<script type="text/javascript" src="https://cdn.bootcss.com/mathjax/2.7.2-beta.0/MathJax.js?config=TeX-AMS-MML_HTMLorMML"></script> <script> MathJax = { tex: { inlineMath: [['$', '$'], ['\\(', '\\)']] }, svg: { fontCache: 'global' } }; </script> 张春^,1,2, 赵小珍^1,2, 庞承珂^1,2, 彭门路^1,2, 王晓东², 陈锋², 张维², 陈松², 彭琦², 易斌³, 孙程明^,2,3,*, 张洁夫^,2,*, 傅廷栋³¹南京农业大学 / 作物遗传与种质创新国家重点实验室, 江苏南京 210095
²江苏省农业科学院经济作物研究所 / 农业农村部长江下游棉花与油菜重点实验室 / 中国江苏省现代作物生产协同创新中心, 江苏南京210014
³华中农业大学植物科学技术学院 / 作物遗传改良国家重点实验室, 湖北武汉 430070

Genome-wide association study of 1000-seed weight in rapeseed (Brassica napus L.)

ZHANG Chun^,1,2, ZHAO Xiao-Zhen^1,2, PANG Cheng-Ke^1,2, PENG Men-Lu^1,2, WANG Xiao-Dong², CHEN Feng², ZHANG Wei², CHEN Song², PENG Qi², YI Bin³, SUN Cheng-Ming^,2,3,*, ZHANG Jie-Fu^,2,*, FU Ting-Dong³¹Nanjing Agricultural University / State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing 210095, Jiangsu, China
²Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences / Key Laboratory of Cotton and Rapeseed (Nanjing), Ministry of Agriculture and Rural Affairs / Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing 210014, Jiangsu, China
³National Key Laboratory of Crop Genetic Improvement / College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China

通讯作者: *张洁夫, E-mail: jiefu_z@163.com; 孙程明, E-mail: suncm8331537@gmail.com

收稿日期:2020-06-22接受日期:2020-09-13网络出版日期:2021-04-12

基金资助:

国家重点研发计划项目.2018YFD0100602 国家现代农业产业技术体系建设专项.CARS-12 国家自然科学基金项目.32001581 江苏省农业科技自主创新基金.CX(19)3055 江苏省基础研究计划(自然科学基金)项目.BK20190260 中央高校基本科研业务费专项资金项目.2662016PY063

Received:2020-06-22Accepted:2020-09-13Online:2021-04-12

Fund supported:

National Key Research and Development Program of China.2018YFD0100602 China Agriculture Research System.CARS-12 National Natural Science Foundation of China.32001581 Jiangsu Agriculture Science and Technology Innovation Fund.CX(19)3055 Natural Fund Project of Jiangsu Basic Research Program.BK20190260 Fundamental Research Funds for the Central Universities.2662016PY063

作者简介 About authors
E-mail: 15662020807@163.com















摘要
千粒重是油菜产量构成的重要因素之一。本研究利用高通量SNP芯片对496份具有代表性的油菜种质资源进行基因型分析, 考察群体在3个环境(14NJ、15TZ、16TZ)中的千粒重表型, 利用混合线性模型(mixed linear model, MLM)和一般线性模型(general linear model, GLM)进行全基因组关联分析。结果表明, 本群体在3个环境中千粒重的广义遗传力为63.12%。MLM模型检测到6个显著位点, 解释28.92%的表型变异; GLM模型检测到61个显著位点, 解释47.08%的表型变异。合并共同位点后得到62个显著位点, 联合解释47.31%的表型变异。这些位点分布在基因组所有染色体上, 在A07、A03和C06染色体上分别检测到数目最多的9、8和7个位点。其中效应最大的位点Bn-scaff_17526_1-p1066214位于C09染色体, 在MLM和GLM模型中表型贡献值分别为5.55%和15.26%。21个位点与前人报道的QTL重叠, 其中8个位点得到至少2个群体的验证。其余41个位点为新鉴定的位点, 其中多个位点效应高且在多环境中被检测到, 如位点Bn-A03-p560769、Bn-scaff_15743_1-p599416和Bn-scaff_15743_1-p590955等。在11个位点附近找到DGAT、EOD3、AGL61、WRI1、DA2、RAV1等拟南芥已报道千粒重基因的同源基因。本研究结果有助于解析甘蓝型油菜千粒重的遗传基础, 为研究千粒重的调控机制、指导千粒重的遗传改良奠定基础。
关键词： 甘蓝型油菜;千粒重;产量;关联分析;SNP标记

Abstract
Thousand-seed weight (TSW) is one of the important component of seed yield. In this study, a collection of 496 representative rapeseed accessions were genotyped by the high-throughput 60K SNP array for TSW in three environments (14NJ, 15TZ, 16TZ). The genome-wide association study (GWAS) of TSW was performed via the MLM (Mixed linear model) and GLM (General linear model). The results showed that the broad sense heritability of TSW was 63.12% in three environments. Six and 61 loci were detected with MLM and GLM, which explained 28.92% and 47.08% of the phenotypic variance, respectively. Combining the common loci between two models, 62 significant loci were obtained and accounted for 47.31% of the phenotypic variance. These loci were distributed on all chromosomes of the genome, with the largest number of 9, 8, and 7 loci detected on Chromosome A07, A03, and C06, respectively. The most significant locus Bn-scaff_17526_1-p1066214 was detected on C09, and accounted for 5.55% and 15.26% of the phenotypic variance in MLM and GLM, respectively. Among them, 21 loci overlapped with previously reported QTLs, of which 8 loci were verified by at least two populations. The remaining 41 loci were newly detected, and many of them had high effects and were detected in multiple environments, such as Bn-A03-p560769, Bn-scaff_15743_1-p599416 and Bn-scaff_15743_1-p590955. Besides, 11 candidates orthologous to documented Arabidopsis seed weight genes, like DGAT, EOD3, AGL61, WRI1, DA2, and RAV1, were found near our GWAS loci. The results are helpful for analyzing the genetic basis of TSW of rapeseed and lay a foundation for studying the regulation mechanism and guiding the genetic improvement of TSW.
Keywords：Brassica napus L.;1000-seed weight;yield;GWAS;SNP


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本文引用格式
张春, 赵小珍, 庞承珂, 彭门路, 王晓东, 陈锋, 张维, 陈松, 彭琦, 易斌, 孙程明, 张洁夫, 傅廷栋. 甘蓝型油菜千粒重全基因组关联分析[J]. 作物学报, 2021, 47(4): 650-659. doi:10.3724/SP.J.1006.2021.04136
ZHANG Chun, ZHAO Xiao-Zhen, PANG Cheng-Ke, PENG Men-Lu, WANG Xiao-Dong, CHEN Feng, ZHANG Wei, CHEN Song, PENG Qi, YI Bin, SUN Cheng-Ming, ZHANG Jie-Fu, FU Ting-Dong. Genome-wide association study of 1000-seed weight in rapeseed (Brassica napus L.)[J]. Acta Agronomica Sinica, 2021, 47(4): 650-659. doi:10.3724/SP.J.1006.2021.04136


甘蓝型油菜(Brassica napus L., 2n = 38, AACC)是世界上广泛种植的油料作物, 也是我国重要的植物油来源之一, 占总食用植物油40%以上。然而我国植物油料自给率不足四成, 且缺口仍呈扩大趋势, 提高油菜产量、保障油料供给安全已迫在眉睫^[1]。千粒重是构成油菜产量的三因素之一, 提高油菜千粒重是增产的直接途径, 因此解析粒重遗传基础和分子机理对油菜产量乃至国家油料供给安全具有重要意义。

千粒重是由多基因控制的数量性状, 受基因型和环境条件共同作用^[2]。从细胞学水平看, 种子器官的发育主要受细胞增殖和细胞膨大调节, 两者又分别决定着细胞数目和细胞大小^[3], Breuninger等^[4]研究认为, 细胞增殖(细胞数目)对种子等器官大小的影响更大。从种子发育进程看, 胚、胚乳及胚珠的协同生长关系着种子重量及大小, 同时发育过程又与母体植株密不可分, 种子大小与母体组织间存在某种内在联系。朱军等^[5]研究认为, 粒重遗传模型为胚、胚乳、细胞质和母体基因型的效应, 其中细胞质和母体基因型效应属母体效应。李娜等^[6]通过对遗传背景广泛的油菜大小粒种质进行遗传分析, 证明了油菜种子大小主要由母体基因型效应调控, 母体效应值达0.93。母体组织可从多种途径对种子大小产生影响: 一是转录因子调控途径, 如AP2通过限制种皮细胞的延伸控制种子生长^[7]; 二是泛素化途径, 如泛素受体DA1和DA2通过限制种皮细胞增殖影响籽粒大小^[8,9]; 三是G蛋白途径, 如G蛋白复合物由异源三聚体的Gα、Gβ和Gγ 3个亚基组成, 超表达Gγ (AGG3)的拟南芥通过细胞增殖促进种子生长发育^[10]; 四是激素途径, 如BZR1通过油菜素内酯途径影响种子大小^[11]; 五是其他类型生长物质, 如EOD3编码细胞色素P450单加氧酶, 通过促进种子株被或种皮细胞的延伸增加种子大小^[12]。

