摘要
【目的】寻找苯磺隆胁迫下油菜种子萌发性状相关的QTL及其耐性基因,为筛选与培育耐苯磺隆油菜种质以及探究油菜种子萌发过程中苯磺隆耐性分子机理奠定基础。【方法】用0.15 mg·kg-1苯磺隆溶液处理由人工合成甘蓝型油菜10D130和甘蓝型油菜常规品种ZS11构建的包含175个株系的高世代重组自交系(RIL)群体,进行种子发芽试验,以蒸馏水为对照,分别测定其相对发芽势、相对发芽率、相对根长和相对干重。然后,利用油菜6K SNP芯片对该RIL群体进行基因分型,通过JoinMap4.0软件构建高密度遗传连锁图谱。基于该遗传图谱,利用MapQTL软件多QTL作图法对4个性状的相对值进行QTL定位,根据各QTL置信区间查找甘蓝型油菜的基因序列,并依次与拟南芥基因组序列进行BLAST,筛选可能与耐苯磺隆胁迫相关的候选基因。【结果】频数分布表明4个相对性状的变异范围较大,且呈连续性分布,符合数量性状表现特征,适宜进行QTL遗传分析。相关分析表明,相对发芽率和相对发芽势呈极显著正相关,相关系数为0.587。构建的遗传图谱包含1 897个多态性SNP标记,覆盖甘蓝型油菜基因组3 214.19 cM,标记之间的平均图距为1.69 cM。利用此图谱共检测到22个相关QTL,表型贡献率变幅为6.4%-12.6%。其中,与相对发芽势、相对发芽率相关的QTL分别有6个和3个,与相对根长和相对干重有关的QTL分别为8个和5个。在A01染色体64.857 cM、55.935 cM和56.645 cM处检测到的相对发芽势与相对发芽率QTL的置信区间完全或者部分重叠。通过分析QTL置信区间上甘蓝型油菜对应的区间序列,筛选到30个可能与油菜耐苯磺隆有关的候选基因,其中包括18个细胞色素P450家族成员、5个糖基转移酶家族基因、1个GSTF相关基因、1个ABC转运蛋白相关基因和1个ALS基因,这些基因均与除草剂抗性机制有关,尤其ALS为磺酰脲类除草剂靶位点酶;另外筛选到1个BHLH和1个JAZ6基因,BHLH与JAZ蛋白可通过相互作用来防御胁迫;检测到1个LSU2蛋白相关基因和1个MATE家族成员,前者参与细胞氧化剂解毒及植物防御反应,后者参与类黄酮、生物碱、金属离子、其他多种代谢物的转运及有毒物质引起的植物胁迫响应。【结论】检测到与相关QTL共22个,筛选出可能与苯磺隆耐性有关的候选基因30个。这些基因通过加速毒性分子的转运与代谢从而响应有毒物质引起的胁迫反应,可能参与植物对苯磺隆的抗性调节与反应机制。
【Objective】The QTLs and tolerance genes related to the germination characters of rape seeds under the stress of tribenuron-methyl were studied, which laid the foundation for screening and cultivating the germplasm of tribenuron-methyl resistant rape and exploring the molecular mechanism of tribenuron-methyl tolerance during the germination of rape seeds.【Method】A high generation RIL population consisted of 175 lines, which were constructed from the synthetic Brassica napus 10 D130 and the conventional variety Brassica napus ZS11, was treated with 0.15 mg·kg-1 tribenuron-methyl solution for seed germination test and the control was under the distilled water. The phenotypic data that including relative germination vigor(RGV), relative germination rate(RGR), relative root length(RRL) and relative dry weight(RDW) were analyzed by Excel software. Then, the RIL population was genotyped with 6 K SNP chip, and the high-density genetic linkage map was constructed by JoinMap4.0 software. Based on the genetic map, the relative values of four characters were mapped by using Multiple QTL mapping method of MapQTL software. And the genes sequence of Brassica napus were searched according to the confidence interval of each QTL, next, blast with Arabidopsis genome sequence in turn and select the candidate genes that may be related to the tolerance to tribenuron-methyl stress.【Result】The frequency distribution of each traits for RIL population’s was continuous with the large variation range, which were consistent with the characteristics of quantitative characters, so it were suitable for the detection of QTL. Correlation analysis showed that there was a significant positive correlation between RGR and RGV, and the correlation coefficient was 0.587. In addition, the constructed genetic map contained 1 897 polymorphic SNP markers covering 3 214.19 cM of the genome of Brassica napus with an average map distance of 1.69 cM. By this map, 22 QTLs related to 4 phenotypic traits were detected and the phenotypic contribution rate was between 6.4% and 12.6%. Among them, there were 6 and 3 QTLs related to RGV and RGR, 8 and 5 QTLs related to RRL and RDW, respectively. Also, the confidence intervals of QTLs for RGV and RGR were found to overlap completely or partially at 64.857 cM, 55.935 cM and 56.645 cM of chromosome A01. Through sequence alignment, 30 candidate genes were screened, including 18 cytochrome P450 family members, 5 glycosyltransferase family genes, 1 GSTF related gene, 1 ABC transporter related gene and 1 ALS gene, all of which were detoxified by accelerating metabolism, and related to the mechanism of herbicide resistance, especially ALS is the target enzyme of sulfonylurea herbicide. Furthermore, others genes were screened, including 1 BHLH gene and 1 JAZ6 gene which could interact to protect against stress;and 1 LSU2 protein gene which was involved in the detoxification of cell oxidants and plant defense response;and 1 MATE family member which was involved in the transport of flavonoids, alkaloids, metal ions, other metabolites and plant stress response caused by toxic substances.【Conclusion】22 QTLs that significantly associated with tribenuron-methyl tolerance related traits and 30 candidate genes for the tolerance to tribenuron-methyl were found. These genes are involved in the stress response caused by toxic substances by accelerating the transport and metabolism of toxic molecules, which may be related to the resistance regulation and response mechanism of plants to tribenuron-methyl.
作者
王刘艳
王瑞莉
叶桑
郜欢欢
雷维
陈柳依
吴家怡
孟丽姣
袁芳
唐章林
李加纳
周清元
崔翠
WANG LiuYan;WANG RuiLi;YE Sang;GAO HuanHuan;LEI Wei;CHEN LiuYi;WU JiaYi;MENG LiJiao;YUAN Fang;TANG ZhangLin;LI JiaNa;ZHOU QingYuan;CUI Cui(College of Agronomy and Biotechnology,Southwest University,Chongqing 400716)
出处
《中国农业科学》
CAS
CSCD
北大核心
2020年第8期1510-1523,共14页
Scientia Agricultura Sinica
基金
国家重点研发计划(2018YFD0100500)
国家农业部现代农业产业技术体系(CARS-12)
重庆市技术创新与应用发展(cstc2019jscx-msxmX0383)。
关键词
苯磺隆
甘蓝型油菜
萌发期
QTL
候选基因
tribenuron-methyl
Brassica napus L.
germination
QTL
candidate genes