甘蓝型油菜每角果粒数全基因组关联分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0824

摘要/Abstract

摘要：

为挖掘甘蓝型油菜每角果粒数显著关联单核苷酸多态性（SNP）位点及相关候选基因。本研究以300份甘蓝型油菜自交系为试验材料，对甘蓝油菜每角果粒数进行一年两地表型考察，并结合该群体前期开发的201 817个SNPs标记，采用一般线性模型（GLM）和混合线性模型（MLM）进行全基因组关联分析（GWAS），此外，对性状显著关联SNP位点两侧100 kb区域内相关候选基因进行功能预测。300份甘蓝型油菜每角果粒数在两地均表现出广泛的表型变异，筛选出2份每角果粒数较多的油菜种质资源。基于GLM模型检测到39个与油菜每角果粒数显著关联SNPs，采用MLM分析发现，两地共检测到的3个每角果粒数显著关联SNPs位点均在GLM检测到。8个位点附近找到CIK，ERF022和EDE1等19个拟南芥已报道角果籽粒发育相关的同源基因。研究结果有助于解析甘蓝型油菜每角果粒数的遗传基础，为研究每角果粒数的调控机制、指导每角果粒数的遗传改良奠定基础。

关键词: 甘蓝型油菜, 全基因组关联分析, 每角果粒数, SNP位点, 同源基因

Abstract:

This study aims to excavate the SNP loci and candidate genes significantly associated with the seed number per silique（SPS）in Brassica napus L. Having 300 B. napus inbreeding lines as experimental materials, we investigated the phenotypes of SPS under two locations（Jiangxi Agricultural University（JXAU）in Nanchang and Jiangxi Institute of Red Soil（JXIRS）in Jinxian in a year. Then we conducted the genome-wide association study via general linear model（GLM）and mixed linear model（MLM）combining 201 817 SNPs markers determined previously. And finally we predicted the functions of relevant candidate genes within the 100 kb region flanking trait significantly associated SNP loci. The results showed that the broad phenotypic variations of SPS in the two locations among the 300 300 B. napus samples existed, and two germplasm with more SNP were screened. Total 39 SNPs loci remarkably associated with SPS were excavated by general linear model（GLM）. Furthermore, 3 SNPs of SPS were detected severally using the mixed linear model（MLM）, and they all were detected using GLM. And 19 candidate orthologous genes to documented Arabidopsis the seed number per silique, like CIK, ERF022 and EDE1, were found near our eight SNPs loci. The results are conducive to analyzing the genetic basis of SPS of rapeseed and would lay a foundation for studying the regulation mechanism and guiding the genetic improvement of SPS.

Key words: Brassica napu, genome-wide association study, seed number per silique, SNP loci, orthologous gene

肖小军, 陈明, 韩德鹏, 余跑兰, 郑伟, 肖国滨, 周庆红, 周会汶. 甘蓝型油菜每角果粒数全基因组关联分析[J]. 生物技术通报, 2023, 39(3): 143-151.

XIAO Xiao-jun, CHEN Ming, HAN De-peng, YU Pao-lan, ZHENG Wei, XIAO Guo-bin, ZHOU Qing-hong, ZHOU Hui-wen. Genome Wide Association Analysis of Thousand Seed Weight in Brassica napus L.[J]. Biotechnology Bulletin, 2023, 39(3): 143-151.

