers	3,487,363	3,485,674	2,483,855	2,506,185
纯合突变SNP Hom SNP number	3,480,115	3,478,433	2,477,251	2,499,464
杂合突变SNP Hete SNP number	7248	7241	6604	6721
纯合突变SNP比率Hom SNP rate (%)	99.79	99.79	99.73	99.73
杂合突变SNP比率Het SNP rate (%)	0.21	0.21	0.27	0.27
转换Ts	2,264,913	2,264,110	1,606,918	1,617,797
颠换Tv	1,218,400	1,217,609	872,951	884,443
转换/颠换Ts/Tv	1.86	1.86	1.84	1.83

序号 No.	基因ID Gene ID	基因注释 Gene annotation	染色体 Chr.	比较组 Comparison group	上调或下调^a Up or down ^a
1	XM_003526153.4	环核苷酸门控离子通道4 Cyclic nucleotide-gated ion channel 4 (CNGC4)	Chr. 6	HR0/HR48 HS0/HS48	Up
2	XM_003533631.4	纤维素合成酶A催化亚基4 (CesA4) Cellulose synthase A catalytic subunit 4 [UDP-forming]	Chr. 9	HR0/HR48 HS0/HS48	Down
3	NM_001250658.2	WRKY16转录因子 WRKY transcription factor 16	Chr. 12	HR0/HR48	Up
4	NM_001255802.1	赤霉素2-β加双氧酶8 Gibberellin 2-beta-dioxygenase 8-like	Chr. 11	HR0/HR48	Up
5	XM_003519602.4	氨基酸透性酶7 (AAP7) Probable amino acid permease 7	Chr. 2	HR0/HR48	Up
6	XM_026126143.1	Mdis1相互受体激酶2 MDIS1-interacting receptor like kinase 2	Chr. 16	HR0/HR48	Up
7	XM_006601656.3	丝氨酸/苏氨酸蛋白激酶 Serine/threonine protein kinase-like protein	Chr. 18	HR0/HR48	Up
8	NM_001248669.2	ZPR1B Protein LITTLE ZIPPER 2-like	Chr. 18	HS0/HS48	Up
9	XM_026126905.1	类枯草杆菌蛋白酶 Subtilisin-like protease Glyma18g48580	Chr. 18	HS0/HR0	Down
10	XM_026125778.1	抗病蛋白RGA3 Putative disease resistance protein RGA3	Chr. 15	HS0/HR0	Down
11	XM_026126161.1	无特征LOC100809946 Uncharacterized LOC100809946	Chr. 16	HS0/HR0	Up
12	XM_026127992.1	硫氰酸酶结构域蛋白酶10 Rhodanese-like domain-containing protein 10	Chr. 3	HS48/HR48 HS0/HR0	Down

基于BSA-Seq和RNA-Seq方法鉴定大豆抗豆卷叶螟候选基因

Identification of the candidate genes of soybean resistance to bean pyralid (Lamprosema indicata Fabricius) by BSA-Seq and RNA-Seq

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