合成小麦抗穗发芽QTL定位及六个籽粒萌发相关基因分子鉴定

发布时间：2018-09-12 08:47

【摘要】：小麦穗发芽是世界性灾害,严重影响小麦的产量和品质。小麦穗发芽受数量性状控制,目前已鉴定穗发芽位点遍布于小麦全部21条染色体上,其中位于第三同源群和第四同源群上的较多。影响小麦穗发芽的因素很多,包括种子休眠性、ABA敏感性、迟熟淀粉酶及籽粒颜色等,尽管已有相关基因的报道,但是目前还缺乏关于这些基因的调控位点与抗穗发芽QTL之间关系的报道。因而挖掘更多的抗穗发芽QTL并鉴定其与这些功能基因的团控位点之间的关系对于有效进行分子辅助育种有更大的价值。本研究利用小麦660K SNP芯片对抗穗发芽人工合成小麦SHW-L1(强休眠节节麦AS60与四川地方四倍体小麦AS2255合成)与易发芽小麦川麦32产生的RILs群体(F10,171个株系)构建高密度遗传图谱。利用该图谱进行抗穗发芽QTL定位并在SHW-L1育种后代中对QTL效应进行评价。同时,对高等电点淀粉酶编码基因TaAmyl及ABA信号相关基因TaVpl、TaSdr4, TaAIP2和TaSnrk2.6进行了表达模式分析及eQTL定位,分析与抗穗发芽QTL重叠的eQTL并挖掘重要eQTL位点的候选基因,获得的主要研究结果如下：1)利用AxiomTM wheat 660k Arrays基因芯片对SHW-L1和川麦32重组自交系构建高密度遗传图谱,平均密度为每个位点0.148cM和143kb(小麦基因组大小参照IWGSC数据)。利用该遗传图谱在2010年-2015年共10个独立环境下进行抗穗发芽QTL定位,定位到9个可在至少两个环境中稳定的QTLs,其中qPHS.sicau-1B和qPHS.sicau-3D.2可在8个环境中重复检测到,同时对该群体籽粒颜色QTL的定位,发现qPHS.sicau-3D.2与种皮颜色QTL qGC.sica-3D重叠,与调控颜色的转录因子Tamyb10-D相近,而另外一个新主效QTL qPHS.sicau-1B与籽粒颜色无关。2)利用SHW-L1与四川主栽品种杂交,创制了一批抗穗发芽新材料,其中小麦新材料蜀麦580(刘登才、张连全等选育,2016年通过云南省区试)的穗发芽抗性水平与SHW-L1相当,7天发芽率比普通小麦降低30%。对蜀麦580的遗传背景进行分析,其中源自节节麦的两个抗穗发芽QTL qPHS.sicau-3D.1和qPHS.sicau-3D.2成功导入蜀麦580中,而另一个主效位点qPHS.sicau-1B可能也导入(正在精细定位验证)。3)控制萌发速率的关键基因TaAmy1属于大麦AMY2-1亚家族,其在SHW-L1和川麦32间存在6个SNPs。TaAmyl基因在川麦32籽粒发育后期的表达显著高于抗穗发芽亲本SHW-L1,通过基因表达差异在1BS上定位到与主效抗穗发芽位点qPHS.sicau-1B位置重叠eQTL位点eqamyl. sicau-DPA30,说明qPHS.sicau-1B在籽粒发育后期可能通过降低TaAny1基因表达水平从而提升穗发芽抗性。4) TaVpl基因在SHW-L1中的表达水平持续高于川麦32,同时TaVpl基因eQTL eqABA.sicau-1B.1与穗发芽QTL qPHS.sicau-1B、α-淀粉酶eQTL eqamy1-DPA30重叠,说明TaVp1基因表达的差异参与了该位点对穗发芽抗性的调控。5)对调控TaVp1基因在开花后15、20天表达的eQTL eqABA.sicau-1D进行了进一步的分析,该区间共线与水稻5号染色体上2.8Mb区间内,根据水稻基因的功能注释,搜索到4个相关基因TaABI1、TaGAox20, TaAkinlO和TaRav1。其中TaRav1基因的表达模式与TaVp1相同,说明TaRav1反式调控TaVp1(与拟南芥中类似)。在开花后15天和20天控制TaRav1表达的QTL定位,与TaVp1、 TaSdr4、TaAIP2、TaSnrk2.6及TaAmy1已定位的eQTL位点重叠,说明TaRav1基因与其它基因可能同时受到上游基因的调控。6)川麦32与SHW-L1中TaRav1-D基因共存在5个SNPs,分别位于-234bp,-91bp,+18bp,+379bp,+584bp处。其中+379bp,+584bp处的SNPs可导致氨基酸的变异。TaRavl基因序列在52份六倍体普通小麦中非常保守,均与川麦32序列完全一致；节节麦AetRavl基因变异类型丰富,单倍型与节节麦的亚群分类相关,其中Ae.strangulata中的一类与小麦相同。
[Abstract]:Ear sprouting is a worldwide disaster, which seriously affects the yield and quality of wheat. Ear sprouting is controlled by quantitative traits. It has been identified that ear sprouting sites are located on all 21 chromosomes of wheat, among which there are many factors affecting ear germination, including seed dormancy and ABA. Sensitivity, late-ripening amylase and grain color, although related genes have been reported, there is no report on the relationship between the regulatory sites of these genes and QTLs for panicle germination resistance. In this study, a high-density genetic map of RILs population (F10,171 lines) produced by strong dormant nodal wheat AS60 and Sichuan endemic tetraploid wheat AS2255 and easy-germinating wheat Chuanmai 32 was constructed by wheat 660K SNP chip. At the same time, expression pattern analysis and eQTL mapping of high-point amylase coding gene TaAmyl and ABA signal-related genes TaVpl, TaSdr4, TaAIP2 and TaSnrk2.6 were carried out, and eQTL overlapping resistance to spike germination QTL was analyzed and candidate genes of important eQTL loci were excavated. The results are as follows: 1) High-density genetic maps of SHW-L 1 and Chuanmai 32 inbred lines were constructed using AxiomTM wheat 660k Arrays gene chip. The average densities were 0.148cM and 143KB per locus (the wheat genome