基于早晚熟突变体的谷子抽穗开花调控分子机制的研究

Adaptation of foxtail millet to local environments and the expansion of their cultivation area is largely determined by optimal heading date or flowering time, which is essential to the success in production of foxtail millet. To better understand the molecular mechanisms of heading date in foxtail millet, we isolated three early maturing and one late maturing mutants from an EMS mutant library. Among them, the mutant ehm1, ehm2 and ehm3 had an early heading date of 60, 30, and 30 days respectively; while the mutant lhm1 delayed heading for about 20 days, compared to the wild type. In addition, the plant height, number of tillers and yield traits of these mutants were also affected. The proposed project is to firstly conduct genetic analysis and phenotype characterization of these mutants to determine the effects of photoperiod and temperature on heading date. The differentially expressed genes will be then identified using RNA-Seq in the mutants, and genetic regulatory network will be subsequently analyzed. Furthermore, the candidate genes will be cloned by high-throughput sequencing based MutMap+ technology.The relationship of these four genes and other key heading or flowering regulating genes will be examined to clarify the functional role of these genes in the regulatory network of heading date in foxtail millet. Finally, functional nucleotide polymorphisms in these genes will be analyzed to elucidate the adaptive genetic variation in heading date control and the underlying molecular mechanisms. The proposed study will not only shed novel insights into the molecular mechanisms controlling heading date, but also lay a theoretical foundation for breeding new foxtail millet varieties with high yield and wide adaptation to the environment.

谷子最佳的抽穗开花时间在很大程度上决定了其对局部环境和栽培地域扩张的适应，对谷子生产至关重要。为了解析谷子抽穗开花调控的分子机制，我们从EMS突变体库中筛选到3个早熟突变体和1个晚熟突变体。其中，早熟突变体ehm1抽穗期提前60天左右，ehm2和ehm3提前30天左右；而晚熟突变体lhm1抽穗期延迟了20天左右。此外，这些突变体株高、分蘖数和产量等也受到了影响。本项目拟首先对这些突变体进行遗传分析和表型分析，明确光周期和温度等因素对其抽穗开花的影响；然后利用RNA-Seq技术分析突变体差异表达的基因，探讨其遗传调控网络；利用基于高通量测序的MutMap+技术克隆候选基因，并探讨其在谷子抽穗开花调控网络中的功能和地位；最后，分析这些基因的功能核苷酸多态性，解析抽穗期控制的适应性遗传变异及分子机制。本项目的实施不仅有助于解析谷子抽穗期调控的分子机制，还将为高产广适谷子新品种的培育奠定理论基础。

抽穗期既是一个重要的农艺性状，又是一个极其复杂的基础理论问题。为了解析谷子抽穗开花调控的分子机制，我们筛选了4个超早熟谷子突变体，1个早熟谷子突变体和1个晚熟谷子突变体。超早熟谷子突变体ehm1（mop1，xiaomi）、ehm3、ehm4和ehm5生育期仅2个月左右，早熟突变体ehm2生育期为110 天左右，而晚熟突变体lhm1生育期约为5个月左右。利用MutMap+的技术克隆了EHM2和EHM3基因，这两个基因分别编码热激蛋白和COPA蛋白。EHM2基因在幼苗中表达量较高，而在根、幼叶和穗中表达量较低。对916分谷子种质资源的测序表明，EHM2基因存在丰富的遗传变异，共检测到12个SNP位点，其中7个位点位于外显子区。这12个SNP位点组成了12个单倍型，为深入发掘优异等位基因提供了丰富的材料。超早熟基因EHM3在幼苗中表达量较高，而在幼叶中表达量较低。该基因在谷子进化过程较为保守，在916谷子种质资源中未发现变异位点。转录组分析表明，ehm1突变体早抽穗表型可能与两个MADS-box家族的基因表达上调有关，ehm2突变体通过上调成花素基因FT和下调硝酸盐转运蛋白NRT基因的表达促进了抽穗；而突变体lhm1的晚抽穗表型可能与MADS15表达量降低有关。构建了早熟谷子GBS和晚熟谷子168的F2群体，利用Supper-BSA技术将早熟性状相关基因定位在第9染色体96 kb的区段。利用单分子测序技术、二代测序技术和Hi-C技术测序和从头组装了ehm1的基因组，组装基因组大小429.94 Mb；注释了35314个蛋白编码基因，2833个假基因，228个miRNA，28260个lncRNA和1318个circRNA；构建了ehm1的全生育期基因表达图谱；建立了基于ehm1的高效遗传转化体系。从而将矮杆极短生育期的ehm1发展成C4禾谷类研究的模式植物。本项目的顺利实施不仅初步解析了谷子抽穗期调控的分子机制，为高产广适谷子新品种的培育奠定了理论基础，而且建立了C4禾谷类研究的模式植物体系，必将大大加快谷子及相关植物功能基因组学研究的进程。