基于集群分离样本总RNA测序快速定位甘蔗梢腐病抗病基因

Sugarcane Pokkah Boeng is one of the most important diseases threatening sugarcane production in China. Sugarcane has excellent resistant resources for Sugarcane Pokkah Boeng. Therefore, the disease can be controlled by breeding sugarcane disease-resistant varieties. Mapping resistance genes is the key to achieve the goal with the help of marker-assisted selection (MAS). At present, no disease resistance genes and molecular markers for this disease have been reported. We have developed a segregating population by crossing a highly resistant variety “YZ 89-7” and a highly susceptible variety “ROC 25”. Based on high-quality disease resistance evaluation, we assembled two contrasting phenotype bulks, resistant and susceptible, for bulked segregant total RNA-Seq. Through comparing bulk frequency ratio of single nucleotide polymorphisms (SNPs) between the two bulks, resistance-related markers can be quickly screened for further analysis. Then the candidate markers will be genotyped in the whole population. Linkage mapping and quantitative trait locus (QTL) mapping will be performed. The flanking markers closely linked to QTLs will be further evaluated in a sugarcane germplasm panel. In addition, analysis of total RNA sequencing data of the parental lines and two bulks will reveal the genes and their interactions between pathogens and sugarcane at different inoculation times. This project will provide molecular tools and theoretical supports for the use of MAS to improve the resistance of sugarcane varieties to Sugarcane Pokkah Boeng, and serve as an important reference study for the rapid mapping of target genes in sugarcane.

梢腐病是危害我国甘蔗生产最重要的病害之一。甘蔗拥有抗梢腐病优异的种质资源，因而可通过选育抗病品种来防控该病害。定位梢腐病抗病基因，是实现该病害分子辅助育种的关键。目前，世界上尚无梢腐病抗病基因及相关分子标记的报导。申请人利用甘蔗高抗病品种云蔗89-7和高感病品种新台糖25杂交获得抗感病分离群体。在此基础上，根据高质量抗病性鉴定数据，构建抗感病集群，通过集群分离样本总RNA测序，比较集群间SNP的等位基因比例差异，快速筛选抗性关联标记，进一步分析候选标记在分离群体的基因型，构建遗传连锁图谱、定位数量性状基因座（QTL），并在甘蔗种质资源中验证与QTL紧密连锁的侧翼标记。另外，通过抗感病亲本及集群的总RNA测序数据分析，可揭示病原菌与甘蔗在不同侵染时间下基因互作及网络。本项目研究结果不仅为利用分子辅助育种途径提高甘蔗梢腐病抗性提供工具和理论支持，而且为在甘蔗中快速定位目标基因提供重要参考。

梢腐病是危害我国甘蔗生产最重要的病害之一。解析甘蔗抗梢腐病的分子机制，定位抗病基因并开发分子标记，是实现针对该病害分子辅助育种的关键。项目组首先系统鉴定了甘蔗NBS-LRR基因，通过单子叶植物中NBS-LRR基因的特征和系统发育分析及保守NBS-LRR基因序列特征及进化分析，揭示了NBS-LRR基因数目与基因组大小无线性关系，基因组复制、基因丢失及扩张影响NBS-LRR基因的数目。利用转录组数据，我们挖掘到数个响应多种甘蔗病害的基因，并且发现割手密对于甘蔗栽品种的抗病性有较大的贡献。同时，项目组通过系统比较滴心、注射、喷雾等三种不同的人工接种方法，综合认为注射接种法效果最好，但只适用于少量样本的精细研究，且与自然发病存在一定差异；我们利用亲本及梢腐病极端抗感子代材料进行转录组分析，通过差异表达基因、WGCNA分析并结合NBS-LRR基因，确定37个可能响应甘蔗梢腐的关键基因；另外，结合简化基因组连锁分析以及转录组分析，我们针对9个候选基因进行qRT-PCR验证，并开发分子标记，在极端材料中验证获得1个与甘蔗梢腐病关联的分子标记。本研究对于甘蔗抗病研究和育种具有一定的指导意义，提供了重要基因资源和分子工具。