苯磺隆胁迫下油菜萌发期相关性状的全基因组关联分析

doi:10.3864/j.issn.0578-1752.2019.03.002

摘要/Abstract

摘要：

目的研究苯磺隆残留对油菜种子萌发的影响,运用全基因组关联分析（genome-wide association analysis,GWAS）揭示苯磺隆胁迫下油菜萌发期相关性状的遗传因子和候选基因,探究油菜在苯磺隆逆境胁迫下的生理形态所反映的基因调控机制,为耐苯磺隆油菜品种的研究提供参考。方法以241份甘蓝型油菜品种（系）为材料、25 mg·L ^-1苯磺隆溶液为处理液、蒸馏水为对照进行发芽试验。发芽7 d测定并计算相对发芽率、相对根长和相对鲜重。结合芸薹属60K SNP芯片分析群体基因型,通过STRUCTURE软件和TASSEL软件分别对该群体进行群体结构分析以及亲缘关系和LD衰减分析。为有效排除假关联的影响,采用一般线性模型（GLM）和混合线性模型（MLM）中的6种模型进行比较,确定每个性状GWAS分析的最优模型。同时,利用TASSEL软件在最优模型下对241份材料的3个性状分别进行全基因组关联分析,根据关联SNP位点的LD区间序列预测候选基因。结果 241份品种（系）群体可分为P1（94份材料）和P2（147份材料）2个亚群,其中约56.28%的材料之间的亲缘关系值为0。全基因组关联分析（K+PCA模型）共检测到16个与性状显著关联的SNP位点,这些位点可解释9.42%—13.14%的表型变异率。通过分析显著SNP位点的LD区间与甘蓝型油菜对应的区间序列,筛选出25个候选基因可能与油菜耐苯磺隆有关,其中9个为细胞色素P450家族基因,5个参与谷胱甘肽合成或代谢过程,2个为多药耐药相关蛋白基因。同时发现与相对发芽率显著相关的基因ATGSTU19编码谷胱甘肽转移酶,参与毒素分解过程,在各种胁迫反应中起重要作用。在相对根长和相对鲜重共同鉴定到的候选基因BnaC02g27690D功能未知。结论共检测到16个SNP位点与耐苯磺隆性状显著关联,筛选出25个候选基因可能与油菜耐苯磺隆有关。

关键词: 油菜, 萌发期, 苯磺隆, 全基因组关联分析

Abstract:

【Objective】 To investigate the effect of residual tribenuron-methyl in soil on seed germination, genome-wide association analysis (GWAS) of 52157 SNPs with genome-wide coverage was used to identify the candidate genes for the germinating traits of rapeseed under tribenuron-methyl stress. The results of this study may provide a theoretical basis for tribenuron-methyl tolerance in oilseed rape.【Method】 In the germination experiment, 241 rape varieties (lines) were treated with tribenuron-methyl solution of 25 mg·L ^-1, and distilled water was added to the control. At the 7th day of germination, the phenotypic data including relative germination rate, relative root length and relative fresh weight were measured and calculated. Using the TASSEL software, tribenuron-methyl tolerance related traits were explored in B. napus under germination with a 60K Brassica Illumina ^? Infinium SNP array. Then, the structure of the population was analyzed by the software STRUCTURE, and the genetic relationship and LD attenuation were analyzed by the software TASSEL, respectively. In order to determine the optimal model for GWAS analysis of each trait, 6 models involved the general linear model and mixed linear model were used to analyze and compare the effects of group structure and relationship. The software TASSEL was employed to analyze the relative values of the 3 traits under the optimal model. Meanwhile, the candidate genes were predicted based on the LD interval sequence of the associated SNP locus. 【Result】 The population structure analysis showed the population could be divided into two subgroups, P1 with 94 materials and P2 with 147 materials. Meanwhile, the result of genetic relationship analysis showed that about 56.28% of the materials had no kinship relationship. In the optimal GWAS model (K+PCA), we found that 16 SNP loci significantly associated with 3 traits including relative root length, relative fresh weight and relative germination rate, and each locus explained phenotypic variations ranging from 9.42% to 13.14%. By analyzing the LD interval of the significant SNP locus and the corresponding interval sequence of Brassica napus, twenty-five candidate genes related to tribenuron-methyl tolerance were screened out in the LD interval of these significant SNP loci, in which nine of them belonged to cytochrome P450 gene families, five were involved in glutathione synthesis or metabolic processes, and two were multidrug-tolerance associated protein. At the same time, it was revealed that the gene ATGSTU19 significantly related to germination rate encodes glutathione transferase, which participates in the process of toxin decomposition and plays an important role in various stress responses. In addition, BnaC02g27690D was identified at relative root length and relative fresh weight. However, its function was not clear. 【Conclusion】 In this study, 16 SNP loci were detected to be significantly associated with tribenuron-methyl tolerance, and 25 candidate genes were screened out.

