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甘蓝型油菜千粒重全基因组关联分析

本站小编 Free考研考试/2021-12-26

<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-Zhen1,2, PANG Cheng-Ke1,2, PENG Men-Lu1,2, 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、WRI1DA2RAV1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DA1DA2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, China2农林42 Nonglin 42日本 Japan
3科里纳 Kelina国外Unknown4淮油6号 Huaiyou 6中国江苏 Jiangsu, China
5炎81-2 Yan 81-2中国重庆Chongqing, China628887中国重庆Chongqing, China
7黔油4号 Qianyou 4中国贵州Guizhou, China8黔油331 Qianyou 331中国贵州 Guizhou, China
9恩油73 Enyou 73-1-2中国湖北 Hubei, China10牛耳朵 Niuerduo中国贵州 Guizhou, China
11880101中国重庆Chongqing, China12SWU40中国重庆 Chongqing, China
13SWU42中国重庆Chongqing, China14SWU43中国重庆 Chongqing, China
15SWU44中国重庆Chongqing, China16SWU45中国重庆 Chongqing, China
17SWU46中国重庆Chongqing, China18SWU47中国重庆 Chongqing, China
19SWU48中国重庆Chongqing, China20SWU52中国重庆 Chongqing, China
21SWU53中国重庆Chongqing, China22SWU56中国重庆 Chongqing, China
23SWU59中国重庆Chongqing, China24SWU65中国重庆 Chongqing, China
25SWU82中国重庆Chongqing, China26SWU83中国重庆 Chongqing, China
27SWU92中国重庆Chongqing, China28SWU101中国重庆 Chongqing, China
29SWU106中国重庆Chongqing, China30SWU108中国重庆 Chongqing, China
31川油20 Chuanyou 20中国四川 Sichuan, China32川油18 Chuanyou 18中国四川Sichuan, China
33CY12NY-7中国四川 Sichuan, China34CY12Q95406中国四川Sichuan, China
35CY12Q8-7中国四川 Sichuan, China36CY12QSZ06中国四川Sichuan, China
37CY12QCWH-1中国四川 Sichuan, China38CY12Q95108中国四川Sichuan, China
39CY12Q21535-N3中国四川 Sichuan, China40CY12PXW-4中国四川Sichuan, China
41CY12PXW-6中国四川 Sichuan, China42CY12PXW-9中国四川Sichuan, China
43CY13PXW-17中国四川 Sichuan, China44CY14PXW-18中国四川Sichuan, China
45CY15PXW-31中国四川 Sichuan, China46CY16PXW-35中国四川 Sichuan, China
47CY17PXW-58中国四川 Sichuan, China48CY18PXW-62中国四川 Sichuan, China
49CY19PXW-65中国四川 Sichuan, China50CY20PXW-66中国四川 Sichuan, China
51CY21PXW-84中国四川 Sichuan, China52CY12GJ-1中国四川 Sichuan, China
53wx1025中国湖南 Hunan, China54wx10213中国湖南 Hunan, China
55wx10296中国湖南 Hunan, China56wx10315中国湖南 Hunan, China
5710-1043中国湖南 Hunan, China5810-1047中国湖南 Hunan, China
5910-1061中国湖南 Hunan, China6010-1070中国湖南 Hunan, China
6110-804中国湖南 Hunan, China6210-1358中国湖南 Hunan, China
631472中国湖南 Hunan, China64湘油13号 Xiangyou 13中国湖南 Hunan, China
65湘油15号 Xiangyou 15中国湖南 Hunan, China66湘油11号 Xiangyou 11中国湖南 Hunan, China
67740中国湖南 Hunan, China68631中国湖南 Hunan, China
69613中国湖南 Hunan, China70783中国湖南 Hunan, China
71782中国湖南 Hunan, China72YB3中国湖南 Hunan, China
731360中国湖南 Hunan, China74563中国湖南 Hunan, China
75WX10329中国湖南 Hunan, China76santana德国 German
771281中国湖南 Hunan, China78509中国湖南 Hunan, China
791368中国湖南 Hunan, China801322中国湖南 Hunan, China
811252中国湖南 Hunan, China821321中国湖南 Hunan, China
8307022中国湖北 Hubei, China8407094中国湖北 Hubei, China
8507016中国湖北 Hubei, China869F087中国湖北 Hubei, China
8797096中国湖北 Hubei, China8897097中国湖北 Hubei, China
8907189中国湖北 Hubei, China9007191中国湖北 Hubei, China
9107037中国湖北 Hubei, China92RQ011中国湖北 Hubei, China
93RR009中国湖北 Hubei, China94RR002中国湖北 Hubei, China
9597177中国湖北 Hubei, China9696021中国湖北 Hubei, China
9796063中国湖北 Hubei, China9801111中国湖北 Hubei, China
9901570中国湖北 Hubei, China1009保22 9Bao 22中国湖北 Hubei, China
10101188中国湖北 Hubei, China10202354中国湖北 Hubei, China
10302359中国湖北 Hubei, China10402365中国湖北 Hubei, China
10593205中国湖北 Hubei, China10693210中国湖北 Hubei, China
107Nca国外 Unknown108中双4号 Zhongshuang 4中国湖北 Hubei, China
109中双9号 Zhongshuang 9中国湖北 Hubei, China110中双11号 Zhongshuang 11中国湖北 Hubei, China
1112011-6200中国湖北 Hubei, China1122011-6308中国湖北 Hubei, China
1132011-7103中国湖北 Hubei, China1142012-11526中国湖北 Hubei, China
1152012-3448中国湖北 Hubei, China1162012-3546中国湖北 Hubei, China
1172012-4531中国湖北 Hubei, China1182012-5086中国湖北 Hubei, China
1192012-5113中国湖北 Hubei, China1202012-8327中国湖北 Hubei, China
1212012-8355中国湖北 Hubei, China1222012-8380中国湖北 Hubei, China
1232012-8998中国湖北 Hubei, China1242012-9323中国湖北 Hubei, China
1252012-9354中国湖北 Hubei, China1262012-9380中国湖北 Hubei, China
1272012-9478中国湖北 Hubei, China1282012-9542中国湖北 Hubei, China
1292012-K8053中国湖北 Hubei, China130R2中国湖北 Hubei, China
131希望106 Xiwang 106中国湖北 Hubei, China132阳光198 Yangguang 198中国湖北 Hubei, China
133阳光2009 Yangguang 2009中国湖北 Hubei, China134中双10号 Zhongshuang 10中国湖北 Hubei, China
135中双12号 Zhongshuang 12中国湖北 Hubei, China136中双6号 Zhongshuang 6中国湖北 Hubei, China
137中双7号 Zhongshuang 7中国湖北 Hubei, China138中油589 Zhongyou 589中国湖北 Hubei, China
139华油2号 Huayou 2中国湖北 Hubei, China140Major法国 France
141华双2号 Huashuang 2中国湖北 Hubei, China142Aurora德国 German
143华油13号 Huayou13中国湖北 Hubei, China144Rucabo德国 German
145华油3号 Huayou 3中国湖北 Hubei, China146华油14 Huayou 14中国湖北 Hubei, China
147宁油1号 Ningyou 1中国江苏 Jiangsu, China148Ceres德国 German
14911-9-700中国湖北 Hubei, China15011-9-701中国湖北 Hubei, China
15111-9-702中国湖北 Hubei, China15211-9-703中国湖北 Hubei, China
15311-9-704中国湖北 Hubei, China15411-9-705中国湖北 Hubei, China
15511-9-706中国湖北 Hubei, China15611-9-707中国湖北 Hubei, China
15711-P63-5育7
11-P63-5 yu 7
中国湖北 Hubei, China15811-P63-8育32
11-P63-8 yu 32
中国湖北 Hubei, China
15911-P63-3育3
11-P63-3 yu 3
中国湖北 Hubei, China16011-P67东 11-P67 Dong中国湖北 Hubei, China
16109-P64-1中国湖北 Hubei, China16210-崇23 10-chong23中国湖北 Hubei, China
16310-崇24 10-chong 24中国湖北 Hubei, China16410-崇25 10-chong25中国湖北 Hubei, China