图/表 7

参考文献 44

[1]	王汉中. 我国油菜产业发展的历史回顾与展望[J]. 中国油料作物学报, 2010, 32(2): 300-302.
	Wang HZ. Review and future development of rapeseed industry in China[J]. Chin J Oil Crop Sci, 2010, 32(2): 300-302.
[2]	王汉中. 以新需求为导向的油菜产业发展战略[J]. 中国油料作物学报, 2018, 40(5): 613-617.
	Wang HZ. New-demand oriented oilseed rape industry developing strategy[J]. Chin J Oil Crop Sci, 2018, 40(5): 613-617.
[3]	刘成, 冯中朝, 肖唐华, 等. 我国油菜产业发展现状、潜力及对策[J]. 中国油料作物学报, 2019, 41(4): 485-489.
	Liu C, Feng ZC, Xiao TH, et al. Development, potential and adaptation of Chinese rapeseed industry[J]. Chin J Oil Crop Sci, 2019, 41(4): 485-489.
[4]	Van CW. Yield enhancement genes: seeds for growth[J]. Curr Opin Biotechnol, 2005, 16(2): 147-153. doi: 10.1016/j.copbio.2005.03.002 URL
[5]	Chen W, Zhang Y, Liu XP, et al. Detection of QTL for six yield-related traits in oilseed rape(Brassica napus)using DH and immortalized F₂ populations[J]. Theor Appl Genet, 2007, 115(6): 849-858. doi: 10.1007/s00122-007-0613-2 pmid: 17665168
[6]	Fan CC, Cai GQ, Qin J, et al. Mapping of quantitative trait loci and development of allele-specific markers for seed weight in Brassica napus[J]. Theor Appl Genet, 2010, 121(7): 1289-1301. doi: 10.1007/s00122-010-1388-4 URL
[7]	Zhu LX, Zhang DX, Fu TD, et al. Analysis of yield and disease resistance traits of new winter rapeseed varieties over the past twenty years in China[J]. Agric Sci ＆ Technol, 2011, 12(6): 842-846.
[8]	易斌, 陈伟, 马朝芝, 等. 甘蓝型油菜产量及相关性状的QTL分析[J]. 作物学报, 2006, 32(5): 676-682.
	Yi B, Chen W, Ma CZ, et al. Mapping of quantitative trait loci for yield and yield components in Brassica napus L[J]. Acta Agron Sin, 2006, 32(5): 676-682.
[9]	张月立. 甘蓝型油菜含油量、角果长和每果粒数的QTL定位[D]. 武汉: 华中农业大学, 2013.
	Zhang YL. Qtl mapping for oil content, silique length and seed number per silique in Brassica napus L[D]. Wuhan: Huazhong Agricultural University, 2013.
[10]	赵卫国, 王灏, 穆建新, 等. 甘蓝型油菜千粒重性状的QTL定位分析[J]. 西北植物学报, 2017, 37(3): 478-485.
	Zhao WG, Wang H, Mu JX, et al. Localization of thousand seed weight trait in Brassica napus by quantitative trait locus analysis[J]. Acta Bot Boreali Occidentalia Sin, 2017, 37(3): 478-485.
[11]	孙程明, 陈松, 彭琦, 等. 甘蓝型油菜角果长度性状的全基因组关联分析[J]. 作物学报, 2019, 45(9): 1303-1310. doi: 10.3724/SP.J.1006.2019.94021
	Sun CM, Chen S, Peng Q, et al. Genome-wide association study of silique length in rapeseed(Brassica napus L.)[J]. Acta Agron Sin, 2019, 45(9): 1303-1310.
[12]	王茹梦, 刘忠松. 甘蓝型油菜开花期全基因组关联分析及开花基因标记开发[J]. 分子植物育种, 2021, 19(10): 3329-3338.
	Wang RM, Liu ZS. Genome-wide association analysis of flowering time and development of flowering gene markers in Brassica napus L[J]. Mol Plant Breed, 2021, 19(10): 3329-3338.
[13]	周庆红, 周灿, 郑伟, 等. 甘蓝型油菜角果长度全基因组关联分析[J]. 中国农业科学, 2017, 50(2): 228-239. doi: 10.3864/j.issn.0578-1752.2017.02.003
	Zhou QH, Zhou C, Zheng W, et al. Genome wide association analysis of silique length in Brassica napus L[J]. Sci Agric Sin, 2017, 50(2): 228-239.
[14]	韩德鹏, 周灿, 郑伟, 等. 甘蓝型油菜主花序性状全基因组关联分析[J]. 核农学报, 2018, 32(3): 463-476. doi: 10.11869/j.issn.100-8551.2018.03.0463
	Han DP, Zhou C, Zheng W, et al. Genome wide association analysis of main inflorescence related traits in Brassica napus L[J]. J Nucl Agric Sci, 2018, 32(3): 463-476.
[15]	Zhao XZ, Yu KJ, Pang CK, et al. QTL analysis of five silique-related traits in Brassica napus L. across multiple environments[J]. Front Plant Sci, 2021, 12: 766271. doi: 10.3389/fpls.2021.766271 URL