Key words: Brassica napus L., germination, tribenuron-methyl tolerance, GWAS

周清元, 王倩, 叶桑, 崔明圣, 雷维, 郜欢欢, 赵愉风, 徐新福, 唐章林, 李加纳, 崔翠. 苯磺隆胁迫下油菜萌发期相关性状的全基因组关联分析[J]. 中国农业科学, 2019, 52(3): 399-413.

ZHOU QingYuan, WANG Qian, YE Sang, CUI MinSheng, LEI Wei, GAO HuanHuan, ZHAO YuFeng, XU XinFu, TANG ZhangLin, LI JiaNa, CUI Cui. Genome-Wide Association Analysis of Tribenuron-Methyl Tolerance Related Traits in Brassica napus L. Under Germination[J]. Scientia Agricultura Sinica, 2019, 52(3): 399-413.

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苯磺隆耐性相关性状的候选基因"

性状 Trait	标记 Marker	染色体 Chr.	LD区间 LD interval (bp)	候选基因 Candidate gene	拟南芥基因 Arabidopsis gene	基因 Gene	参考文献 Reference
相对根长 Relative root length	Bn-A03-p21744993	A03	20397711-20657711	BnaA03g40900D	AT3G50740	UGT72E1	[33]
	Bn-A05-p23188406	A05	21196681-21496681	BnaA05g30790D	AT3G06590	AIF2	[34]
	Bn-A02-p5442618	C02	4040991-6340991	BnaC02g07650D	AT5G17960	未知Unknown
				BnaC02g08720D	AT5G19450	CDPK19	[35]
				BnaC02g10110D	AT1G01190	CYP78A8	[36]
				BnaC02g10430D	AT5G58860	CYP86A1	[37]
	Bn-A05-p9277011	C02	25214667-27514667	BnaC02g27690D	AT4G01210	未知Unknown
				BnaC02g28290D	AT4G12320	CYP706A6
	Bn-scaff_17799_ 1-p1082380	C06	35150328-35890328	BnaC06g38000D	AT1G77290	未知Unknown
相对鲜重 Relative fresh weight	Bn-A01-p11163980	A01	9083493-9383493	BnaA01g17570D	AT4G16190	未知Unknown
相对鲜重 Relative fresh weight	Bn-scaff_22749 _1-p462527	C02	23961534-26261534	BnaC02g27690D	AT4G01210	未知Unknown
				BnaC02g27750D	AT4G01070	GT72B1	[38]
	Bn-scaff_17457 _1-p187316	C03	53987603-54727603	BnaC03g64930D	AT4G12560	CPR1	[39]
				BnaC03g65100D	AT4G22690	CYP706A1	[40]
				BnaC03g65140D	AT4G23100	GSH1	[41]
	Bn-scaff_17457 _1-p82664	C03	54093472-54833472	BnaC03g65260D	AT1G49860	GSTF14	[42]
	Bn-scaff_15798 _1-p152131	C04	36198548-37318548	BnaC04g35220D	AT1G13080	CYP71B2	[43]
				BnaC04g35620D	AT1G78490	CYP708A3
相对发芽率 Relative germination rate	Bn-A07-p5710530	A07	7546960-7826960	BnaA07g07310D	AT1G30410	ATMRP13	[41]
相对发芽率 Relative germination rate				BnaA07g07330D	AT1G30420	ATMRP12	[44]
	Bn-scaff_16300 _1-p1228965	C02	21415646-23715646	BnaC02g24410D	AT1G47620	CYP96A8	[45]
				BnaC02g24920D	AT1G78270	UGT85A4	[46]
				BnaC02g24980D	AT1G78380	ATGSTU19	[47]
	Bn-A09-p9040117	C09	10675455-12195455	BnaC09g14070D	AT3G26160	CYP71B17	[48]
				BnaC09g14150D	AT3G26170	CYP71B19	[49]
				BnaC09g15080D	AT1G59700	GSTU16	[50]