分子标记、SNP芯片等技术的出现大大推进了对复杂数量性状如千粒重基因的定位。多数科研工作者利用遗传差异大的双亲构建连锁群体, 进行千粒重QTL定位。Quijada等^[13]利用2个DH群体定位到3个千粒重QTL位点, 分别位于A07、C07和C09染色体上。Basunanda等^[14]同样利用DH群体, 结合SSR标记和AFLP标记, 检测到34个千粒重QTL, 表型变异贡献率范围为2.4%~23.0%, 其中7个位点能重复检测到。Yang等^[15]考察186个RIL群体在2环境下的表型, 检测到9个与千粒重相关的QTL, 分布在5个连锁群A01、A06、A07、A09和C03, 其中贡献率最大的QTL cqSW.A9位于A09染色体, 解释28.2%的表型变异。Zhao等^[16]考察DH群体在5个环境下的表型, 定位到40个千粒重QTL位点, 其中位于A06的主效QTL qSN.A6贡献率为20.1%。Fan等^[17]考察DH和F2两群体在2环境下的表型, 共检测到9个千粒重QTL, 分别位于在A01、A02、A05、A07、A10和C04, 解释的表型变异范围为27.6%~37.9%。Sun等^[18]利用高密度SNP图谱, 考察189个RIL群体在5个环境下的表型, 定位到25个千粒重相关的QTL, 其中QTL uqC2-1解释6.6%~7.6%的表型变异。目前甘蓝型油菜中已精细定位的粒重基因很少, Liu等^[19]成功克隆了第1个影响千粒重的基因ARF18, 该基因是控制角果长和千粒重的双效调控因子, 通过延长角果皮细胞长度促进角果伸长, 增加角果皮光合面积, 促进千粒重增加。Shi等^[20]利用图位克隆得到1个影响千粒重的基因BnaA9.CYP78A9D, 该基因编码1个P450单加氧酶, 通过促进角果瓣中的细胞伸长来调节角果长度, 影响种子大小。

随着高密度单核苷酸多态性(single nucleotide polymorphism, SNP)基因分型芯片和全基因组测序等技术的发展, 全基因组关联分析(genome-wide association study, GWAS)已广泛应用于油菜复杂性状的遗传结构解析^[21,22]。本研究选用496份具有代表性的油菜种质资源为关联群体, 利用60K SNP芯片对群体进行基因型分析, 结合3个环境考察的千粒重表型数据, 对该性状进行全基因组关联分析, 基于检测到的显著SNP位点挖掘候选基因。本研究中筛选出千粒重大的优异种质资源, 为今后的性状改良提供材料; 基于检测到的显著位点, 为开发粒重分子标记提供信息; 挖掘候选基因, 为千粒重基因克隆和调控机理研究奠定基础。

1 材料与方法

1.1 试验材料

496份甘蓝型油菜为国内外的地方品种、育成品种及高世代育种材料(附表1)。其中国内资源444份, 主要来自湖北、重庆、江苏、湖南、四川、陕西等油菜主产省市; 国外资源52份, 主要来自德国、瑞典、朝鲜、加拿大等国家。所有材料均由华中农业大学国家油菜工程技术研究中心提供。