16510-崇29 10-chong 29中国湖北 Hubei, China16610-崇32 10-chong 32中国湖北 Hubei, China
16710-崇33 10-chong 33中国湖北 Hubei, China16810-崇34 10-chong 34中国湖北 Hubei, China
16910-江棚2 10-jiangpeng 2中国湖北 Hubei, China17010-江棚3 10-jiangpeng 3中国湖北 Hubei, China
17111-育7-103 11-yu 7-103中国湖北 Hubei, China17211-育7-117 11-yu7-117中国湖北 Hubei, China
17311-育7-125 11-yu 7-125中国湖北 Hubei, China17411-P74-8中国湖北 Hubei, China
17511-P74-13中国湖北 Hubei, China1767-7766-74中国湖北 Hubei, China
177P18中国湖北 Hubei, China17864棚-1064 peng-10中国湖北 Hubei, China
179圣光77 Shengguang 77中国湖北 Hubei, China180甲预17棚 Jiayu17peng中国湖北 Hubei, China
181甲预25棚 Jiayu25peng中国湖北 Hubei, China182甲预16棚 Jiayu16peng中国湖北 Hubei, China
183甲预31棚 Jiayu31peng中国湖北 Hubei, China184华双5号 Huashuang5中国湖北 Hubei, China
185华双4号 Huashuang 4中国湖北 Hubei, China186甲972Jia972中国湖北 Hubei, China
187华双128 Huashuang 128中国湖北 Hubei, China188甲904 Jia904中国湖北 Hubei, China
189甲908 Jia 908中国湖北 Hubei, China190甲PF190棚 Jia PF190peng中国湖北 Hubei, China
191甲915 Jia 915中国湖北 Hubei, China192甲922 Jia 922中国湖北 Hubei, China
193甲951棚 Jia951peng中国湖北 Hubei, China194甲917 Jia 917中国湖北 Hubei, China
195甲923 Jia923中国湖北 Hubei, China196甲931 Jia 931中国湖北 Hubei, China
197甲预05棚 Jiayu05peng中国湖北 Hubei, China198甲963棚 Jia963peng中国湖北 Hubei, China
199沪油12号 Huyou 12中国上海 Shanghai, China200宁油16号 Ningyou 16中国江苏 Jiangsu, China
201史力佳 Shilijia中国江苏 Jiangsu, China202杨油6号 Yangyou 6中国江苏 Jiangsu, China
203扬油5号 Yangyou 5中国江苏 Jiangsu, China204镇油3号 Zhenyou 3中国江苏 Jiangsu, China
205苏油1号 Suyou 1中国江苏 Jiangsu, China206浙油18 Zheyou18中国浙江 Zhejiang, China
207浙双72 Zheshuang 72中国浙江 Zhejiang, China208浙双8号 Zheshuang 8中国浙江 Zhejiang, China
209浙油758 Zheyou 758中国浙江 Zhejiang, China210沪油14 Huyou 14中国上海 Shanghai, China
211沪油18 Huyou 18-2中国上海 Shanghai, China212沪油19 Huyou 19中国上海 Shanghai, China
213浙油19 Zheyou 19中国浙江 Zhejiang, China214浙油21 Zheyou 21中国浙江 Zhejiang, China
215浙双6号 Zheshuang 6中国浙江 Zhejiang, China216皖油15号 Wanyou 15中国安徽 Anhui, China
217皖油16号 Wanyou 16中国安徽 Anhui, China218皖油29 Wanyou 29中国安徽 Anhui, China
219AGREV012德国 German220AGREV019德国 German
221AGREV021德国 German222Topas瑞典 Sweden
223cyclon丹麦 Denmark224伟杰 Weijie加拿大 Canada
225四达 Sida加拿大 Canada226至尊 Zhizun加拿大 Canada
227海神 Haishen加拿大 Canada228D2丹麦 Denmark
229D3丹麦 Denmark230Qu美国 America
23111-985中国青海 Qinghai, China23211-504中国青海 Qinghai, China
23311-997中国青海 Qinghai, China23411-540中国青海 Qinghai, China
23511-1184中国青海 Qinghai, China23610-758中国青海 Qinghai, China
23710-1230中国青海 Qinghai, China23810-847中国青海 Qinghai, China
23911-1124中国青海 Qinghai, China240垦C1 KenC1中国陕西 Shaanxi, China
241P113中国陕西 Shaanxi, China242P158中国陕西 Shaanxi, China
243P310中国陕西 Shaanxi, China244P312中国陕西 Shaanxi, China
245P668中国陕西 Shaanxi, China246P685中国陕西 Shaanxi, China
247A117中国陕西 Shaanxi, China248A172中国陕西 Shaanxi, China
249B250中国陕西 Shaanxi, China250B265中国陕西 Shaanxi, China
251A109中国陕西 Shaanxi, China252B285中国陕西 Shaanxi, China
253C052中国陕西 Shaanxi, China254GY270中国陕西 Shaanxi, China
255GY282中国陕西 Shaanxi, China256GY284中国陕西 Shaanxi, China
257B262中国河南 Henan, China258A82中国江西 Jiangxi, China
259A98中国陕西 Shaanxi, China260B414中国新疆 Xinjiang, China
261B308瑞典 Sweden262A97中国四川 Sichuan, China
263A148瑞典 Sweden264B431丹麦 Denmark
26508-P35中国湖北 Hubei, China26608-P36中国湖北 Hubei, China
26709-P32中国湖北 Hubei, China26809-P36中国湖北 Hubei, China
26909-P37中国湖北 Hubei, China27010-P10中国湖北 Hubei, China
27110-P29中国湖北 Hubei, China27211-P30中国湖北 Hubei, China
27312-P24中国湖北 Hubei, China27412-P25中国湖北 Hubei, China
27503ⅡB中国湖北 Hubei, China27603Ⅱ3B中国湖北 Hubei, China
27703Ⅰ32B中国湖北 Hubei, China278964中国湖北 Hubei, China
279DD1中国湖北 Hubei, China2809L302-1中国湖北 Hubei, China
28106T9F中国湖北 Hubei, China28203Ⅱ4B中国湖北 Hubei, China
28303LF1中国湖北 Hubei, China2849852中国湖北 Hubei, China
2859801C中国湖北 Hubei, China286986中国湖北 Hubei, China
287876中国湖北 Hubei, China288武164 Wu164中国湖北 Hubei, China
2899889中国湖北 Hubei, China29006H7中国湖北 Hubei, China
291湖北白花油菜
Hubeibaihuayoucai
中国湖北 Hubei, China292南川长角
Nanchuanchangjiao
中国四川 Sichuan, China
293IMC103中国重庆 Chongqing, China294Oscar澳大利亚 Australia
295Sophia德国 German296campina德国 German
297Conny瑞典 Sweden298Wesreo澳大利亚 Australia
299Wase Chousen朝鲜 North Korea300Gogatsuna日本 Japan
301Nakaee Chousen朝鲜 North Korea302Cat.No.117德国 German
30390750中国重庆 Chongqing, China304农林43 Nonglin 43日本
305西藏油菜 Xizangyoucai中国西藏 Tibet, China306油研2号 Youyan 2中国贵州
307矮架早 Aijiazao中国四川 Sichuan, China308SWU41中国重庆 Chongqing, China
309SWU49中国重庆 Chongqing, China310SWU54中国重庆 Chongqing, China
311SWU57中国重庆 Chongqing, China312SWU60中国重庆 Chongqing, China
313SWU61中国重庆 Chongqing, China314SWU62中国重庆 Chongqing, China
315SWU63中国重庆 Chongqing, China316SWU64中国重庆 Chongqing, China
317SWU66中国重庆 Chongqing, China318SWU67中国重庆 Chongqing, China
319SWU68中国重庆 Chongqing, China320SWU69中国重庆 Chongqing, China
321SWU70中国重庆Chongqing, China322SWU71中国重庆 Chongqing, China
323SWU74中国重庆 Chongqing, China324SWU75中国重庆 Chongqing, China
325SWU76中国重庆 Chongqing, China326SWU77中国重庆 Chongqing, China
327SWU80中国重庆 Chongqing, China328SWU81中国重庆 Chongqing, China
329SWU84中国重庆 Chongqing, China330SWU85中国重庆 Chongqing, China
331SWU87中国重庆 Chongqing, China332SWU88中国重庆 Chongqing, China
333SWU89中国重庆 Chongqing, China334SWU90中国重庆 Chongqing, China
335SWU93中国重庆 Chongqing, China336SWU94中国重庆 Chongqing, China
337SWU95中国重庆 Chongqing, China338SWU96中国重庆 Chongqing, China
