基于SNPs技术的甘蔗种质资源与脱叶性的遗传关联分析

The establishment of platform for molecular marker-assisted selection is one of the difficult in sugarcane molecular breeding. And it is one of the emphases in sugarcane breeding in Guangxi province. Guangxi has become the largest producer of sugarcane, and the center of cane sugar producing in China. Selective breeding of new sugarcane varieties has become one of the most effective measures to solve the problems of single species, bad resistance to adversity, and the weak of capability for defense against natural disasters. Recently, the sugarcane varieties bred in China are limited in the narrowed hereditary basis of sugarcane germplasm resource and the diminution of the blood of wild sugarcane germplasm. With the development and application of molecular markers rapidly in sugarcane assisted selection, the research of sugarcane molecular breeding has entered a new breeding stage characterized by modern biotechnology. In this project, screening genetic markers of important agronomic traits (such as deciduous leaf) of sugarcane by using single nucleotide polymorphism (SNP). Total eighteen sugarcane germplasm resources were selected, twelve germplasm resources were easy deciduous, and six germplasm resources were holds onto its leaves. All the germplasm resources were studied by using 30 combinations. Sugarcane germplasm resources and F1 segregation population of all combinations were selected by using sugarcane breeding program five nursery systems. In this project, more and more SNPs linked tightly to important agronomic traits (such as deciduous leaf) will be acquired and obtained, and the objectives are to enhance the capability manipulating SNPs molecular markers assisted-selected breeding for breeders, and provide theory basis for further important agronomic traits SNPs screening, cloning and application.

分子标记辅助育种平台的建立是甘蔗分子育种研究的难点，也是未来广西甘蔗育种工作的重点。广西是我国甘蔗最大的生产基地和产糖中心。新品种选育成为解决甘蔗品种单一、抗逆性差以及抵御自然风险最有效的措施之一。近年来，甘蔗亲本种质资源遗传基础狭窄和野生种血缘减少，已成为常规育种取得重大突破的重要限制因素。随着分子标记技术在甘蔗辅助育种上的发展和应用，甘蔗分子育种研究也进入以现代生物工程为主要特征的育种新阶段。本项目将应用SNP技术发掘更多与甘蔗重要农艺性状（即脱叶性）相连锁的SNPs位点。项目组已筛选出12个易脱叶亲本和6个难脱叶亲本，并配成30个常规杂交组合，利用甘蔗"五圃制"育种技术分阶段筛选F1分离群体。本项目拟通过SNP技术获得与甘蔗脱叶性相连锁的SNP位点，应用生物信息学技术分析甘蔗种质与脱叶性状的遗传关联性。

甘蔗是一种广泛种植于热带和亚热带地区的重要糖料和生物燃料作物。随着蔗糖和生物燃料需要的日益增加，要求选育在成熟期叶片容易脱落的甘蔗品种，降低收获成本。利用分子技术研究甘蔗脱叶机制，为辅助常规育种早期筛选脱叶性甘蔗品种有重要意义。完成126份甘蔗脱叶遗传群体构建，通过EST-SNP技术获得2025个SNP位点。此外，还以易脱叶和不易脱叶典型表型分离性状的亲本及其F1作为研究对象，利用高通量第二代测序技术Illumina HiSeq 2000平台对样本进行转录物测序及分析，获得3,722个SNPs，由于受甘蔗全基因未公布的限制，无法完全解读，因此，利用转录组测序技术尚未发现与脱叶基因的高度关联性的SNP。利用高通量第二代测序技术TruSeq平台测序及分析，研究识别96个甘蔗miRNAs共135个SNPs。分析结果表明，miR5564和miRN396与甘蔗miRNAs具有很大的关联性。正常和变异miRN396分享76个靶基因包括CSLD4（纤维素合酶-类蛋白质D4）和CDKA2（细胞周期蛋白-激酶A-2）。如预测变异miRN396缺少11个靶基因包括WEX（沃纳综合征-类核酸外切酶也得到等11个新的靶基因，包括POL2（297转位子逆转录酶病毒相关Pol多蛋白）和E139（葡聚糖-1，3-beta-葡糖苷酶9）。由于尚不知以上相关靶基因的具体功能，但是这些miRNAs和甘蔗脱叶有很高的关联性。研究结果发现，96条miRNA有135个SNP位点，其中2个变异miRNAs（sof-miR5564b-3p和sof-miRN396-3p）在样本中高表达量而值得关注，他们可能影响甘蔗miRNAs在脱叶过程中的生源和功能。与不易脱叶甘蔗相比，我们成功鉴定93条保守的RNAs和547个甘蔗miRNA，易脱叶甘蔗有25个miRNA上调，13个miRNA下调。首次在甘蔗上鉴定了被称作“miR5384”的禾本科特有miRNA。而且还提供了一些甘蔗miRNA-mRNA模式，包括miR156-SPL，miR319-TPR2，miR396-GRF和miR408- LAC，这些模式可能参与调控甘蔗成熟期叶片脱落。基于miRNAs和靶因子的保守性，推测miR160和miR167可能通过调控生长素响应因子参与甘蔗脱叶。发表研究论文9篇（SCI4篇），申请实用新型1项。