Table S1
附表1
附表1496份油菜的信息
Table S1Information of 496 rapeseed accessions

材料号 Line No.	材料名称 Name	来源 Origin	材料号Line No.	材料名称 Name	来源 Origin
1	甘油5号 Ganyou 5	中国湖北Hubei, China	2	农林42 Nonglin 42	日本 Japan
3	科里纳 Kelina	国外Unknown	4	淮油6号 Huaiyou 6	中国江苏 Jiangsu, China
5	炎81-2 Yan 81-2	中国重庆Chongqing, China	6	28887	中国重庆Chongqing, China
7	黔油4号 Qianyou 4	中国贵州Guizhou, China	8	黔油331 Qianyou 331	中国贵州 Guizhou, China
9	恩油73 Enyou 73-1-2	中国湖北 Hubei, China	10	牛耳朵 Niuerduo	中国贵州 Guizhou, China
11	880101	中国重庆Chongqing, China	12	SWU40	中国重庆 Chongqing, China
13	SWU42	中国重庆Chongqing, China	14	SWU43	中国重庆 Chongqing, China
15	SWU44	中国重庆Chongqing, China	16	SWU45	中国重庆 Chongqing, China
17	SWU46	中国重庆Chongqing, China	18	SWU47	中国重庆 Chongqing, China
19	SWU48	中国重庆Chongqing, China	20	SWU52	中国重庆 Chongqing, China
21	SWU53	中国重庆Chongqing, China	22	SWU56	中国重庆 Chongqing, China
23	SWU59	中国重庆Chongqing, China	24	SWU65	中国重庆 Chongqing, China
25	SWU82	中国重庆Chongqing, China	26	SWU83	中国重庆 Chongqing, China
27	SWU92	中国重庆Chongqing, China	28	SWU101	中国重庆 Chongqing, China
29	SWU106	中国重庆Chongqing, China	30	SWU108	中国重庆 Chongqing, China
31	川油20 Chuanyou 20	中国四川 Sichuan, China	32	川油18 Chuanyou 18	中国四川Sichuan, China
33	CY12NY-7	中国四川 Sichuan, China	34	CY12Q95406	中国四川Sichuan, China
35	CY12Q8-7	中国四川 Sichuan, China	36	CY12QSZ06	中国四川Sichuan, China
37	CY12QCWH-1	中国四川 Sichuan, China	38	CY12Q95108	中国四川Sichuan, China
39	CY12Q21535-N3	中国四川 Sichuan, China	40	CY12PXW-4	中国四川Sichuan, China
41	CY12PXW-6	中国四川 Sichuan, China	42	CY12PXW-9	中国四川Sichuan, China
43	CY13PXW-17	中国四川 Sichuan, China	44	CY14PXW-18	中国四川Sichuan, China
45	CY15PXW-31	中国四川 Sichuan, China	46	CY16PXW-35	中国四川 Sichuan, China
47	CY17PXW-58	中国四川 Sichuan, China	48	CY18PXW-62	中国四川 Sichuan, China
49	CY19PXW-65	中国四川 Sichuan, China	50	CY20PXW-66	中国四川 Sichuan, China
51	CY21PXW-84	中国四川 Sichuan, China	52	CY12GJ-1	中国四川 Sichuan, China
53	wx1025	中国湖南 Hunan, China	54	wx10213	中国湖南 Hunan, China
55	wx10296	中国湖南 Hunan, China	56	wx10315	中国湖南 Hunan, China
57	10-1043	中国湖南 Hunan, China	58	10-1047	中国湖南 Hunan, China
59	10-1061	中国湖南 Hunan, China	60	10-1070	中国湖南 Hunan, China
61	10-804	中国湖南 Hunan, China	62	10-1358	中国湖南 Hunan, China
63	1472	中国湖南 Hunan, China	64	湘油13号 Xiangyou 13	中国湖南 Hunan, China
65	湘油15号 Xiangyou 15	中国湖南 Hunan, China	66	湘油11号 Xiangyou 11	中国湖南 Hunan, China
67	740	中国湖南 Hunan, China	68	631	中国湖南 Hunan, China
69	613	中国湖南 Hunan, China	70	783	中国湖南 Hunan, China
71	782	中国湖南 Hunan, China	72	YB3	中国湖南 Hunan, China
73	1360	中国湖南 Hunan, China	74	563	中国湖南 Hunan, China
75	WX10329	中国湖南 Hunan, China	76	santana	德国 German
77	1281	中国湖南 Hunan, China	78	509	中国湖南 Hunan, China
79	1368	中国湖南 Hunan, China	80	1322	中国湖南 Hunan, China
81	1252	中国湖南 Hunan, China	82	1321	中国湖南 Hunan, China
83	07022	中国湖北 Hubei, China	84	07094	中国湖北 Hubei, China
85	07016	中国湖北 Hubei, China	86	9F087	中国湖北 Hubei, China
87	97096	中国湖北 Hubei, China	88	97097	中国湖北 Hubei, China
89	07189	中国湖北 Hubei, China	90	07191	中国湖北 Hubei, China
91	07037	中国湖北 Hubei, China	92	RQ011	中国湖北 Hubei, China
93	RR009	中国湖北 Hubei, China	94	RR002	中国湖北 Hubei, China
95	97177	中国湖北 Hubei, China	96	96021	中国湖北 Hubei, China
97	96063	中国湖北 Hubei, China	98	01111	中国湖北 Hubei, China
99	01570	中国湖北 Hubei, China	100	9保22 9Bao 22	中国湖北 Hubei, China
101	01188	中国湖北 Hubei, China	102	02354	中国湖北 Hubei, China
103	02359	中国湖北 Hubei, China	104	02365	中国湖北 Hubei, China
105	93205	中国湖北 Hubei, China	106	93210	中国湖北 Hubei, China
107	Nca	国外 Unknown	108	中双4号 Zhongshuang 4	中国湖北 Hubei, China
109	中双9号 Zhongshuang 9	中国湖北 Hubei, China	110	中双11号 Zhongshuang 11	中国湖北 Hubei, China
111	2011-6200	中国湖北 Hubei, China	112	2011-6308	中国湖北 Hubei, China
113	2011-7103	中国湖北 Hubei, China	114	2012-11526	中国湖北 Hubei, China
115	2012-3448	中国湖北 Hubei, China	116	2012-3546	中国湖北 Hubei, China
117	2012-4531	中国湖北 Hubei, China	118	2012-5086	中国湖北 Hubei, China
119	2012-5113	中国湖北 Hubei, China	120	2012-8327	中国湖北 Hubei, China
121	2012-8355	中国湖北 Hubei, China	122	2012-8380	中国湖北 Hubei, China
123	2012-8998	中国湖北 Hubei, China	124	2012-9323	中国湖北 Hubei, China
125	2012-9354	中国湖北 Hubei, China	126	2012-9380	中国湖北 Hubei, China
127	2012-9478	中国湖北 Hubei, China	128	2012-9542	中国湖北 Hubei, China
129	2012-K8053	中国湖北 Hubei, China	130	R2	中国湖北 Hubei, China
131	希望106 Xiwang 106	中国湖北 Hubei, China	132	阳光198 Yangguang 198	中国湖北 Hubei, China
133	阳光2009 Yangguang 2009	中国湖北 Hubei, China	134	中双10号 Zhongshuang 10	中国湖北 Hubei, China
135	中双12号 Zhongshuang 12	中国湖北 Hubei, China	136	中双6号 Zhongshuang 6	中国湖北 Hubei, China
137	中双7号 Zhongshuang 7	中国湖北 Hubei, China	138	中油589 Zhongyou 589	中国湖北 Hubei, China
139	华油2号 Huayou 2	中国湖北 Hubei, China	140	Major	法国 France
141	华双2号 Huashuang 2	中国湖北 Hubei, China	142	Aurora	德国 German
143	华油13号 Huayou13	中国湖北 Hubei, China	144	Rucabo	德国 German
145	华油3号 Huayou 3	中国湖北 Hubei, China	146	华油14 Huayou 14	中国湖北 Hubei, China
147	宁油1号 Ningyou 1	中国江苏 Jiangsu, China	148	Ceres	德国 German
149	11-9-700	中国湖北 Hubei, China	150	11-9-701	中国湖北 Hubei, China
151	11-9-702	中国湖北 Hubei, China	152	11-9-703	中国湖北 Hubei, China
153	11-9-704	中国湖北 Hubei, China	154	11-9-705	中国湖北 Hubei, China
155	11-9-706	中国湖北 Hubei, China	156	11-9-707	中国湖北 Hubei, China
157	11-P63-5育7 11-P63-5 yu 7	中国湖北 Hubei, China	158	11-P63-8育32 11-P63-8 yu 32	中国湖北 Hubei, China
159	11-P63-3育3 11-P63-3 yu 3	中国湖北 Hubei, China	160	11-P67东 11-P67 Dong	中国湖北 Hubei, China
161	09-P64-1	中国湖北 Hubei, China	162	10-崇23 10-chong23	中国湖北 Hubei, China
163	10-崇24 10-chong 24	中国湖北 Hubei, China	164	10-崇25 10-chong25	中国湖北 Hubei, China
165	10-崇29 10-chong 29	中国湖北 Hubei, China	166	10-崇32 10-chong 32	中国湖北 Hubei, China
167	10-崇33 10-chong 33	中国湖北 Hubei, China	168	10-崇34 10-chong 34	中国湖北 Hubei, China
169	10-江棚2 10-jiangpeng 2	中国湖北 Hubei, China	170	10-江棚3 10-jiangpeng 3	中国湖北 Hubei, China
171	11-育7-103 11-yu 7-103	中国湖北 Hubei, China	172	11-育7-117 11-yu7-117	中国湖北 Hubei, China
173	11-育7-125 11-yu 7-125	中国湖北 Hubei, China	174	11-P74-8	中国湖北 Hubei, China
175	11-P74-13	中国湖北 Hubei, China	176	7-7766-74	中国湖北 Hubei, China
177	P18	中国湖北 Hubei, China	178	64棚-1064 peng-10	中国湖北 Hubei, China
179	圣光77 Shengguang 77	中国湖北 Hubei, China	180	甲预17棚 Jiayu17peng	中国湖北 Hubei, China
181	甲预25棚 Jiayu25peng	中国湖北 Hubei, China	182	甲预16棚 Jiayu16peng	中国湖北 Hubei, China
183	甲预31棚 Jiayu31peng	中国湖北 Hubei, China	184	华双5号 Huashuang5	中国湖北 Hubei, China
185	华双4号 Huashuang 4	中国湖北 Hubei, China	186	甲972Jia972	中国湖北 Hubei, China
187	华双128 Huashuang 128	中国湖北 Hubei, China	188	甲904 Jia904	中国湖北 Hubei, China
189	甲908 Jia 908	中国湖北 Hubei, China	190	甲PF190棚 Jia PF190peng	中国湖北 Hubei, China
191	甲915 Jia 915	中国湖北 Hubei, China	192	甲922 Jia 922	中国湖北 Hubei, China
193	甲951棚 Jia951peng	中国湖北 Hubei, China	194	甲917 Jia 917	中国湖北 Hubei, China
195	甲923 Jia923	中国湖北 Hubei, China	196	甲931 Jia 931	中国湖北 Hubei, China
197	甲预05棚 Jiayu05peng	中国湖北 Hubei, China	198	甲963棚 Jia963peng	中国湖北 Hubei, China
199	沪油12号 Huyou 12	中国上海 Shanghai, China	200	宁油16号 Ningyou 16	中国江苏 Jiangsu, China
201	史力佳 Shilijia	中国江苏 Jiangsu, China	202	杨油6号 Yangyou 6	中国江苏 Jiangsu, China
203	扬油5号 Yangyou 5	中国江苏 Jiangsu, China	204	镇油3号 Zhenyou 3	中国江苏 Jiangsu, China
205	苏油1号 Suyou 1	中国江苏 Jiangsu, China	206	浙油18 Zheyou18	中国浙江 Zhejiang, China
207	浙双72 Zheshuang 72	中国浙江 Zhejiang, China	208	浙双8号 Zheshuang 8	中国浙江 Zhejiang, China
209	浙油758 Zheyou 758	中国浙江 Zhejiang, China	210	沪油14 Huyou 14	中国上海 Shanghai, China
211	沪油18 Huyou 18-2	中国上海 Shanghai, China	212	沪油19 Huyou 19	中国上海 Shanghai, China
213	浙油19 Zheyou 19	中国浙江 Zhejiang, China	214	浙油21 Zheyou 21	中国浙江 Zhejiang, China
215	浙双6号 Zheshuang 6	中国浙江 Zhejiang, China	216	皖油15号 Wanyou 15	中国安徽 Anhui, China
217	皖油16号 Wanyou 16	中国安徽 Anhui, China	218	皖油29 Wanyou 29	中国安徽 Anhui, China
219	AGREV012	德国 German	220	AGREV019	德国 German
221	AGREV021	德国 German	222	Topas	瑞典 Sweden
223	cyclon	丹麦 Denmark	224	伟杰 Weijie	加拿大 Canada
225	四达 Sida	加拿大 Canada	226	至尊 Zhizun	加拿大 Canada
227	海神 Haishen	加拿大 Canada	228	D2	丹麦 Denmark
229	D3	丹麦 Denmark	230	Qu	美国 America
231	11-985	中国青海 Qinghai, China	232	11-504	中国青海 Qinghai, China
233	11-997	中国青海 Qinghai, China	234	11-540	中国青海 Qinghai, China
235	11-1184	中国青海 Qinghai, China	236	10-758	中国青海 Qinghai, China
237	10-1230	中国青海 Qinghai, China	238	10-847	中国青海 Qinghai, China
239	11-1124	中国青海 Qinghai, China	240	垦C1 KenC1	中国陕西 Shaanxi, China
241	P113	中国陕西 Shaanxi, China	242	P158	中国陕西 Shaanxi, China
243	P310	中国陕西 Shaanxi, China	244	P312	中国陕西 Shaanxi, China