339SWU99中国重庆 Chongqing, China340SWU100中国重庆 Chongqing, China
341SWU102中国重庆 Chongqing, China342SWU103中国重庆 Chongqing, China
343SWU104中国重庆 Chongqing, China344SWU105中国重庆 Chongqing, China
345SWU107中国重庆 Chongqing, China346SWU110中国重庆 Chongqing, China
347SWU111中国重庆 Chongqing, China348SWU112中国重庆 Chongqing, China
349SWU113中国重庆 Chongqing, China350SWU114中国重庆 Chongqing, China
351辐油4号 Fuyou 4中国重庆 Chongqing, China352镇3736 Zhen3736中国江苏 Jiangsu, China
353镇2609 Zhen 2609中国江苏 Jiangsu, China354HX0352中国江苏 Jiangsu, China
355阳光2009 Yangguang 2009中国江苏 Jiangsu, China356沪油21 Huyou 21中国上海 Shanghai, China
357浙双3号 Zheshuang 3中国浙江 Zhejiang, China358浙油21 Zheyou 21中国浙江 Zhejiang, China
359皖油20 Wanyou 20中国安徽 Anhui, China360皖油12 Wanyou 12中国安徽 Anhui, China
361皖油7号 Wanyou 7中国安徽 Anhui, China362红油3号 Hongyou 3中国江苏 Jiangsu, China
363镇油5号 Zhenyou5中国江苏 Jiangsu, China364扬油4号 Yangyou 4中国上海 Shanghai, China
365沪油15 Huyou 15中国上海 Shanghai, China366沪油16 Huyou 16中国上海 Shanghai, China
367沪油17 Huyou17中国上海 Shanghai, China368浙双72 Zheshuang 72中国浙江 Zhejiang, China
369浙双8号 Zheshuang 8中国浙江 Zhejiang, China370浙油50 Zheyou 50中国浙江 Zhejiang, China
371中双11 Zhongshuang 11中国湖北 Hubei, China372阳光198 Yangguang198中国湖北 Hubei, China
373华航901 Huahang 901中国湖北 Hubei, China374扬J6711 Yang J6711中国江苏 Jiangsu, China
375盐6055 Yan 6055中国江苏 Jiangsu, China376杨鉴8号 Yangjian8中国江苏 Jiangsu, China
377希望106 Xiwang 106中国湖北 Hubei, China378浙油17号 Zheyou 17中国浙江 Zhejiang, China
379中双5号 Zhongshuang 5中国湖北 Hubei, China380中油821 Zhongyou 821中国湖北 Hubei, China
381秦油1号 Qinyou 1中国陕西 Shaanxi, China382纬隆88 Weilong 88中国陕西 Shaanxi, China
383盐油2号 Yanyou 2中国江苏 Jiangsu, China384秦油5号 Qinyou 5中国陕西 Shaanxi, China
387德68-12 De 68-12中国四川 Sichuan, China388Monty澳大利亚 Australia
389Oscar澳大利亚 Australia390宁油12 Ningyou 12-1中国江苏 Jiangsu, China
391宁油14 Ningyou 14中国江苏 Jiangsu, China392史力丰-1 Shilifeng-1中国江苏 Jiangsu, China
393宁油18 Ningyou 18中国江苏 Jiangsu, China394Helios丹麦 Denmark
395Hector加拿大 Canada396棉96-203 Mian96-203中国青海 Qinghai, China
397青662A Qing662A中国青海 Qinghai, China398699中国湖北 Hubei, China
399加拿大2号 Jianada 2加拿大 Canada400中双2号 Zhongshuang 2中国湖北 Hubei, China
401WH-12中国湖北 Hubei, China402WH-15中国湖北 Hubei, China
403WH-17中国湖北 Hubei, China404WH-19中国湖北 Hubei, China
405WH-20中国湖北 Hubei, China406WH-23中国湖北 Hubei, China
407WH-24中国湖北 Hubei, China408WH-25中国湖北 Hubei, China
409WH-26中国湖北 Hubei, China410WH-27中国湖北 Hubei, China
411WH-28中国湖北 Hubei, China412WH-29中国湖北 Hubei, China
413WH-30中国湖北 Hubei, China414WH-31中国湖北 Hubei, China
415WH-33中国湖北 Hubei, China416WH-37中国湖北 Hubei, China
417WH-38中国湖北 Hubei, China418WH-41中国湖北 Hubei, China
419WH-42中国湖北 Hubei, China420WH-43中国湖北 Hubei, China
421WH-45中国湖北 Hubei, China422WH-49中国湖北 Hubei, China
423WH-50中国湖北 Hubei, China424WH-55中国湖北 Hubei, China
425WH-56中国湖北 Hubei, China426WH-57中国湖北 Hubei, China
427WH-58中国湖北 Hubei, China428WH-59中国湖北 Hubei, China
429WH-60中国湖北 Hubei, China430WH-61中国湖北 Hubei, China
431WH-62中国湖北 Hubei, China432WH-63中国湖北 Hubei, China
433WH-81中国湖北 Hubei, China434WH-83中国湖北 Hubei, China
435WH-85中国湖北 Hubei, China436WH-88中国湖北 Hubei, China
437WH-93中国湖北 Hubei, China438WH-95中国湖北 Hubei, China
439WH-100中国湖北 Hubei, China440WH-127中国湖北 Hubei, China
441豫油1号 Yuyou 1中国河南 Henan, China442COBRA德国 German
443宁油7号 Ningyou 7中国江苏 Jiangsu, China444Tapidor法国 France
445华油6号 Huayou 6中国湖北 Hubei, China446华油12 Huayou 12中国湖北 Hubei, China
447Cubs root朝鲜 North Korea448华油10 Huayou 10中国湖北 Hubei, China
449Bienvenu法国 France450胜利油菜 Shengliyoucai中国四川 Sichuan, China
451ERAKE波兰 Poland452Taisetsu日本 Japan
453cresor加拿大 Canada454Daichousen朝鲜 North Korea
455comet瑞典 Sweden456Niklas丹麦 Denmark
457Askari德国 German458chuosenshu朝鲜 North Korea
459WESBROOK澳大利亚 Australia460Suigenshu朝鲜 North Korea
461华油4号 Huayou 4中国湖北 Hubei, China462胜利青梗 Shengliqinggeng中国上海 Shanghai, China
463密角多头油菜
Mijiaoduotouyoucai
中国上海 Shanghai, China464矮箕胜利 Aijishengli中国上海 Shanghai, China
465漕泾胜利 Caojingshengli中国上海 Shanghai, China466沪油三号 Huyou 3中国上海 Shanghai, China
467勺叶青 Shaoyeqing中国上海 Shanghai, China468非洲油菜乳黄花
Feizhouyoucairuhuanghua
中国上海 Shanghai, China
469沪激早 Hujizao中国上海 Shanghai, China470漕油2号 Caoyou 2中国上海 Shanghai, China
471封顶240 Fengding 240中国江苏 Jiangsu, China472全紫油菜 Quanziyoucai中国江苏 Jiangsu, China
473大花球 Dahuaqiu中国江苏 Jiangsu, China474荣选 Rongxuan中国江苏 Jiangsu, China
475宁油10号 Ningyou 10中国江苏 Jiangsu, China476宁油8号 Ningyou 8中国江苏 Jiangsu, China
477宁油六号 Ningyouliu中国江苏 Jiangsu, China478垛油一号 Duoyou 1中国江苏 Jiangsu, China
479淮油12号 Huaiyou 12中国江苏 Jiangsu, China480沛选170 Peixuang 170中国江苏 Jiangsu, China
481广德138 Guangde 138中国安徽 Anhui, China482广德8104 Guangde 8104中国安徽 Anhui, China
483当油早1号Dangyouzao 1中国安徽 Anhui, China484广德761 Guangde 761中国安徽 Anhui, China
485铜陵花叶 Tonglinghuaye中国安徽 Anhui, China486滁610 Chu610中国安徽 Anhui, China
487滁107 Chu 107中国安徽 Anhui, China488宿84-6 Su 84-6中国安徽 Anhui, China
489皖油早 Wanyouzao中国安徽 Anhui, China490滁油1号 Chuyou 1中国安徽 Anhui, China
491滁县白花 Chuxianbaihua中国安徽 Anhui, China492芥65-1 Jie 65-1中国浙江 Zhejiang, China
493申黄1号 Shenhuang 1中国上海 Shanghai, China494浙油601 Zheyou 601中国浙江 Zhejiang, China
495三高油菜Three high rape中国浙江 Zhejiang, China496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Nanjing2.277.634.27±0.880.21
2014/2015 泰州Taizhou1.645.773.65±0.580.16
2015/2016 泰州Taizhou1.506.133.51±0.640.18