表5

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性状 Traits	均值±标准差 Mean±SD	变幅 Change amplitude	变异系数 CV (%)
相对根长Relative root length	0.136±0.066**	0.050-0.588	48.950
相对鲜重Relatively fresh weight	0.980±0.202**	0.411-2.357	20.593
相对发芽率Relative germination rate	0.942±0.097**	0.333-1.000	10.276

染色体 Chr.	SNP数目 No.	长度 Length (bp)	SNP密度 SNP density (1 SNP/kb)	染色体 Chr.	SNP数目 No.	长度 Length (bp)	SNP密度 SNP density (1 SNP/kb)
A01	1533	23213190	15.14	C01	2321	38751959	16.70
A02	1266	24751063	19.55	C02	2225	46171227	20.75
A03	2206	29727584	13.48	C03	2707	60554466	22.37
A04	1444	19097304	13.23	C04	3005	48891192	16.27
A05	1623	22986419	14.16	C05	1011	43165527	42.70
A06	1519	24371484	16.04	C06	1287	37161109	28.87
A07	1882	23921822	12.71	C07	1683	44305541	26.33
A08	1085	18658096	17.20	C08	1527	38226084	25.03
A09	1637	33803997	20.65	C09	1006	48440856	48.15
A10	1526	17348295	11.37
A染色体SNP 的平均分布 Mean A genome	1572	23787925	15.13	C染色体SNP 的平均分布 Mean C genome	1864	45074218	24.19

性状 Trait	标记 Marker	等位基因 Allele	染色体 Chr.	位置 Position (bp)	阈值 P-value	贡献率 R²(%)
相对根长 Relative root length	Bn-A03-p21744993	A/G	A03	20527711	4.37×10^-6	12.19
	Bn-A05-p23188406	A/C	A05	21346681	1.18×10^-5	11.22
	Bn-A02-p5442618	A/G	C02	5190991	4.35×10^-6	12.20
	Bn-A05-p9277011	A/C	C02	26364667	2.94×10^-5	10.35
	Bn-scaff_18322_1-p2518158	T/C	C03	6708755	4.26×10^-6	12.22
	Bn-scaff_17799_1-p1082380	A/G	C06	35520328	9.75×10^-6	11.42
相对鲜重 Relative fresh weight	Bn-A01-p11163980	A/G	A01	9233493	1.48×10^-5	11.12
	Bn-scaff_22749_1-p462527	T/C	C02	25111534	8.44×10^-6	11.66
	Bn-scaff_17457_1-p187316	A/G	C03	54357603	1.34×10^-5	11.22
	Bn-scaff_17457_1-p82664	T/G	C03	54463472	2.31×10^-5	10.69
	Bn-scaff_15798_1-p152131	T/G	C04	36758548	2.28×10^-5	9.42
	Bn-scaff_15794_3-p286350	A/C	C03	55318298	3.12×10^-5	10.40
相对发芽率 Relative germination rate	Bn-A04-p10628643	T/C	A04	11766577	1.88×10^-6	13.14
	Bn-A07-p5710530	T/G	A07	7686960	2.52×10^-6	12.85
	Bn-scaff_16300_1-p1228965	A/C	C02	22565646	1.75×10^-5	10.95
	Bn-A09-p9040117	A/C	C09	11435455	1.28×10^-5	11.26