245	P668	中国陕西 Shaanxi, China	246	P685	中国陕西 Shaanxi, China
247	A117	中国陕西 Shaanxi, China	248	A172	中国陕西 Shaanxi, China
249	B250	中国陕西 Shaanxi, China	250	B265	中国陕西 Shaanxi, China
251	A109	中国陕西 Shaanxi, China	252	B285	中国陕西 Shaanxi, China
253	C052	中国陕西 Shaanxi, China	254	GY270	中国陕西 Shaanxi, China
255	GY282	中国陕西 Shaanxi, China	256	GY284	中国陕西 Shaanxi, China
257	B262	中国河南 Henan, China	258	A82	中国江西 Jiangxi, China
259	A98	中国陕西 Shaanxi, China	260	B414	中国新疆 Xinjiang, China
261	B308	瑞典 Sweden	262	A97	中国四川 Sichuan, China
263	A148	瑞典 Sweden	264	B431	丹麦 Denmark
265	08-P35	中国湖北 Hubei, China	266	08-P36	中国湖北 Hubei, China
267	09-P32	中国湖北 Hubei, China	268	09-P36	中国湖北 Hubei, China
269	09-P37	中国湖北 Hubei, China	270	10-P10	中国湖北 Hubei, China
271	10-P29	中国湖北 Hubei, China	272	11-P30	中国湖北 Hubei, China
273	12-P24	中国湖北 Hubei, China	274	12-P25	中国湖北 Hubei, China
275	03ⅡB	中国湖北 Hubei, China	276	03Ⅱ3B	中国湖北 Hubei, China
277	03Ⅰ32B	中国湖北 Hubei, China	278	964	中国湖北 Hubei, China
279	DD1	中国湖北 Hubei, China	280	9L302-1	中国湖北 Hubei, China
281	06T9F	中国湖北 Hubei, China	282	03Ⅱ4B	中国湖北 Hubei, China
283	03LF1	中国湖北 Hubei, China	284	9852	中国湖北 Hubei, China
285	9801C	中国湖北 Hubei, China	286	986	中国湖北 Hubei, China
287	876	中国湖北 Hubei, China	288	武164 Wu164	中国湖北 Hubei, China
289	9889	中国湖北 Hubei, China	290	06H7	中国湖北 Hubei, China
291	湖北白花油菜 Hubeibaihuayoucai	中国湖北 Hubei, China	292	南川长角 Nanchuanchangjiao	中国四川 Sichuan, China
293	IMC103	中国重庆 Chongqing, China	294	Oscar	澳大利亚 Australia
295	Sophia	德国 German	296	campina	德国 German
297	Conny	瑞典 Sweden	298	Wesreo	澳大利亚 Australia
299	Wase Chousen	朝鲜 North Korea	300	Gogatsuna	日本 Japan
301	Nakaee Chousen	朝鲜 North Korea	302	Cat.No.117	德国 German
303	90750	中国重庆 Chongqing, China	304	农林43 Nonglin 43	日本
305	西藏油菜 Xizangyoucai	中国西藏 Tibet, China	306	油研2号 Youyan 2	中国贵州
307	矮架早 Aijiazao	中国四川 Sichuan, China	308	SWU41	中国重庆 Chongqing, China
309	SWU49	中国重庆 Chongqing, China	310	SWU54	中国重庆 Chongqing, China
311	SWU57	中国重庆 Chongqing, China	312	SWU60	中国重庆 Chongqing, China
313	SWU61	中国重庆 Chongqing, China	314	SWU62	中国重庆 Chongqing, China
315	SWU63	中国重庆 Chongqing, China	316	SWU64	中国重庆 Chongqing, China
317	SWU66	中国重庆 Chongqing, China	318	SWU67	中国重庆 Chongqing, China
319	SWU68	中国重庆 Chongqing, China	320	SWU69	中国重庆 Chongqing, China
321	SWU70	中国重庆Chongqing, China	322	SWU71	中国重庆 Chongqing, China
323	SWU74	中国重庆 Chongqing, China	324	SWU75	中国重庆 Chongqing, China
325	SWU76	中国重庆 Chongqing, China	326	SWU77	中国重庆 Chongqing, China
327	SWU80	中国重庆 Chongqing, China	328	SWU81	中国重庆 Chongqing, China
329	SWU84	中国重庆 Chongqing, China	330	SWU85	中国重庆 Chongqing, China
331	SWU87	中国重庆 Chongqing, China	332	SWU88	中国重庆 Chongqing, China
333	SWU89	中国重庆 Chongqing, China	334	SWU90	中国重庆 Chongqing, China
335	SWU93	中国重庆 Chongqing, China	336	SWU94	中国重庆 Chongqing, China
337	SWU95	中国重庆 Chongqing, China	338	SWU96	中国重庆 Chongqing, China
339	SWU99	中国重庆 Chongqing, China	340	SWU100	中国重庆 Chongqing, China
341	SWU102	中国重庆 Chongqing, China	342	SWU103	中国重庆 Chongqing, China
343	SWU104	中国重庆 Chongqing, China	344	SWU105	中国重庆 Chongqing, China
345	SWU107	中国重庆 Chongqing, China	346	SWU110	中国重庆 Chongqing, China
347	SWU111	中国重庆 Chongqing, China	348	SWU112	中国重庆 Chongqing, China
349	SWU113	中国重庆 Chongqing, China	350	SWU114	中国重庆 Chongqing, China
351	辐油4号 Fuyou 4	中国重庆 Chongqing, China	352	镇3736 Zhen3736	中国江苏 Jiangsu, China
353	镇2609 Zhen 2609	中国江苏 Jiangsu, China	354	HX0352	中国江苏 Jiangsu, China
355	阳光2009 Yangguang 2009	中国江苏 Jiangsu, China	356	沪油21 Huyou 21	中国上海 Shanghai, China
357	浙双3号 Zheshuang 3	中国浙江 Zhejiang, China	358	浙油21 Zheyou 21	中国浙江 Zhejiang, China
359	皖油20 Wanyou 20	中国安徽 Anhui, China	360	皖油12 Wanyou 12	中国安徽 Anhui, China
361	皖油7号 Wanyou 7	中国安徽 Anhui, China	362	红油3号 Hongyou 3	中国江苏 Jiangsu, China
363	镇油5号 Zhenyou5	中国江苏 Jiangsu, China	364	扬油4号 Yangyou 4	中国上海 Shanghai, China
365	沪油15 Huyou 15	中国上海 Shanghai, China	366	沪油16 Huyou 16	中国上海 Shanghai, China
367	沪油17 Huyou17	中国上海 Shanghai, China	368	浙双72 Zheshuang 72	中国浙江 Zhejiang, China
369	浙双8号 Zheshuang 8	中国浙江 Zhejiang, China	370	浙油50 Zheyou 50	中国浙江 Zhejiang, China
371	中双11 Zhongshuang 11	中国湖北 Hubei, China	372	阳光198 Yangguang198	中国湖北 Hubei, China
373	华航901 Huahang 901	中国湖北 Hubei, China	374	扬J6711 Yang J6711	中国江苏 Jiangsu, China
375	盐6055 Yan 6055	中国江苏 Jiangsu, China	376	杨鉴8号 Yangjian8	中国江苏 Jiangsu, China
377	希望106 Xiwang 106	中国湖北 Hubei, China	378	浙油17号 Zheyou 17	中国浙江 Zhejiang, China
379	中双5号 Zhongshuang 5	中国湖北 Hubei, China	380	中油821 Zhongyou 821	中国湖北 Hubei, China
381	秦油1号 Qinyou 1	中国陕西 Shaanxi, China	382	纬隆88 Weilong 88	中国陕西 Shaanxi, China
383	盐油2号 Yanyou 2	中国江苏 Jiangsu, China	384	秦油5号 Qinyou 5	中国陕西 Shaanxi, China
387	德68-12 De 68-12	中国四川 Sichuan, China	388	Monty	澳大利亚 Australia
389	Oscar	澳大利亚 Australia	390	宁油12 Ningyou 12-1	中国江苏 Jiangsu, China
391	宁油14 Ningyou 14	中国江苏 Jiangsu, China	392	史力丰-1 Shilifeng-1	中国江苏 Jiangsu, China
393	宁油18 Ningyou 18	中国江苏 Jiangsu, China	394	Helios	丹麦 Denmark
395	Hector	加拿大 Canada	396	棉96-203 Mian96-203	中国青海 Qinghai, China
397	青662A Qing662A	中国青海 Qinghai, China	398	699	中国湖北 Hubei, China
399	加拿大2号 Jianada 2	加拿大 Canada	400	中双2号 Zhongshuang 2	中国湖北 Hubei, China
401	WH-12	中国湖北 Hubei, China	402	WH-15	中国湖北 Hubei, China
403	WH-17	中国湖北 Hubei, China	404	WH-19	中国湖北 Hubei, China
405	WH-20	中国湖北 Hubei, China	406	WH-23	中国湖北 Hubei, China
407	WH-24	中国湖北 Hubei, China	408	WH-25	中国湖北 Hubei, China
409	WH-26	中国湖北 Hubei, China	410	WH-27	中国湖北 Hubei, China
411	WH-28	中国湖北 Hubei, China	412	WH-29	中国湖北 Hubei, China
413	WH-30	中国湖北 Hubei, China	414	WH-31	中国湖北 Hubei, China
415	WH-33	中国湖北 Hubei, China	416	WH-37	中国湖北 Hubei, China
417	WH-38	中国湖北 Hubei, China	418	WH-41	中国湖北 Hubei, China
419	WH-42	中国湖北 Hubei, China	420	WH-43	中国湖北 Hubei, China
421	WH-45	中国湖北 Hubei, China	422	WH-49	中国湖北 Hubei, China
423	WH-50	中国湖北 Hubei, China	424	WH-55	中国湖北 Hubei, China
425	WH-56	中国湖北 Hubei, China	426	WH-57	中国湖北 Hubei, China
427	WH-58	中国湖北 Hubei, China	428	WH-59	中国湖北 Hubei, China
429	WH-60	中国湖北 Hubei, China	430	WH-61	中国湖北 Hubei, China
431	WH-62	中国湖北 Hubei, China	432	WH-63	中国湖北 Hubei, China
433	WH-81	中国湖北 Hubei, China	434	WH-83	中国湖北 Hubei, China
435	WH-85	中国湖北 Hubei, China	436	WH-88	中国湖北 Hubei, China
437	WH-93	中国湖北 Hubei, China	438	WH-95	中国湖北 Hubei, China
439	WH-100	中国湖北 Hubei, China	440	WH-127	中国湖北 Hubei, China
441	豫油1号 Yuyou 1	中国河南 Henan, China	442	COBRA	德国 German
443	宁油7号 Ningyou 7	中国江苏 Jiangsu, China	444	Tapidor	法国 France
445	华油6号 Huayou 6	中国湖北 Hubei, China	446	华油12 Huayou 12	中国湖北 Hubei, China
447	Cubs root	朝鲜 North Korea	448	华油10 Huayou 10	中国湖北 Hubei, China
449	Bienvenu	法国 France	450	胜利油菜 Shengliyoucai	中国四川 Sichuan, China
451	ERAKE	波兰 Poland	452	Taisetsu	日本 Japan
453	cresor	加拿大 Canada	454	Daichousen	朝鲜 North Korea
455	comet	瑞典 Sweden	456	Niklas	丹麦 Denmark
457	Askari	德国 German	458	chuosenshu	朝鲜 North Korea
459	WESBROOK	澳大利亚 Australia	460	Suigenshu	朝鲜 North Korea
461	华油4号 Huayou 4	中国湖北 Hubei, China	462	胜利青梗 Shengliqinggeng	中国上海 Shanghai, China
463	密角多头油菜 Mijiaoduotouyoucai	中国上海 Shanghai, China	464	矮箕胜利 Aijishengli	中国上海 Shanghai, China
465	漕泾胜利 Caojingshengli	中国上海 Shanghai, China	466	沪油三号 Huyou 3	中国上海 Shanghai, China
467	勺叶青 Shaoyeqing	中国上海 Shanghai, China	468	非洲油菜乳黄花 Feizhouyoucairuhuanghua	中国上海 Shanghai, China
469	沪激早 Hujizao	中国上海 Shanghai, China	470	漕油2号 Caoyou 2	中国上海 Shanghai, China
471	封顶240 Fengding 240	中国江苏 Jiangsu, China	472	全紫油菜 Quanziyoucai	中国江苏 Jiangsu, China
473	大花球 Dahuaqiu	中国江苏 Jiangsu, China	474	荣选 Rongxuan	中国江苏 Jiangsu, China
475	宁油10号 Ningyou 10	中国江苏 Jiangsu, China	476	宁油8号 Ningyou 8	中国江苏 Jiangsu, China
477	宁油六号 Ningyouliu	中国江苏 Jiangsu, China	478	垛油一号 Duoyou 1	中国江苏 Jiangsu, China
479	淮油12号 Huaiyou 12	中国江苏 Jiangsu, China	480	沛选170 Peixuang 170	中国江苏 Jiangsu, China
481	广德138 Guangde 138	中国安徽 Anhui, China	482	广德8104 Guangde 8104	中国安徽 Anhui, China
483	当油早1号Dangyouzao 1	中国安徽 Anhui, China	484	广德761 Guangde 761	中国安徽 Anhui, China
485	铜陵花叶 Tonglinghuaye	中国安徽 Anhui, China	486	滁610 Chu610	中国安徽 Anhui, China
487	滁107 Chu 107	中国安徽 Anhui, China	488	宿84-6 Su 84-6	中国安徽 Anhui, China
489	皖油早 Wanyouzao	中国安徽 Anhui, China	490	滁油1号 Chuyou 1	中国安徽 Anhui, China
491	滁县白花 Chuxianbaihua	中国安徽 Anhui, China	492	芥65-1 Jie 65-1	中国浙江 Zhejiang, China
493	申黄1号 Shenhuang 1	中国上海 Shanghai, China	494	浙油601 Zheyou 601	中国浙江 Zhejiang, China
495	三高油菜Three high rape	中国浙江 Zhejiang, China	496	早丰一号 Zaofeng 1	中国四川 Sichuan, China