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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-p23472380A0119,346,1965.346.0316TZ
Bn-A02-p6300284A023,473,3194.815.4115TZ
Bn-A02-p6564861A023,784,9815.386.0614NJ[32]
Bn-A03-p22125583A0320,956,9674.775.36[32,35]
Bn-scaff_16874_1-p411591C0631,817,6214.334.8616TZ[30,36,37]
Bn-scaff_17526_1-p1066214C091,488,2834.935.5516TZ[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-p1156979A01760,6566.375.6115TZ[35]
Bn-A01-p1440009A011,036,0325.965.21
Bn-A01-p9621623A018,234,3436.435.66[17,30,36]
Bn-A01-p12222806A0110,407,9897.027.0314NJ
Bn-A01-p15496639A0112,941,3946.646.6716TZ
Bn-A02-p6300284A023,473,3197.227.2314TZ
Bn-A02-p6564861A023,784,9818.598.5314NJ/16TZ[32,35]
Bn-A03-p560769A03445,7069.169.0814NJ/16TZ
Bn-A03-p4352837A033,885,1806.186.22
Bn-A03-p6586436A035,884,5088.478.4316TZ
Bn-A03-p8851142A038,156,2997.887.8614NJ/15TZ/16TZ[32]
Bn-A03-p13758392A0312,893,5446.566.6015TZ
Bn-A03-p14747263A0313,916,0476.576.6014NJ[35]
Bn-A03-p22125583A0320,956,9675.645.60[32,35]
Bn-A03-p24494224A0322,999,8256.056.10[32]
Bn-A04-p13705636A0414,344,6698.848.7715TZ[35]
Bn-A05-p2610006A052,720,7635.695.75
Bn-scaff_27198_1-p445589A0615,593,1478.017.9914NJ/15TZ
Bn-A06-p23759352A0622,715,8145.976.01
Bn-Scaffold002856-p361A07592,9825.845.8914NJ/16TZ[14,17,31,36]
Bn-A10-p11601681A072,573,0876.956.1516TZ[33]
Bn-A07-p5877587A077,824,4427.246.4214NJ/15TZ[13,32]
Bn-A07-p10557557A0711,738,1665.715.7716TZ
Bn-A02-p305007A0712,930,6795.664.9314NJ/15TZ/16TZ
Bn-A07-p11611255A0713,756,1705.956.00[35]
Bn-A07-p16095589A0717,996,5535.966.01
Bn-scaff_15743_1-p590955A0718,512,7868.648.5814NJ/15TZ/16TZ
Bn-A07-p17804261A0719,646,0536.205.45[32,38]
Bn-A08-p2675098A082,098,8086.816.8316TZ
Bn-A08-p10443959A088,372,6286.306.34[32]
Bn-A08-p13284369A0811,051,8285.895.94
Bn-A08-p20343735A0817,811,8868.827.9015TZ
Bn-A09-p7329993A095,542,3616.206.2414NJ/15TZ[14,32]
Bn-A10-p15021776A1014,967,1285.785.8315TZ
Bn-A10-p15167470A1015,100,3926.676.6915TZ
Bn-scaff_15803_1-p837307C0114,780,3205.585.6414NJ
Bn-scaff_20942_1-p52095C0211,201,0486.546.5814NJ
Bn-scaff_16002_1-p1803014C0312,573,7566.956.9715TZ
Bn-scaff_26320_1-p269450C0330,924,9885.614.89
Bn-scaff_16182_1-p296671C0351,992,9676.416.4414NJ
Bn-scaff_15794_3-p89166C0355,716,4418.287.3914NJ/15TZ
Bn-scaff_17119_1-p148890C0356,814,5879.289.1914NJ/15TZ[35]
Bn-scaff_16217_1-p597569C0421,919,4125.684.9614NJ
Bn-scaff_18062_1-p229981C0431,112,5206.235.4714NJ/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-p1089867C0444,500,2435.885.1416TZ
Bn-scaff_18903_1-p371596C0447,531,6766.876.8916TZ
Bn-scaff_16414_1-p1774629C05279,0597.337.3314NJ
Bn-scaff_20901_1-p2029631C051,973,9736.476.5014NJ/16TZ
Bn-scaff_20901_1-p1279675C052,707,5647.367.3614NJ
Bn-scaff_15763_1-p596396C0511,699,1365.714.9816TZ
Bn-scaff_15763_1-p588874C0620,160,2985.855.9016TZ
Bn-scaff_16064_1-p938130C0624,703,44111.5711.3114NJ/15TZ/16TZ[35]
Bn-scaff_15743_1-p599416C0627,808,9719.808.8014NJ/15TZ/16TZ
Bn-scaff_18807_1-p747016C0630,110,3945.694.96[36]
Bn-scaff_16874_1-p411591C0631,817,6216.666.6914NJ/16TZ[30,36,37]
Bn-scaff_17799_1-p2391172C0634,110,5827.716.8714NJ/16TZ
Bn-scaff_17799_1-p853567C0635,737,8777.707.6914NJ[35]
Bn-scaff_15705_1-p2279820C0735,285,0376.126.1616TZ
Bn-A08-p10452462C0816,678,5226.886.0814NJ/15TZ/16TZ
Bn-scaff_17526_1-p1066214C091,488,28315.9815.2614NJ/15TZ/16TZ[32]
Bn-scaff_15576_1-p614226C0941,707,7209.038.9614NJ/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 TSWA7bqSW.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-tsm07TH-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-p15496639BnaA01g20420DA0112,306,073NPC6AT3G48610
Bn-A03-p22125583BnaA03g41350DA0320,713,211DGATAT3G51520
Bn-A03-p8851142BnaA03g17130DA038,038,181TTG2AT2G37260
Bn-A05-p2610006BnaA05g36830DA052,713,236AGL61AT5G60440
Bn-A07-p11611255BnaA07g16350DA0713,870,810WRI1AT3G54320
Bn-A09-p7329993BnaA09g12210DA096,402,925GW5AT1G64500
Bn-scaff_20942_1-p52095BnaA08g15580DC0212,923,783CLV3AT2G27250
Bn-scaff_18903_1-p371596BnaC04g50960DC0448,343,005EOD3AT2G46660
Bn-scaff_18062_1-p229981BnaC04g33070DC0431,436,211RAV1AT1G13260
Bn-scaff_17799_1-p853567BnaC06g38800DC0636,244,755GRF2AT1G78300
Bn-scaff_17799_1-p853567BnaC06g38900DC0636,295,477DA2AT1G78420
Bn-A08-p10452462BnaC08g12050DC0817,299,420SHB1AT4G25350

