【作者】 李磊；

【导师】 尹钧；

【作者基本信息】 河南农业大学 ， 作物学， 2023， 博士

【摘要】 小麦春化作用是其重要的发育特性,也是影响小麦种植区划、品种改良、引种用种、栽培技术优化等的主要因素。利用现代组学技术,深入探究小麦春化作用,明确小麦春化特性调控机理,为小麦发育特性改良和品种布局提供理论指导和技术支撑。本研究在对不同小麦品种春化发育特性观测、群体QTL定位研究的基础上,通过冬性小麦品种京841的4个春化处理,开展转录组、miRNA和降解组测序研究,系统分析了小麦春化过程中的差异表达基因、差异表达miRNA及其与靶基因的调控关系,探索miRNA参与小麦春化发育特性调控机理。主要研究结果如下:1.鉴定了10个代表性小麦品种VRN1基因在A、B、D三个基因组水平上的显隐性,基因型为vrn-A1/vrn-B1/vrn-D1(全隐性)的有7个,其中春化发育特性为强冬性1个,半冬性6个;基因型为vrn-A1/Vrn-B1/vrn-D1(B基因组显性)的1个,春化发育特性为弱春性;基因型为vrn-A1/vrn-B1/Vrn-D1(D基因组显性)的有2个,春化发育特性均为弱春性。VRN1三个等位基因全隐性类型的小麦品种,其春化特性存在明显差异,需要利用现代组学技术,进一步挖掘新的春化调控基因。2.利用小麦品种周麦22(ZM22)和中国春(CS)构建的RIL群体,开展了两年多点的穗分化进程观测,并利用小麦55K SNP芯片成功构建了ZM22/CS RIL群体的高密度遗传图谱,该图谱全长14458.31 c M,包含2827个标记,覆盖了小麦21条染色体,标记间平均距离为5.11c M。通过单环境、多环境联合分析,检测到9个稳定的加性QTL,它们控制小麦幼穗二棱期的发育进程,其中,位于4D染色体的a QDR.henau-4D.1在两年间被重复检测到,该QTL位于256.5-257.5 c M区间内,两侧标记为AX-111402771至AX-109410812,其为DR的主效QTL。3.为了筛选鉴定春化调控关键基因,从京841的4个春化处理测序样本中得到了3.82亿条原始读序(raw reads)和114.57Gb比对读序(clean reads),从中鉴定发现22,928个新基因,其中15,242个具有功能注释。春化处理初期(CK-D10)的样本间差异表达基因(DEGs)数量最少,春化处理后期(CK-D30)的样本间DEGs最多。在所有的DEGs中,检测到854个独特的差异表达转录因子(TFs)。b HLH、C2H2、MYB和NAC家族在响应春化发育中发挥了相对重要的作用。4.为了鉴定春化发育过程中表达模式相似的DEGs,对12,926个DEGs进行了K-means聚类检验,得到了8个最优聚类。淀粉和蔗糖的代谢(ko00500)和苯丙醇生物合成(ko00940)参与了8个簇中的6个。谷胱甘肽代谢(ko00480)参与了8个聚类中的3个。对12,926个差异基因进行了WGCNA分析,聚类树图显示了7个用不同的颜色标记的模块,绿松石模块与CK、红色模块与D10、绿色模块与D20、蓝色模块与D30的相关系数最高。鉴定得到Traes CS7B01G016800、Traes CS6B01G383200、Traes CS6B01G274200、Traes CS1D01G189600、Traes CS1D01G190100、Traes CS5B01G426300、Traes CS7D01G030800、Traes CS7D01G050800共8个高度相关的核心基因。5.为探索miRNA对小麦春化特性的调控作用,利用miRNA高通量测序,总共产生25.3亿条原始读序(raw reads)和20.08M的比对读序(clean reads),鉴定得到873个miRNA,其中已知miRNA有107个,新的miRNA(novel＿miRNA)有766个;预测到miRNA靶基因18,664个,主要集中在生物过程、细胞组成和分子功能。降解组测序共鉴定到68个miRNA,其中包括15个已知miRNA,53个新的miRNA;分别靶向129个靶基因,形成331对miRNA与靶基因作用对,从中筛选到8个关键miRNA(tae-miR159a、tae-miR160、tae-miR164、tae-miR167a、tae-miR396、tae-miR5200、novel＿miR320和novel＿miR646),分别靶向8个春化发育相关基因(ZFP1、ARF8、NAC100、ARF12、GRF10、VRN3、AP2和VRN2)。转基因功能验证结果表明,tae-miR396b通过靶向抑制GRFs家族成员,影响小麦体内激素平衡,促进了小麦发育进程;novel＿miR646通过靶向抑制VRN2的表达,而促进了小麦发育进程。上述8个关键miRNA共同参与了小麦春化发育调控,揭示了miRNAs参与小麦春化发育的调控网络和作用机理。更多还原