[16]	Wang XD, Chen L, Wang AN, et al. Quantitative trait loci analysis and genome-wide comparison for silique related traits in Brassica napus[J]. BMC Plant Biol, 2016, 16: 71. doi: 10.1186/s12870-016-0759-7 URL
[17]	任义英, 崔翠, 王倩, 等. 甘蓝型油菜籽粒着生密度及其相关性状全基因组关联分析[J]. 植物遗传资源学报, 2018, 19(2): 314-325.
	Ren YY, Cui C, Wang Q, et al. Genome-wide association analysis of seed density within per silique and its related traits in Brassica napus L[J]. J Plant Genet Resour, 2018, 19(2): 314-325.
[18]	Zhou QH, Zhou C, Zheng W, et al. Genome-wide SNP markers based on SLAF-seq uncover breeding traces in rapeseed(Brassica napus L.)[J]. Front Plant Sci, 2017, 8: 648. doi: 10.3389/fpls.2017.00648 URL
[19]	Sun XW, Liu DY, Zhang XF, et al. SLAF-seq: an efficient method of large-scale de novo SNP discovery and genotyping using high-throughput sequencing[J]. PLoS One, 2013, 8(3): e58700. doi: 10.1371/journal.pone.0058700 URL
[20]	伍晓明, 陈碧云, 陆光远. 油菜种质资源描述规范和数据标准[M]. 北京: 中国农业出版社, 2007.
	Wu XM, Chen BY, Lu GY. Descriptors and data standard for rapeseed(Brassica spp.)[M]. Beijing: Chinese Agriculture Press, 2007.
[21]	Cui YW, Hu C, Zhu YF, et al. CIK receptor kinases determine cell fate specification during early anther development in Arabidopsis[J]. Plant Cell, 2018, 30(10): 2383-2401. doi: 10.1105/tpc.17.00586 URL
[22]	Nowak K, Wójcikowska B, Gaj MD. ERF022 impacts the induction of somatic embryogenesis in Arabidopsis through the ethylene-related pathway[J]. Planta, 2015, 241(4): 967-985. doi: 10.1007/s00425-014-2225-9 URL
[23]	Pignocchi C, Minns GE, Nesi N, et al. ENDOSPERM DEFECTIVE1 is a novel microtubule-associated protein essential for seed development in Arabidopsis[J]. Plant Cell, 2009, 21(1): 90-105. doi: 10.1105/tpc.108.061812 pmid: 19151224
[24]	Wang YP, Wu YY, Yu BY, et al. EXTRA-LARGE G PROTEINs interact with E3 ligases PUB4 and PUB2 and function in cytokinin and developmental processes[J]. Plant Physiol, 2017, 173(2): 1235-1246. doi: 10.1104/pp.16.00816 pmid: 27986866
[25]	Xiong F, Ren JJ, Yu Q, et al. AtBUD13 affects pre-mRNA splicing and is essential for embryo development in Arabidopsis[J]. Plant J, 2019, 98(4): 714-726. doi: 10.1111/tpj.14268
[26]	Toujani W, Muñoz-Bertomeu J, Flores-Tornero M, et al. Identification of the phosphoglycerate dehydrogenase isoform EDA9 as the essential gene for embryo and male gametophyte development in Arabidopsis[J]. Plant Signal Behav, 2013, 8(11): e27207. doi: 10.4161/psb.27207 URL
[27]	Portereiko MF, Sandaklie-Nikolova L, Lloyd A, et al. NUCLEAR FUSION DEFECTIVE1 encodes the Arabidopsis RPL21M protein and is required for karyogamy during female gametophyte development and fertilization[J]. Plant Physiol, 2006, 141(3): 957-965. doi: 10.1104/pp.106.079319 pmid: 16698901
[28]	Louvet R, Cavel E, Gutierrez L, et al. Comprehensive expression profiling of the pectin methylesterase gene family during silique development in Arabidopsis thaliana[J]. Planta, 2006, 224(4): 782-791. doi: 10.1007/s00425-006-0261-9 URL
[29]	Richmond TA, Bleecker AB. A defect in beta-oxidation causes abnormal inflorescence development in Arabidopsis[J]. Plant Cell, 1999, 11(10): 1911-1924. pmid: 10521521
[30]	Wang XM, Xie B, Zhu MS, et al. Nucleostemin-like 1 is required for embryogenesis and leaf development in Arabidopsis[J]. Plant Mol Biol, 2012, 78(1-2): 31-44. doi: 10.1007/s11103-011-9840-7 URL