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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NPC6AGL61SHB1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TTG2AGL61WRI1RAV1GRF2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MINI3IKU2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、AGL61WRI1DA2RAV1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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DOI:10.1016/S0070-2153(10)91007-7URLPMID:20705183 [本文引用: 1]
Plant organs grow to characteristic, species-specific sizes and shapes. At the cellular level, organ growth is initially characterized by cell proliferation, which gives way to cell expansion at later stages. Using mainly Arabidopsis thaliana as a model species, a number of factors have been isolated in recent years that promote or restrict organ growth, with the altered organ size being associated with changes in cell number, in cell size, or in both. However, cells in an organ do not appear to follow a strictly autonomous program of proliferation and expansion, and their behavior is coordinated in at least three different respects: normally sized organs can be formed consisting of altered numbers of cells with compensatory changes in the size of the individual cells, suggesting that cellular behavior is subject to organ-wide control; the growth of cells derived from more than one clonal origin is coordinated within a plant lateral organ with its different histological layers; and growth of cells in different regions of an organ is coordinated to generate a reasonably flat leaf or floral organ. Organ growth is strongly modulated by environmental factors, and the molecular basis for this regulation is beginning to be understood. Given the complexity of organ growth as a dynamic four-dimensional process, precise quantification of growth parameters and mathematical modeling are increasingly used to understand this fascinating problem of plant biology.