新窗口打开|下载CSV

1.2 田间试验与表型调查

试验材料于2014年在江苏南京(14NJ)以及2015年、2016年在泰州(15TZ和16TZ)种植。采用完全随机区组设计, 设置3个重复, 单个材料每重复种2行, 每行15株, 行宽1.5 m, 行距40 cm, 株距15~17 cm。每年10月上旬大田直播, 田间管理按当地常规方式进行。田间材料均是自然成熟时收获, 每小区选择具有代表性的6个单株, 在挂藏室内自然阴干后进行考种分析。千粒重具体测量方法为: 单株全株脱粒后, 随机选择其中500粒种子称重, 重复取样3次, 保证3次称重结果差异小于0.1 g (5%以内)为有效的3个数据, 取平均数后加倍即为测量值。利用RStudio软件对各个环境的千粒重表型进行统计分析和相关性分析, 并计算广义遗传力和最佳线性无偏预测值(best linear unbiased prediction, BLUP) ^[23,24,25]。

1.3 基因型检测与分析

利用Illumina 60K SNP芯片对496份甘蓝型油菜资源进行基因型分析。在Genome Studio软件中对SNP进行质量控制。将过滤后的33,218个SNP序列比对至油菜Darmor-bzh基因组, e-value阈值设为10^-12, 得到19,167个单拷贝且位置清楚的SNP标记用于关联分析^[26]。

1.4 全基因组关联分析

利用软件Structure 2.3.4基于贝叶斯模型进行群体结构, 得到Q矩阵^[27], 利用软件SPAGeDi计算不同材料间的亲缘关系, 得到K矩阵^[28], 在软件Tassel 5.0中导入基因型、表型、Q矩阵和K矩阵数据文件。以Q矩阵和Q+K矩阵作协变量, 分别进行MLM和GLM 2种模型的全基因组关联分析^[29]。对MLM模型设置的显著性阈值(bonferroni threshold)为1/总标记数, 即-lg (P) = 4.28; 对GLM模型设置更为严格的显著性阈值为0.05/总标记数, 即-lg (P) = 5.58。当1 Mb区间内存在多个显著SNP时, 若两两间的r²≥0.1, 则将这些SNP归为1个关联位点, 以最小P值的SNP作为显著性位点代表。用RStudio软件包qqman绘制曼哈顿图和QQ (Quantile-Quantile)图。用RStudio软件lm功能分析关联位点解释的总表型变异。

1.5 已报道QTL的信息整理

目前有4篇千粒重关联分析研究, 4个群体分别包含157、192、427和521份油菜资源^{[30,31,32,33]}, 整理其检测到的显著位点并与本研究结果进行比较。针对利用SNP芯片进行基因分型的连锁群体, 将检测到的QTL两侧的SNP探针序列比对至油菜Darmor-bzh参考基因组, 以确定QTL的物理区间^[34]。对于以SSR和AFLP等传统标记进行基因分型的研究, 搜集QTL两侧标记的引物序列并将其比对至参考基因组, 以确定QTL的物理区间。

1.6 候选基因挖掘

对于显著关联位点, 以r² = 0.1作为衰减阈值, 用软件Tassel 5.0计算显著关联位点的LD衰减距离作为置信区间, 提取置信区间内的基因CDS序列, 与模式植物拟南芥中已报道的千粒重相关基因CDS进行BLAST比对, 设e-value阈值为10^-10, 以相似度最高的拟南芥基因信息来注释油菜基因。

2 结果与分析

2.1 千粒重表型统计分析

496份油菜种质资源表型千粒重在3个环境中具有广泛的变异, 单个环境的千粒重极差范围为4.63~ 5.36 g。单个环境表型平均值范围为(3.51±0.64)~ (4.27±0.88) g, 变异系数范围为0.16~ 0.21 (表1和图1)。3个环境间的相关系数为0.55, 达到极显著水平(P≤0.001)。这表明本研究考察的表型数据具有较高的可靠性和重复性。利用RStudio软件包lem4计算, 油菜千粒重在3个环境下的广义遗传力为63.12%。

Table 1
表1
表1关联群体千粒重性状的统计分析
Table 1Statistical analysis of 1000-seed weight of the association panel

年份/环境 Year/Environment	最小值 Minimum	最大值 Maximum	平均值±标准差 Average ± SD	变异系数 Coefficient of variation
2013/2014 南京Nanjing	2.27	7.63	4.27±0.88	0.21
2014/2015 泰州Taizhou	1.64	5.77	3.65±0.58	0.16
2015/2016 泰州Taizhou	1.50	6.13	3.51±0.64	0.18

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图1

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图1关联群体在3个环境(14NJ、15TZ、16TZ)中的千粒重分布

Fig. 11000-seed weight of the association panel in three environments (14NJ, 15TZ, 16TZ)

2.2 千粒重全基因组关联分析

利用MLM模型对千粒重BLUP值进行关联分析, 显著性阈值为-lg (P) = 4.28时, 共检测到8个显著位点。合并存在连锁不平衡的SNP (r² ≥ 0.1), 得到6个显著位点, 分布在A01、A02、A03、C06和C09染色体(表2和图2)。6个显著位点的-lg (P)值范围是4.33~5.38, 可解释4.86%~6.06%的表型变异。利用一个简单加性模型计算, 6个位点联合解释28.92%的表型变异。与单环境的关联结果比较, 5个位点被重复检测到。

Table 2
表2
表2MLM千粒重显著关联位点(BLUP)
Table 2Significant GWAS loci of 1000-seed weight in MLM (BLUP)

标记 Marker	染色体 Chr.	位置 Position (bp)	-lg (P)	表型变异 R2 (%)	环境 Environment	已报道QTL Reported QTL
Bn-A01-p23472380	A01	19,346,196	5.34	6.03	16TZ
Bn-A02-p6300284	A02	3,473,319	4.81	5.41	15TZ
Bn-A02-p6564861	A02	3,784,981	5.38	6.06	14NJ	[32]
Bn-A03-p22125583	A03	20,956,967	4.77	5.36		[32,35]
Bn-scaff_16874_1-p411591	C06	31,817,621	4.33	4.86	16TZ	[30,36,37]
Bn-scaff_17526_1-p1066214	C09	1,488,283	4.93	5.55	16TZ	[32]

缩写同图1。
Abbreviations are the same as those given in Fig. 1.

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图2

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图2油菜千粒重全基因组关联分析(BLUP)

A: 千粒重MLM曼哈顿图; B: 千粒重MLM QQ图; C: 千粒重GLM曼哈顿图; D: 千粒重GLM QQ图。
Fig. 2Genome-wide association study of 1000-seed weight in rapeseed (BLUP)

A: Manhattan plot of MLM for 1000-seed weight; B: quantile-quantile plot of MLM for 1000-seed weight; C: Manhattan plot of GLM for 1000-seed weight; D: quantile-quantile plot of GLM for 1000-seed weight.