size was compared with IWGSC data). QTL mapping of resistance to spike germination was carried out in 10 independent environments from 2010 to 2015. Nine stable QTLs were identified in at least two environments, of which qPHS. sicau-1B and qPHS. sicau-3D.2 could be detected repeatedly in eight environments. At the same time, qPHS. sicau-3D.2 was found to overlap with seed coat color QTL qGC. sica-3D, similar to transcription factor Tamyb10-D and another new major QT. L qPHS.sicau-1B has nothing to do with grain color.2) A number of new materials for ear germination resistance were developed by crossing SHW-L1 with Sichuan main cultivars. Among them, Shumai 580 (Liu Dengcai, Zhang Lianquan, etc.) was selected and bred, and its ear germination resistance level was similar to that of SHW-L1 in 2016. The 7-day germination rate was 30% lower than that of common wheat. Genetic background analysis showed that two QTLs qPHS.sicau-3D.1 and qPHS.sicau-3D.2 derived from Arthropus japonicus were successfully introduced into Shumai 580, while another major locus qPHS.sicau-1B might also be introduced into (being fine-mapping verified). 3) TaAmy1, a key gene controlling the germination rate, belonged to the barley AMY2-1 subfamily, and it was found in SHW-L 1 and Chuanmai 3. Six SNPs. TaAmyl genes were significantly higher than SHW-L1 in the late grain development stage of Chuanmai 32, and the overlapping eQTL site eqamyl. sicau-DPA30 with the main resistant sprouting site qPHS. sicau-1B was located on 1BS by gene expression difference, suggesting that qPHS. sicau-1B might reduce the Any1 base in the late grain development stage. The expression level of TaVpl gene in SHW-L 1 was consistently higher than that in Chuanmai 32, and the expression level of TaVpl gene eQTL eqABA.sicau-1B.1 overlapped with QTL qPHS.sicau-1B and alpha-amylase eQTL eqamy1-DPA30 in spike germination, indicating that the difference of TaVp1 gene expression was involved in the regulation of spike germination resistance. The eQTL eqABA.sicau-1D expressed by aVp1 gene at 15 and 20 days after anthesis was further analyzed. According to the functional annotations of rice genes, four related genes, TaABI1, TaGAox20, TaAkinlO and TaRav1, were found. The expression pattern of TaRav1 gene was the same as that of TaVp1, indicating that TaRav1 was antigenic. TaVp1 (similar to that in Arabidopsis) was regulated by the expression of TaRav1. The QTL mapping of TaRav1 was controlled at 15 and 20 days after anthesis and overlapped with the eQTL loci of TaVp1, TaSdr4, TaAIP2, TaSnrk2.6 and TaAmy1, indicating that TaRav1 gene and other genes might be regulated by the upstream genes at the same time. 6) TaRav1-D gene of Chuanmai 32 and SHW-L1 shared five SNPs, respectively. SNPs located at - 234 bp, - 91 bp, + 18 bp, + 379 bp, + 584 BP could induce amino acid mutation. TaRavl gene sequence was very conserved in 52 hexaploid common wheat, which was completely consistent with Chuanmai 32 sequence; AetRavl gene had abundant variation types, and haplotypes were related to the subgroup classification of Arthroid wheat, including Ae. strangula. One of TA is the same as wheat.
【学位授予单位】：四川农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S512.1

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