朱军, 许馥华. 胚乳性状的遗传模型及其分析方法
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Li N. Maternal Control of Seed Weight in Rapeseed (Brassica napus L.)
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Li S, Liu Y, Zheng L, Chen L, Li N, Corke F, Lu Y, Fu X, Zhu Z, Bevan M W, Li Y H. The plant-specific G protein γ subunit AGG3 influences organ size and shape in Arabidopsis thaliana
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Basunanda P, Radoev M, Ecke W, Friedt W, Becker H, Snowdon R. Comparative mapping of quantitative trait loci involved in heterosis for seedling and yield traits in oilseed rape ( Brassica napus L.)
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Yang P, Shu C, Chen L, Xu J S, Wu J S, Liu K D. Identification of a major QTL for silique length and seed weight in oilseed rape (Brassica napus L.)
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Zhao W, Wang X, Wang H, Tian J, Li B, Chen L, Chao H, Long Y, Xiang J, Gan J, Liang W, Li M. Genome-wide identification of QTL for seed yield and yield-related traits and construction of a high-density consensus map for QTL comparison in Brassica napus
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Sun L J, Wang X D, Yu K J, Li W J, Peng Q, Chen F, Zhang W, Fu S X, Xiong D Q, Chu P, Guan R Z, Zhang J F. Mapping of QTLs controlling seed weight and seed-shape traits in Brassica napus L. using a high-density SNP map
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Liu J, Hua W, Hu Z, Yang H, Zhang L, Li R, Deng L, Sun X, Wang X, Wang H. Natural variation in ARF18 gene simultaneously affects seed weight and silique length in polyploid rapeseed
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DOI:10.1073/pnas.1423244112URLPMID:25838284 [本文引用: 1]
Among the variety of tissue-resident NK-like populations recently distinguished from recirculating classical NK (cNK) cells, liver innate lymphoid cells (ILC) type 1 (ILC1s) have been shown to represent a distinct lineage that originates from a novel promyelocytic leukaemia zinc finger (PLZF)-expressing ILC precursor (ILCP) strictly committed to the ILC1, ILC2, and ILC3 lineages. Here, using PLZF-reporter mice and cell transfer assays, we studied the developmental progression of ILC1s and demonstrated substantial overlap with stages previously ascribed to the cNK lineage, including pre-pro-NK, pre-NK precursor (pre-NKP), refined NKP (rNKP), and immature NK (iNK). Although they originated from different precursors, the ILC1 and cNK lineages followed a parallel progression at early stages and diverged later at the iNK stage, with a striking predominance of ILC1s over cNKs early in ontogeny. Although a limited set of ILC1 genes depended on PLZF for expression, characteristically including Il7r, most of these genes were also differentially expressed between ILC1s and cNKs, indicating that PLZF together with other, yet to be defined, factors contribute to the divergence between these lineages.