[31]	Chantha SC, Gray-Mitsumune M, Houde J, et al. The MIDASIN and NOTCHLESS genes are essential for female gametophyte development in Arabidopsis thaliana[J]. Physiol Mol Biol Plants, 2010, 16(1): 3-18. doi: 10.1007/s12298-010-0005-y URL
[32]	Töller A, Brownfield L, Neu C, et al. Dual function of Arabidopsis glucan synthase-like genes GSL8 and GSL10 in male gametophyte development and plant growth[J]. Plant J, 2008, 54(5): 911-923. doi: 10.1111/j.1365-313X.2008.03462.x URL
[33]	Boavida LC, Shuai B, Yu HJ, et al. A collection of Ds insertional mutants associated with defects in male gametophyte development and function in Arabidopsis thaliana[J]. Genetics, 2009, 181(4): 1369-1385. doi: 10.1534/genetics.108.090852 pmid: 19237690
[34]	Dean PJ, Siwiec T, Waterworth WM, et al. A novel ATM-dependent X-ray-inducible gene is essential for both plant meiosis and gametogenesis[J]. Plant J, 2009, 58(5): 791-802. doi: 10.1111/tpj.2009.58.issue-5 URL
[35]	Cheng Y, Cao L, Wang S, et al. Downregulation of multiple CDK inhibitor ICK/KRP genes upregulates the E2F pathway and increases cell proliferation, and organ and seed sizes in Arabidopsis[J]. Plant J, 2013, 75(4): 642-655. doi: 10.1111/tpj.2013.75.issue-4 URL
[36]	Berg M, Rogers R, Muralla R, et al. Requirement of aminoacyl-tRNA synthetases for gametogenesis and embryo development in Arabidopsis[J]. Plant J, 2005, 44(5): 866-878. doi: 10.1111/tpj.2005.44.issue-5 URL
[37]	Feng QN, Liang X, Li S, et al. The ADAPTOR PROTEIN-3 complex mediates pollen tube growth by coordinating vacuolar targeting and organization[J]. Plant Physiol, 2018, 177(1): 216-225. doi: 10.1104/pp.17.01722 URL
[38]	Kamata N, Okada H, Komeda Y, et al. Mutations in epidermis-specific HD-ZIP IV genes affect floral organ identity in Arabidopsis thaliana[J]. Plant J, 2013, 75(3): 430-440. doi: 10.1111/tpj.2013.75.issue-3 URL
[39]	Ahmad A. 甘蓝型油菜初始胚珠数和每角果粒数数量性状位点(QTL)的研究[D]. 武汉: 华中农业大学, 2018.
	Ahmad A. Quantitative trait loci(QTL)studies for the number of ovules and seeds per pod in Brassica napus L.[D]. Wuhan: Huazhong Agricultural University, 2018.
[40]	钟婷婷, 郭诗芬, 卢文斌, 等. 甘蓝型油菜抗根肿病KASP标记开发和利用[J]. 华北农学报, 2021, 36(4): 184-190. doi: 10.7668/hbnxb.20192042
	Zhong TT, Guo SF, Lu WB, et al. Development and utilization of KASP marker for Brassica napus clubroot resistance breeding[J]. Acta Agric Boreali Sin, 2021, 36(4): 184-190.
[41]	Jiao YM, Zhang KP, Cai GQ, et al. Fine mapping and candidate gene analysis of a major locus controlling ovule abortion and seed number per silique in Brassica napus L[J]. Theor Appl Genet, 2021, 134(8): 2517-2530. doi: 10.1007/s00122-021-03839-6
[42]	Zhai YG, Cai SL, Hu LM, et al. CRISPR/Cas9-mediated genome editing reveals differences in the contribution of INDEHISCENT homologues to pod shatter resistance in Brassica napus L[J]. Theor Appl Genet, 2019, 132(7): 2111-2123. doi: 10.1007/s00122-019-03341-0
[43]	Zheng M, Zhang L, Tang M, et al. Knockout of two BnaMAX1 homologs by CRISPR/Cas9-targeted mutagenesis improves plant architecture and increases yield in rapeseed(Brassica napus L.)[J]. Plant Biotechnol J, 2020, 18(3): 644-654. doi: 10.1111/pbi.13228 pmid: 31373135
[44]	高谢旺, 谭安琪, 胡信畅, 等. 利用CRISPR/Cas9技术创制高油酸甘蓝型油菜新种质[J]. 植物遗传资源学报, 2020, 21(4): 1002-1008.
	Gao XW, Tan AQ, Hu XC, et al. Creation of new germplasm of high-oleic rapeseed using CRISPR/Cas9[J]. J Plant Genet Resour, 2020, 21(4): 1002-1008.