利用GLM模型对千粒重BLUP值进行关联分析, 显著性阈值为-lg (P) = 5.58时, 共检测到226个显著位点。合并存在连锁不平衡的SNP (r² ≥ 0.1), 得到61个显著位点, 分布在全基因组所有染色体(表3和图2)。其中显著性最高位点Bn-scaff_17526_ 1-p1066214的-lg (P) = 15.98, 对表型变异的贡献率为15.26%。其余位点的-lg (P)值范围是5.58~11.57, 可解释4.89%~11.31%的表型变异。利用一个简单加性模型计算, 61个位点联合解释47.08%的表型变异。与单环境的关联结果比较, 47个位点被检测到, 13个位点在2环境中重复检测到, 7个位点在3个环境中被检测到。

Table 3
表3
表3GLM千粒重显著关联位点(BLUP)
Table 3Significant GWAS loci of 1000-seed weight in GLM (BLUP)

标记 Marker	染色体 Chr.	位置 Position (bp)	-lg (P)	表型变异 R2 (%)	环境 Environment	已报道QTL Reported QTL
Bn-A01-p1156979	A01	760,656	6.37	5.61	15TZ	[35]
Bn-A01-p1440009	A01	1,036,032	5.96	5.21
Bn-A01-p9621623	A01	8,234,343	6.43	5.66		[17,30,36]
Bn-A01-p12222806	A01	10,407,989	7.02	7.03	14NJ
Bn-A01-p15496639	A01	12,941,394	6.64	6.67	16TZ
Bn-A02-p6300284	A02	3,473,319	7.22	7.23	14TZ
Bn-A02-p6564861	A02	3,784,981	8.59	8.53	14NJ/16TZ	[32,35]
Bn-A03-p560769	A03	445,706	9.16	9.08	14NJ/16TZ
Bn-A03-p4352837	A03	3,885,180	6.18	6.22
Bn-A03-p6586436	A03	5,884,508	8.47	8.43	16TZ
Bn-A03-p8851142	A03	8,156,299	7.88	7.86	14NJ/15TZ/16TZ	[32]
Bn-A03-p13758392	A03	12,893,544	6.56	6.60	15TZ
Bn-A03-p14747263	A03	13,916,047	6.57	6.60	14NJ	[35]
Bn-A03-p22125583	A03	20,956,967	5.64	5.60		[32,35]
Bn-A03-p24494224	A03	22,999,825	6.05	6.10		[32]
Bn-A04-p13705636	A04	14,344,669	8.84	8.77	15TZ	[35]
Bn-A05-p2610006	A05	2,720,763	5.69	5.75
Bn-scaff_27198_1-p445589	A06	15,593,147	8.01	7.99	14NJ/15TZ
Bn-A06-p23759352	A06	22,715,814	5.97	6.01
Bn-Scaffold002856-p361	A07	592,982	5.84	5.89	14NJ/16TZ	[14,17,31,36]
Bn-A10-p11601681	A07	2,573,087	6.95	6.15	16TZ	[33]
Bn-A07-p5877587	A07	7,824,442	7.24	6.42	14NJ/15TZ	[13,32]
Bn-A07-p10557557	A07	11,738,166	5.71	5.77	16TZ
Bn-A02-p305007	A07	12,930,679	5.66	4.93	14NJ/15TZ/16TZ
Bn-A07-p11611255	A07	13,756,170	5.95	6.00		[35]
Bn-A07-p16095589	A07	17,996,553	5.96	6.01
Bn-scaff_15743_1-p590955	A07	18,512,786	8.64	8.58	14NJ/15TZ/16TZ
Bn-A07-p17804261	A07	19,646,053	6.20	5.45		[32,38]
Bn-A08-p2675098	A08	2,098,808	6.81	6.83	16TZ
Bn-A08-p10443959	A08	8,372,628	6.30	6.34		[32]
Bn-A08-p13284369	A08	11,051,828	5.89	5.94
Bn-A08-p20343735	A08	17,811,886	8.82	7.90	15TZ
Bn-A09-p7329993	A09	5,542,361	6.20	6.24	14NJ/15TZ	[14,32]
Bn-A10-p15021776	A10	14,967,128	5.78	5.83	15TZ
Bn-A10-p15167470	A10	15,100,392	6.67	6.69	15TZ
Bn-scaff_15803_1-p837307	C01	14,780,320	5.58	5.64	14NJ
Bn-scaff_20942_1-p52095	C02	11,201,048	6.54	6.58	14NJ
Bn-scaff_16002_1-p1803014	C03	12,573,756	6.95	6.97	15TZ
Bn-scaff_26320_1-p269450	C03	30,924,988	5.61	4.89
Bn-scaff_16182_1-p296671	C03	51,992,967	6.41	6.44	14NJ
Bn-scaff_15794_3-p89166	C03	55,716,441	8.28	7.39	14NJ/15TZ
Bn-scaff_17119_1-p148890	C03	56,814,587	9.28	9.19	14NJ/15TZ	[35]
Bn-scaff_16217_1-p597569	C04	21,919,412	5.68	4.96	14NJ
Bn-scaff_18062_1-p229981	C04	31,112,520	6.23	5.47	14NJ/16TZ
标记 Marker	染色体 Chr.	位置 Position (bp)	-lg (P)	表型变异 R2 (%)	环境 Environment	已报道QTL Reported QTL
Bn-scaff_15585_1-p1089867	C04	44,500,243	5.88	5.14	16TZ
Bn-scaff_18903_1-p371596	C04	47,531,676	6.87	6.89	16TZ
Bn-scaff_16414_1-p1774629	C05	279,059	7.33	7.33	14NJ
Bn-scaff_20901_1-p2029631	C05	1,973,973	6.47	6.50	14NJ/16TZ
Bn-scaff_20901_1-p1279675	C05	2,707,564	7.36	7.36	14NJ
Bn-scaff_15763_1-p596396	C05	11,699,136	5.71	4.98	16TZ
Bn-scaff_15763_1-p588874	C06	20,160,298	5.85	5.90	16TZ
Bn-scaff_16064_1-p938130	C06	24,703,441	11.57	11.31	14NJ/15TZ/16TZ	[35]
Bn-scaff_15743_1-p599416	C06	27,808,971	9.80	8.80	14NJ/15TZ/16TZ
Bn-scaff_18807_1-p747016	C06	30,110,394	5.69	4.96		[36]
Bn-scaff_16874_1-p411591	C06	31,817,621	6.66	6.69	14NJ/16TZ	[30,36,37]
Bn-scaff_17799_1-p2391172	C06	34,110,582	7.71	6.87	14NJ/16TZ
Bn-scaff_17799_1-p853567	C06	35,737,877	7.70	7.69	14NJ	[35]
Bn-scaff_15705_1-p2279820	C07	35,285,037	6.12	6.16	16TZ
Bn-A08-p10452462	C08	16,678,522	6.88	6.08	14NJ/15TZ/16TZ
Bn-scaff_17526_1-p1066214	C09	1,488,283	15.98	15.26	14NJ/15TZ/16TZ	[32]
Bn-scaff_15576_1-p614226	C09	41,707,720	9.03	8.96	14NJ/16TZ

缩写同图1。
Abbreviations are the same as those given in Fig. 1.

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综合2个模型关联结果, 5个MLM模型的显著位点与GLM重叠, 合并重叠位点后共得到62个显著位点, 联合解释47.31%的表型变异。这些位点分布在全基因组所有染色体上, 其中A07染色体上检测到的数目最多, 达9个位点, A03和C06染色体上分别检测到8个和7个位点。分析显著位点的分布, 58.06%的位点分布在A亚基因组, 41.94%位于C亚基因组(表2和表3)。

2.3 与前人报道 QTL 的比较

综合MLM和GLM模型检测结果, 与已报道的千粒重QTL结果比较, 本研究中21个显著位点与前人研究结果重叠, 其中有8个位点得到至少2个群体的验证(表2和表3)。位于A02染色体的位点Bn-A02-p6564861位置为3.78 Mb, 与Shahid等^[32]通过关联分析检测到的位点Bn-A02-p6564861和Luo等^[35]通过DH群体定位到的千粒重QTL qSW.A2-4 (位置: 3.78~3.83 Mb)重叠。位于A07染色体的位点Bn-Scaffold002856-p361位置为0.59 Mb, 与Cai等^[31]、Fan等^[17]和Shi等^[36]采用不同策略检测到位点BrGMS3983、QTL TSWA7b和qSW.A7-2 (位置: 0.83 Mb)重叠。位于A09染色体的位点Bn-A09- p7329993位置为5.54 Mb, 与Basunanda等^[14]定位到QTL TH-tsm07、TH-tsm06和Shahid等^[32]检测到位点Bn-A09-p7327691 (位置: 5.54~5.74 Mb)重叠。位于C06染色体的位点Bn-scaff_16874_1-p411591位置为31.82 Mb, 与Dong等^[30]、Shi等^[36]和Li等^[37]通过不同策略检测到的位点(位置: 31.18 Mb)重叠。GLM模型中效应最大的位点Bn-scaff_ 17526_1-p1066214位于C09染色体1.49 Mb, -lg (P) = 15.98, 解释15.26%的表型变异, 与Shahid等^[32]检测到的效应最大位点Bn-scaff_17526_1-p1063974 (位置: 1.49 Mb)一致。该模型中效应次高的位点Bn-scaff_16064_1-p938130位于C06染色体24.70 Mb, -lg (P) = 11.57, 解释11.31%的表型变异, 与Luo等^[35]定位到的千粒重QTL qSW.C6-3 (位置: 24.52 Mb)重叠。此外有16个位点与前人检测的QTL位点重叠或相邻(表3), 以上QTL比较验证了本研究结果的可靠性。