Shi L L, Song J R, Guo C C, Wang B, Guan Z L, Yang P, Chen X, Zhang Q H, Graham J K, Wang J, Liu K D. A CACTA-like transposable element in the upstream region of BnaA9.CYP78A9 acts as an enhancer to increase silique length and seed weight in rapeseed
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Front Plant Sci, 2016,7:1102.

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孙程明, 陈锋, 陈松, 彭琦, 张维, 易斌, 张洁夫, 傅廷栋. 甘蓝型油菜每角粒数的全基因组关联分析
作物学报, 2020,46:147-153.

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Sun C M, Chen F, Chen S, Peng Q, Zhang W, Yi B, Zhang J F, Fu T D. Genome-wide association study of seed number per silique in rapeseed ( Brassica napus L.)
Acta Agron Sin, 2020,46:147-153 (in Chinese with English abstract).

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孙程明, 陈松, 彭琦, 张维, 易斌, 张洁夫, 傅廷栋. 甘蓝型油菜角果长度性状的全基因组关联分析
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Sun C M, Chen S, Peng Q, Zhang W, Yi B, Zhang J F, Fu T D. Genome-wide association study of silique length in rapeseed ( Brassica napus L.)
Acta Agron Sin, 2019,45:1303-1310 (in Chinese with English abstract).

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Dong H L, Tan C D, Li Y Z, He Y, Wei S, Cui Y X, Chen Y G, Wei D Y, Fu Y, He Y G, Wan H F, Liu H, Xiong Q, Lu K, Li J N, Qian W. Genome-wide association study reveals both overlapping and independent genetic loci to control seed weight and silique length in Brassica napus
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Cai D F, Xiao Y J, Yang W, Ye W, Wang B, Muhammad Y, Wu J S, Liu K D. Association mapping of six yield-related traits in rapeseed ( Brassica napus L.)
Thero Appl Genet, 2014,127:85-96.

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Shahid U K, Jiao Y M, Liu S, Zhang K P, Muhammad H U K, Zhai Y G, Amoo O, Fan C C, Zhou Y M. Genome-wide association studies in the genetic dissection of ovule number, seed number, and seed weight in Brassica napus L
Ind Crops Prod, 2019,142:UNSP111877.

[本文引用: 4]

Li F, Chen B, Xu K, Wu J, Wu X. Genome-wide association study dissects the genetic architecture of seed weight and seed quality in rapeseed ( Brassica napus L.)
DNA Res, 2014,21:355-367.

URLPMID:24510440 [本文引用: 1]

Atwell S, Huang Y S, Vilhjálmsson B J, Willems G, Horton M, Li Y, Meng D, Platt A, Tarone A M, Hu T T, Jiang R, Muliyati N W, Zhang X, Amer M A, Baxter I, Brachi B, Chory J, Dean C, Debieu M, Meaux J, Ecker J R, Faure N, Kniskern J M, Jones J D, Michael T, Nemri A, Roux F, Salt D E, Tang C, Todesco M, TrawM B, Weigel D, Marjoram P, Borevitz J O, Bergelson J, Nordborg M. Genome-wide association study of 107 phenotypes in a common set of Arabidopsis thaliana inbred lines
Nature, 2010,465:627-631.

DOI:10.1038/nature08800URLPMID:20336072 [本文引用: 1]
Although pioneered by human geneticists as a potential solution to the challenging problem of finding the genetic basis of common human diseases, genome-wide association (GWA) studies have, owing to advances in genotyping and sequencing technology, become an obvious general approach for studying the genetics of natural variation and traits of agricultural importance. They are particularly useful when inbred lines are available, because once these lines have been genotyped they can be phenotyped multiple times, making it possible (as well as extremely cost effective) to study many different traits in many different environments, while replicating the phenotypic measurements to reduce environmental noise. Here we demonstrate the power of this approach by carrying out a GWA study of 107 phenotypes in Arabidopsis thaliana, a widely distributed, predominantly self-fertilizing model plant known to harbour considerable genetic variation for many adaptively important traits. Our results are dramatically different from those of human GWA studies, in that we identify many common alleles of major effect, but they are also, in many cases, harder to interpret because confounding by complex genetics and population structure make it difficult to distinguish true associations from false. However, a-priori candidates are significantly over-represented among these associations as well, making many of them excellent candidates for follow-up experiments. Our study demonstrates the feasibility of GWA studies in A. thaliana and suggests that the approach will be appropriate for many other organisms.