【Abstract】 Vernalization of wheat is an important developmental characteristic of wheat,and it is also the main factor affecting wheat planting division,variety improvement,seed introduction and cultivation technology optimization.The modern omics technology was used to clarify the regulation mechanism of vernalization in wheat(Triticum aestivum L.),so as to provide theoretical guidance and technical support for the improvement of development characteristics and variety layout.In this study,based on the observation of vernalization development characteristics of different wheat varieties and population QTL mapping,transcriptomic,mi RNA and degradation sequencing were carried out through four vernalization treatments of winter wheat variety Jing 841,and the differential expression genes,differential expression mi RNAs and their regulatory relationships with target genes were systematically analyzed during the vernalization.To explore the regulation mechanism of mi RNA involved in vernalization in wheat.The main results were as follows:1. The VRN1 genotype of 10 main wheat varieties were identified,and 8 of them were vrn-A1/vrn-B1/vrn-D1,among which 1 was strong winter,6 was half-winter.There were 1varieties with vrn-A1/Vrn-B1/vrn-D1 genotype,and the developmental characteristics were weak spring.There were 2 varieties with vrn-A1/vrn-B1/Vrn-D1 genotype,and the developmental characteristics were all weak spring.The VRN1 genotype could not distinguish the vernalization characteristics of wheat varieties completely.It is necessary to use multi-omics technology to further explore new marker genes.2. Panicle differentitation was investigated using ZM22/CS RIL population,and a high-density genetic map of the ZM22/CS RIL population was constructed using wheat 55K SNP chip.The total length of map was 14458.31 c M,containing 2827 markers and covering21 chromosomes of wheat.The average distance between markers was 5.11 c M.Through a combination analysis of single-environment and multi-environment,9 stable additive QTLs were detected,which controlled the developmental process of young panicle in double range(DR)stage of wheat.a QDR.henau-4D1 was located in the 256.5.07-257.5 c M range,labeled AX-111402771 to AX-109410812 on both sides,which is the dominant QTL for DR.3. To screen and identify key genes in vernalization,transcriptome sequencing was performed.Total 382 million raw reads and 114.57Gb clean reads were obtained from 4sequenced samples.Based on the mapping results,22,928 new genes were found,15,242 of which had functional annotations.The amount of differentially expressed genes(DEGs)between samples at the early stage of vernalization(CK-D10)was the lowest,and the amount of DEGs between samples at the late stage of vernalization(CK-D30)was the highest.Of all the DEGs,854 unique differentially expressed transcription factors(TFs)were detected.The b HLH,C2H2,MYB and NAC families played a relatively important role in response to vernalization.4. To identify DEGs with similar expression patterns during vernalization,we performed a K-means clustering test on 12,926 DEGs and obtained 8 optimal clusters.Starch and sucrose Metabolism(ko00500)and phenylpropanoid biosynthesis(ko00940)were involved in 6 of the 8 clusters.Glutathione metabolomics(ko00480)was involved in 3of the 8 clusters.WGCNA analysis was performed on 12,926 differential genes.The cluster tree showed 7 different modules labeled with different colors.Turquoise module had the highest correlation coefficient with CK,red module with D10,green module with D20,and blue module with D30.Traes CS7B01G016800,Traes CS6B01G383200,Traes CS6B01G274200,Traes CS1D01G189600,Traes CS1D01G190100,Traes CS5B01G426300,Traes CS7D01G030800,Traes CS7D01G050800 were selected as the highly related hub genes.5. A total of 2.53 billion raw reads and 20.08M clean reads were generated by high-throughput mi RNA sequencing.And 873 mi RNAs were identified,including 107known mi RNAs and 766 novel mi RNAs.Total 18,664 target genes were predicted,focusing on biological processes,cell composition,and molecular function.A total of 68 mi RNAs were identified by degradation group sequencing,including 15 known mi RNAs and 53 new mi RNAs.They targeted 129 target genes,and 331 pairs of mi RNAs interacting with target genes were formed.Among 331 mi RNA and target gene interaction pairs,8 key mi RNA and target gene interaction pairs were screened and predicted to be involved in the regulation of vernalization development.The results of transgenic functional verification showed that tae-mi R396b affected hormone metabolism in wheat and promoted the development of wheat through targeted inhibition of GRFs family members.Novel_mi R646 promoted wheat development by targeting VRN2 expression inhibition.The 8 key mi RNAs mentioned above are involved in the regulation of wheat vernalization development,which reveals the regulatory effect of mi RNAs on wheat vernalization development.更多还原