性状 Trait	环境 Environment	平均值±标准差 Mean±SD	最小值 Min	50%分位数 50% quantile	最大值 Max	变异系数 CV /%	Kolmogorov-Smirnov
每角果粒数 SPS	JXAU	17.23±2.89	7.40	17.41	24.48	16.77	P=0.729 58
每角果粒数 SPS	JXIRS	15.79±3.36	5.54	15.88	27.78	21.29	P=0.346 01

性状 Trait	环境 Environment	平均值±标准差 Mean±SD	最小值 Min	50%分位数 50% quantile	最大值 Max	变异系数 CV /%	Kolmogorov-Smirnov
每角果粒数 SPS	JXAU	17.23±2.89	7.40	17.41	24.48	16.77	P=0.729 58
每角果粒数 SPS	JXIRS	15.79±3.36	5.54	15.88	27.78	21.29	P=0.346 01

变异来源Source of variation	自由度DF（degree of freedom）	方差及占总方差的比例MS（SS/SST）/%
区组 Block	1	52.12**（7.02）
处理间 Treatment	299	20.66**（2.78）
环境 Environment/E	1	621.99**（83.80）
基因型 Genotype /G	299	26.56**（3.58）
基因型×环境 G×E	299	12.75*（1.72）
误差 Error	599	8.17（1.10）

变异来源Source of variation	自由度DF（degree of freedom）	方差及占总方差的比例MS（SS/SST）/%
区组 Block	1	52.12**（7.02）
处理间 Treatment	299	20.66**（2.78）
环境 Environment/E	1	621.99**（83.80）
基因型 Genotype /G	299	26.56**（3.58）
基因型×环境 G×E	299	12.75*（1.72）
误差 Error	599	8.17（1.10）

环境Environment	染色体 Chromosome	位置 Locus	P值P value	贡献率 R²/%	GLM	MLM
JXAU	A04	11830197	2.40E-06	11.07	●
	A05	10733464	2.32E-06	8.65	●
	A09	16100373	1.11E-06	8.87	●
	A09	16100374	1.55E-06	8.60	●
	A09	16100386	1.65E-06	8.56	●
	A09	16100422	1.39E-06	8.67	●
	A09	16100439	1.35E-06	8.76	●
	A09	16299217	1.68E-06	9.45	●
	A09	16299389	1.49E-06	9.71	●
	A09	16299462	1.13E-06	9.87	●
	A09	16299495	4.31E-07	10.38	●
	A09	16299626	4.29E-08	12.92	●
	A09	16303740	3.33E-07	9.69	●
	A09	16303768	1.98E-06	8.59	●
	A09	16366473	2.84E-06	9.82	●
	A09	17453710	1.10E-07	17.80	●
	A09	17453729	1.29E-07	17.42	●
	A09	17453891	4.02E-07	16.69	●
	A09	17471305	2.81E-06	8.66	●
	A09	17581807	3.44E-06	9.45	●
	A09	17581835	3.73E-06	9.77	●
	A09	17582057	3.16E-06	8.96	●
	C02	27389727	2.58E-06	7.91	●
	C02	27389750	1.90E-06	8.06	●
	C02	27389775	2.39E-06	7.93	●
	C02	34901810	4.35E-06	8.04	●
	C03	18955107	4.53E-06	8.52	●
	C03	21284972	3.40E-08-4.29E-06	10.85-8.9	●	○
	C03	21342259	4.40E-06	7.67	●
	C07	32557367	4.69E-06	7.48	●
	C07	42323087	8.09E-07	14.77	●
	C07	42323117	2.00E-07-3.28E-06	15.23-14.05	●	○
	C07	42323137	3.24E-06	12.97	●
	C07	42323279	4.80E-06	11.83	●
	C07	42323280	4.90E-06	11.88	●
	C08	27721260	3.47E-06	8.13	●
	C08	37461613	2.43E-06	11.73	●
JXIRS	A05	659981	5.92E-07-4.95E-06	16.00-16.75	●	○
JXIRS	A07	7482038	3.89E-06	9.63	●