Luo Z L, Wang M, Long Y, Huang Y J, Shi L, Zhang C Y, Liu X, Bruce D L F, Xiang J X, Mason A S, Snowdon R J, Liu P F, Meng J L, Zou J. Incorporating pleiotropic quantitative trait loci in dissection of complex traits: seed yield in rapeseed as an example
Theor Appl Genet, 2017,130:1569-1585.

URLPMID:28455767 [本文引用: 2]

Shi J Q, Li R Y, Qiu D, Jiang C C, Long Y, Morgan C. Unraveling the complex trait of crop yield with quantitative trait loci mapping in Brassica napus
Genetics, 2009,182:851-861.

URLPMID:19414564 [本文引用: 2]

Li N, Shi J Q, Wang X H, Liu G H, Wang H Z. A combined linkage and regional association mapping validation and fine mapping of two major pleiotropic QTLs for seed weight and silique length in rapeseed (Brassica napus L.)
. BMC Plant Biol, 2014,14:114.

URLPMID:24779415 [本文引用: 1]

Zhao W, Wang X, Wang H, Tian J, Li B, Chen L, Chao H, Xiang J, Gan J. Genome-wide identification of QTL for seed yield and yield-related traits and construction of a high-density consensus map for QTL comparison in Brassica napus
Front Plant Sci, 2016,7:17.



Jako C, Kumar A, Wei Y, Zou J, Barton D L, Giblin E M, Covello P S, Taylor D C. Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight
Plant Physiol, 2001,126:861-874.

DOI:10.1104/pp.126.2.861URLPMID:11402213 [本文引用: 2]
We recently reported the cloning and characterization of an Arabidopsis (ecotype Columbia) diacylglycerol acyltransferase cDNA (Zou et al., 1999) and found that in Arabidopsis mutant line AS11, an ethyl methanesulfonate-induced mutation at a locus on chromosome II designated as Tag1 consists of a 147-bp insertion in the DNA, which results in a repeat of the 81-bp exon 2 in the Tag1 cDNA. This insertion mutation is correlated with an altered seed fatty acid composition, reduced diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) activity, reduced seed triacylglycerol content, and delayed seed development in the AS11 mutant. The effect of the insertion mutation on microsomal acyl-coenzyme A-dependent DGAT is examined with respect to DGAT activity and its substrate specificity in the AS11 mutant relative to wild type. We demonstrate that transformation of mutant AS11 with a single copy of the wild-type Tag1 DGAT cDNA can complement the fatty acid and reduced oil phenotype of mutant AS11. More importantly, we show for the first time that seed-specific over-expression of the DGAT cDNA in wild-type Arabidopsis enhances oil deposition and average seed weight, which are correlated with DGAT transcript levels. The DGAT activity in developing seed of transgenic lines was enhanced by 10% to 70%. Thus, the current study confirms the important role of DGAT in regulating the quantity of seed triacylglycerols and the sink size in developing seeds.

Johnson C S, Ben K, Smyth D R. TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor
Plant Cell, 2002,14:1359-1375.

DOI:10.1105/tpc.001404URLPMID:12084832 [本文引用: 1]
Mutants of a new gene, TRANSPARENT TESTA GLABRA2 (TTG2), show disruptions to trichome development and to tannin and mucilage production in the seed coat. The gene was tagged by the endogenous transposon Tag1 and shown to encode a WRKY transcription factor. It is the first member of this large, plant-specific family known to control morphogenesis. The functions of all other WRKY genes revealed to date involve responses to pathogen attack, mechanical stress, and senescence. TTG2 is strongly expressed in trichomes throughout their development, in the endothelium of developing seeds (in which tannin is later generated) and subsequently in other layers of the seed coat, and in the atrichoblasts of developing roots. TTG2 acts downstream of the trichome initiation genes TTG1 and GLABROUS1, although trichome expression of TTG2 continues to occur if they are inactivated. Later, TTG2 shares functions with GLABRA2 in controlling trichome outgrowth. In the seed coat, TTG2 expression requires TTG1 function in the production of tannin. Finally, TTG2 also may be involved in specifying atrichoblasts in roots redundantly with other gene(s) but independently of TTG1 and GLABRA2.

Steffen J G, Kang I, Portereiko M F, Lloyd A, Drews G N. AGL61 interacts with AGL80 and is required for central cell development in Arabidopsis
Plant Physiol, 2008,148:259-268.

[本文引用: 1]

An D, Suh M C. Overexpression of Arabidopsis WRI1 enhanced seed mass and storage oil content in Camelina sativa
Plant Biotechnol Rep, 2015,9:137-148.

DOI:10.1007/s11816-015-0351-xURL [本文引用: 1]

Hyun-Young S, Hee N K. RAV1 negatively regulates seed development by directly repressing MINI3 and IKU2 in Arabidopsis
Mol Cells, 2018,41:1072-1080.

URLPMID:30518173 [本文引用: 1]

Cai G Q, Fan C C, Liu S, Yang Q Y, Liu D, Wu J, Li J W, Zhou Y M, Guo L, Wang X M. Nonspecific phospholipase C6 increases seed oil production in oilseed Brassica ceae plants
New Phytol, 2020,226:1055-1073.

DOI:10.1111/nph.16473URLPMID:32176333 [本文引用: 1]
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.

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.

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.

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.

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.

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]

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.

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]

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