2.4 候选基因挖掘

基于油菜基因组的注释信息, 在Bn-A03-p221 25583位点下游244 kb处找到候选基因BnaA03g4 1350D, 该基因的拟南芥同源基因DGAT编码二酰甘油酰基转移酶, 其过表达促进种子中甘油三酯含量和种子重量增加^[39] (表4)。在Bn-A03-p8851142位点下游118 kb处找到候选基因BnaA03g17130D, 该基因的拟南芥同源基因TTG2编码WRKY转录因子, 通过增加种皮细胞延伸促进种子粒重增加^[40]。在Bn-A05-p2610006位点下游7.5 kb处找到候选基因BnaA05g36830D, 该基因的拟南芥同源基因AGL61编码I型MADS结构域蛋白, 在中央细胞发育过程和种子胚乳形成过程中起调节作用, 影响种子大小^[41]。在Bn-A07-p11611255位点上游115 kb处找到候选基因BnaA07g16350D, 该基因的拟南芥同源基因WRI1调节编码糖酵解途径和脂肪酸相关酶基因的表达, WRI1通过增大种子体积引起种子重量增加^[42]。在Bn-scaff_18062_1-p229981位点上游323 kb找到候选基因BnaC04g33070D, 该基因的拟南芥同源基因RAV1是植物生长的负调控因子, 其高表达的转基因植株表现出异常胚珠, 导致种子大小、重量和数量减少^[43]。此外, 本研究还找到其他6个候选基因, 包括拟南芥已知调控粒重相关基因NPC6^[44]、CLV3^[45]、GRF2^[46]、EOD3^[12]、GW5^[47]、DA2^[9]、SHB1^[48]等在油菜中的同源拷贝。

Table 4
表4
表4千粒重关联位点候选基因信息
Table 4Information of candidate genes of 1000-seed weight GWAS loci

标记 Marker	油菜基因 Rapeseed gene	染色体 Chr.	位置 Position	拟南芥同源基因Arabidopsis homolog gene
				基因名称Gene name	基因号Gene ID
Bn-A01-p15496639	BnaA01g20420D	A01	12,306,073	NPC6	AT3G48610
Bn-A03-p22125583	BnaA03g41350D	A03	20,713,211	DGAT	AT3G51520
Bn-A03-p8851142	BnaA03g17130D	A03	8,038,181	TTG2	AT2G37260
Bn-A05-p2610006	BnaA05g36830D	A05	2,713,236	AGL61	AT5G60440
Bn-A07-p11611255	BnaA07g16350D	A07	13,870,810	WRI1	AT3G54320
Bn-A09-p7329993	BnaA09g12210D	A09	6,402,925	GW5	AT1G64500
Bn-scaff_20942_1-p52095	BnaA08g15580D	C02	12,923,783	CLV3	AT2G27250
Bn-scaff_18903_1-p371596	BnaC04g50960D	C04	48,343,005	EOD3	AT2G46660
Bn-scaff_18062_1-p229981	BnaC04g33070D	C04	31,436,211	RAV1	AT1G13260
Bn-scaff_17799_1-p853567	BnaC06g38800D	C06	36,244,755	GRF2	AT1G78300
Bn-scaff_17799_1-p853567	BnaC06g38900D	C06	36,295,477	DA2	AT1G78420
Bn-A08-p10452462	BnaC08g12050D	C08	17,299,420	SHB1	AT4G25350

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3 讨论

本研究对496份油菜资源千粒重进行考察, 分析其在3个环境下的千粒重表型, 其中材料2012-8998、甲904、WH-59和CY19PXW-65等千粒重大且稳定, 其平均千粒重分别为5.63、5.59、5.09和5.06 g, 上述材料值得作为改良千粒重的优良亲本。比较MLM和GLM模型的关联分析, MLM将群体结构Q矩阵作为固定效应, 亲缘关系K矩阵作为随机效应, 很好地控制了假阳性, 位点可靠性更高。MLM模型检测到6个显著位点, 联合解释了28.92%的表型变异, 其中4个位点得到前人验证。但MLM检测功效偏低, 容易造成假阴性。因此, 引入了检测功效高, 同时假阳性更高的模型GLM; 但对GLM模型设置更为严格的显著性阈值为0.05/总标记数, 以减小GLM中可能存在的假阳性位点。GLM检测到61个显著位点, 联合解释了47.08%的表型变异, 其中21个位点得到前人验证。合并两模型位点后共得到62个显著位点, 联合解释47.31%的表型变异。21个位点与前人定位的QTL重叠, 包括Bn-scaff_ 17526_1-p1066214和Bn-scaff_16064_1-p938130等大效应位点, 其中Bn-A01-p9621623、Bn-Scaffold 002856-p361和Bn-scaff_16874_1-p411591等位点得到多个群体的验证。在后续研究中值得对上述可靠性高的位点可进行精细定位和基因克隆, 为油菜千粒重的改良提供基因资源。

本研究共收集了26篇已发表的油菜千粒重QTL信息, 包含了3~159个QTL, 分布在19条染色体上, 解释的表型变异范围为2.40%~41.46%。与前人QTL位点比较, 本研究中41个显著位点尚未得到验证(表2和表3)。多个位点效应值较高且在多环境中被检测到, 如位点Bn-A03-p560769、Bn-scaff_ 15743_1-p599416和Bn-scaff_15743_1-p590955的表型贡献值分别为9.08%、8.80%和8.58%, 以上位点在3个单环境中均被检测到。此外, 在多个新位点附近找到候选基因, 如在Bn-A01-p15496639、Bn- A05-p2610006和Bn-A08-p10452462等位点附近分别找到拟南芥粒重已知基因NPC6、AGL61和SHB1等的同源基因。这些新位点可靠性高, 值得进一步研究与验证。鉴于千粒重是受多基因调控的复杂数量性状, 在育种上可针对上述位点设计分子标记, 通过分子标记辅助选择聚合有利等位基因, 选育出高产油菜新品种。

此外, 本研究在11个显著位点附近找到千粒重相关的候选基因及其拟南芥同源基因(表4), 根据基因功能注释, 多数候选基因归类于转录因子, 如TTG2、AGL61、WRI1、RAV1和GRF2等。这些基因还影响除粒重外的其他性状, 如EOD3功能缺失后对粒重和角果长均有抑制作用^[12]; DGAT编码二酰甘油酰基转移酶, 对籽粒含油量和籽粒均有重要调节作用^[39]; CLV3双突变产生多室角果, 每角粒数和千粒重均增加^[45], 这些基因调控范围广, 利用价值大, 后期可重点关注。目前很多研究通过CRISPR/Cas9基因编辑技术改良油菜的性状, 如改良抗裂角^[49]、株高和分枝数^[50]、高油酸^[51]等。本研究在Bn-scaff_18062_1-p229981位点附近找到候选基因BnaC04g33070D, 其拟南芥同源基因RAV1为负调控因子, 可与MINI3和IKU2的启动子直接结合, 导致后者表达受抑制, 进而影响粒重, 后续研究可通过CRISPR/Cas9敲除提高油菜的千粒重。

4 结论

本研究利用496份油菜种质资源对千粒重进行全基因组关联分析, 群体在3个环境中千粒重的广义遗传力为63.12%。MLM模型检测到6个显著位点, GLM模型检测到61个显著位点, 合并共同位点后得到62个显著位点, 联合解释47.31%的表型变异。此外, 21个位点与前人研究定位的QTL重叠, 其中8个位点得到至少2个群体验证, 其余41个为新检测到的位点。在11个位点附近发现拟南芥粒重基因DGAT、EOD3、AGL61、WRI1、DA2和RAV1等的同源拷贝。

附表请见网络版: 1) 本刊网站http://zwxb.chinacrops.org/; 2) 中国知网http://www.cnki.net/; 3) 万方数据http://c.wanfangdata.com.cn/Periodical-zuowxb.aspx。

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Plant oils are valuable commodities for food, feed, renewable industrial feedstocks and biofuels. To increase vegetable oil production, here we show that the nonspecific phospholipase C6 (NPC6) promotes seed oil production in the Brassicaceae seed oil species Arabidopsis, Camelina and oilseed rape. Overexpression of NPC6 increased seed oil content, seed weight and oil yield both in Arabidopsis and Camelina, whereas knockout of NPC6 decreased seed oil content and seed size. NPC6 is associated with the chloroplasts and microsomal membranes, and hydrolyzes phosphatidylcholine and galactolipids to produce diacylglycerol. Knockout and overexpression of NPC6 decreased and increased, respectively, the flux of fatty acids from phospholipids and galactolipids into triacylglycerol production. Candidate-gene association study in oilseed rape indicates that only BnNPC6.C01 of the four homeologues NPC6s is associated with seed oil content and yield. Haplotypic analysis indicates that the BnNPC6.C01 favorable haplotype can increase both seed oil content and seed yield. These results indicate that NPC6 promotes membrane glycerolipid turnover to accumulate TAG production in oil seeds and that NPC6 has a great application potential for oil yield improvement.

[45] Yang Y, Zhu K Y, Li H L, Han S Q, Meng Q W, Shahid U K, Fan C C, Xie K B, Zhou Y M. Precise editing of CLAVATA genes in Brassica napus L. regulates multilocular silique development
Plant Biotechnol J, 2018,16:1322-1335.
DOI:10.1111/pbi.12872URLPMID:29250878 [本文引用: 2]
Multilocular silique is a desirable agricultural trait with great potential for the development of high-yield varieties of Brassica. To date, no spontaneous or induced multilocular mutants have been reported in Brassica napus, which likely reflects its allotetraploid nature and the extremely low probability of the simultaneous random mutagenesis of multiple gene copies with functional redundancy. Here, we present evidence for the efficient knockout of rapeseed homologues of CLAVATA3 (CLV3) for a secreted peptide and its related receptors CLV1 and CLV2 in the CLV signalling pathway using the CRISPR/Cas9 system and achieved stable transmission of the mutations across three generations. Each BnCLV gene has two copies located in two subgenomes. The multilocular phenotype can be recovered only in knockout mutations of both copies of each BnCLV gene, illustrating that the simultaneous alteration of multiple gene copies by CRISPR/Cas9 mutagenesis has great potential in generating agronomically important mutations in rapeseed. The mutagenesis efficiency varied widely from 0% to 48.65% in T0 with different single-guide RNAs (sgRNAs), indicating that the appropriate selection of the sgRNA is important for effectively generating indels in rapeseed. The double mutation of BnCLV3 produced more leaves and multilocular siliques with a significantly higher number of seeds per silique and a higher seed weight than the wild-type and single mutant plants, potentially contributing to increased seed production. We also assessed the efficiency of the horizontal transfer of Cas9/gRNA cassettes by pollination. Our findings reveal the potential for plant breeding strategies to improve yield traits in currently cultivated rapeseed varieties.

[46] Liu J, Hua W, Yang H, Li Z, Han Z. TheBnGRF2 gene(GRF2-like gene from Brassica napus) enhances seed oil production through regulating cell number and plant photosynthesis
J Exp Bot, 2012,63:3727-3740.
DOI:10.1093/jxb/ers066URL [本文引用: 1]
Seed yield and oil content are two important agricultural characteristics in oil crop breeding, and a lot of functional gene research is being concentrated on increasing these factors. In this study, by differential gene expression analyses between rapeseed lines (zy036 and 51070) which exhibit different levels of seed oil production, BnGRF2 (Brassica napus growth-regulating factor 2-like gene) was identified in the high oil-producing line zy036. To elucidate the possible roles of BnGRF2 in seed oil production, the cDNA sequences of the rapeseed GRF2 gene were isolated. The Blastn result showed that rapeseed contained BnGRF2a/2b which were located in the A genome (A1 and A3) and C genome (Cl and C6), respectively, and the dominantly expressed gene BnGRF2a was chosen for transgenic research. Analysis of 35S-BnGRF2a transgenic Arabidopsis showed that overexpressed BnGRF2a resulted in an increase in seed oil production of >50%. Moreover, BnGRF2a also induced a >20% enlargement in extended leaves and >40% improvement in photosynthetic efficiency because of an increase in the chlorophyll content. Furthermore, transcriptome analyses indicated that some genes associated with cell proliferation, photosynthesis, and oil synthesis were up-regulated, which revealed that cell number and plant photosynthesis contributed to the increased seed weight and oil content. Because of less efficient self-fertilization induced by the longer pistil in the 35S-BnGRF2a transgenic line, Napin-BnGRF2a transgenic lines were further used to identify the function of BnGRF2, and the results showed that seed oil production also could increase >40% compared with the wild-type control. The results suggest that improvement to economically important characteristics in oil crops may be achieved by manipulation of the GRF2 expression level.

[47] Weng J F, Gu S H, Wan X Y, Gao H, Guo T, Su N, Lei C L, Zhang X, Cheng Z J, Guo X P, Wang J L, Jiang L, Zhai H Q, Wan J M. Isolation and initial characterization of GW5, a major QTL associated with rice grain width and weight
Cell Res, 2008,18:1199-1209.
DOI:10.1038/cr.2008.307URLPMID:19015668 [本文引用: 1]
Grain weight is a major determinant of crop grain yield and is controlled by naturally occurring quantitative trait loci (QTLs). We earlier identified a major QTL that controls rice grain width and weight, GW5, which was mapped to a recombination hotspot on rice chromosome 5. To gain a better understanding of how GW5 controls rice grain width, we conducted fine mapping of this locus and uncovered a 1 212-bp deletion associated with the increased grain width in the rice cultivar Asominori, in comparison with the slender grain rice IR24. In addition, genotyping analyses of 46 rice cultivars revealed that this deletion is highly correlated with the grain-width phenotype, suggesting that the GW5 deletion might have been selected during rice domestication. GW5 encodes a novel nuclear protein of 144 amino acids that is localized to the nucleus. Furthermore, we show that GW5 physically interacts with polyubiquitin in a yeast two-hybrid assay. Together, our results suggest that GW5 represents a major QTL underlying rice width and weight, and that it likely acts in the ubiquitin-proteasome pathway to regulate cell division during seed development. This study provides novel insights into the molecular mechanisms controlling rice grain development and suggests that GW5 could serve as a potential tool for high-yield breeding of crops.Cell Research (2008) 18:1199-1209. doi: 10.1038/cr.2008.307; published online 18 November 2008.

[48] Zhou Y, Zhang X, Kang X, Zhao X, Zhang X, Ni M. SHORT HYPOCOTYL UNDER BLUE1 associates with MINISEED3 and HAIKU2 promoters in vivo to regulate Arabidopsis seed development
Plant Cell, 2009,21:106-117.
DOI:10.1105/tpc.108.064972URLPMID:19141706 [本文引用: 1]
Seed development in Arabidopsis thaliana undergoes an initial phase of endosperm proliferation followed by a second phase in which the embryo grows at the expense of the endosperm. As mature seed size is largely attained during the initial phase, seed size is coordinately determined by the growth of the maternal ovule, endosperm, and embryo. Here, we identify SHORT HYPOCOTYL UNDER BLUE1 (SHB1) as a positive regulator of Arabidopsis seed development that affects both cell size and cell number. shb1-D, a gain-of-function overexpression allele, increases seed size, and shb1, a loss-of-function allele, reduces seed size. SHB1 is transmitted zygotically. The increase in shb1-D seed size is associated with endosperm cellurization, chalazal endosperm enlargement, and embryo development. SHB1 is required for the proper expression of two other genes that affect endosperm development, MINISEED3 (MINI3) and HAIKU2 (IKU2), a WRKY transcription factor gene and a leucine-rich repeat receptor kinase gene. SHB1 associates with both MINI3 and IKU2 promoters in vivo. SHB1 may act with other proteins that bind to MINI3 and IKU2 promoters to promote a large seed cavity and endosperm growth in the early phase of seed development. In the second phase, SHB1 enhances embryo cell proliferation and expansion through a yet unknown IKU2-independent pathway.

[49] Zhai Y G, Cai S G, Hu L M, Yang Y, Amoo O, Fan C C, Zhou Y M. CRISPR/Cas9-mediated genome editing reveals differences in the contribution of INDEHISCENT homologues to pod shatter resistance in Brassica napus L
Thero Appl Genet, 2019,132:2111-2123.
[本文引用: 1]

[50] Zheng M, Zhang L, Tang M, Liu J L, Liu H F, Yang H L, Fan S H, Terzaghi W, Wang H Z, Hua W. Knockout of two BnaMAX1 homologs by CRISPR/Cas9-targeted mutagenesis improves plant architecture and increases yield in rapeseed (Brassica napus L.)
. Plant Biotechnol J, 2020,18:644-654.
DOI:10.1111/pbi.13228URLPMID:31373135 [本文引用: 1]
Plant height and branch number are essential components of rapeseed plant architecture and are directly correlated with its yield. Presently, improvement of plant architecture is a major challenge in rapeseed breeding. In this study, we first verified that the two rapeseed BnaMAX1 genes had redundant functions resembling those of Arabidopsis MAX1, which regulates plant height and axillary bud outgrowth. Therefore, we designed two sgRNAs to edit these BnaMAX1 homologs using the CRISPR/Cas9 system. The T0 plants were edited very efficiently (56.30%-67.38%) at the BnaMAX1 target sites resulting in homozygous, heterozygous, bi-allelic and chimeric mutations. Transmission tests revealed that the mutations were passed on to the T1 and T2 progeny. We also obtained transgene-free lines created by the CRISPR/Cas9 editing, and no mutations were detected in potential off-target sites. Notably, simultaneous knockout of all four BnaMAX1 alleles resulted in semi-dwarf and increased branching phenotypes with more siliques, contributing to increased yield per plant relative to wild type. Therefore, these semi-dwarf and increased branching characteristics have the potential to help construct a rapeseed ideotype. Significantly, the editing resources obtained in our study provide desirable germplasm for further breeding of high yield in rapeseed.

[51] 高谢旺, 谭安琪, 胡信畅, 祝孟洋, 阮颖, 刘春林. 利用CRISPR/Cas9技术创制高油酸甘蓝型油菜新种质
植物遗传资源学报, 2020,21:1002-1008.
[本文引用: 1]

Gao X W, Tan A Q, Hu X C, Zhu M Y, Ruan Y, Liu C L. Creation of new germplasm of high-oleic rapeseed using CRISPR/Cas9
J Plant Genetic Res, 2020,21:1002-1008 (in Chinese with English abstract).
[本文引用: 1]