柑桔线虫抗性主基因座Tyr1的特异标记开发与遗传作图改进(英文)

Developing Specific Markers and Improving Genetic Mapping for a Major Locus Tyr1 of Citrus Nematode Resistance

本文利用先期从BAC文库获得的NBS-LRR类候选抗病基因克隆序列Pt8a和Pt9a,进一步开发与柑桔线虫抗性主效基因位点Tyr1连锁的分子标记。以Pt8a和Pt9a序列作探针,通过高密度克隆印迹杂交,从BAC文库筛选出200个以上的阳性克隆,以阳性克隆插入序列设计引物,对柑桔抗线虫材料和感线虫材料开展以PCR扩增为基础的集群分离分析,发现一部分克隆序列与柑桔线虫抗性主效基因位点Tyr1紧密连锁;再通过染色体步行测序,分别从3个克隆(7A4,4L17和29F20)获得3个完整的NBS-LRR类候选抗病基因序列。从此类序列开发更高特异性的分子标记,并在利用原有分子标记的基础上,对柑桔线虫抗性杂交后代群体(9145family)构建较高密度的遗传图谱;同时,将新开发的分子标记应用于柑桔衰退病抗性杂交后代群体(9401family),以初步估算柑桔线虫抗性主效基因位点Tyr1与柑桔衰退病抗性基因Ctv的遗传距离。本文利用先期从BAC文库获得的NBS-LRR类候选抗病基因克隆序列Pt8a和Pt9a,进一步开发与柑桔线虫抗性主效基因位点Tyr1连锁的分子标记。以Pt8a和Pt9a序列作探针,通过高密度克隆印迹杂交,从BAC文库筛选出200个以上的阳性克隆,以阳性克隆插入序列设计引物,对柑桔抗线虫材料和感线虫材料开展以PCR扩增为基础的集群分离分析,发现一部分克隆序列与柑桔线虫抗性主效基因位点Tyr1紧密连锁;再通过染色体步行测序,分别从3个克隆(7A4,4L17和29F20)获得3个完整的NBS-LRR类候选抗病基因序列。从此类序列开发更高特异性的分子标记,并在利用原有分子标记的基础上,对柑桔线虫抗性杂交后代群体(9145family)构建较高密度的遗传图谱;同时,将新开发的分子标记应用于柑桔衰退病抗性杂交后代群体(9401family),以初步估算柑桔线虫抗性主效基因位点Tyr1与柑桔衰退病抗性基因Ctv的遗传距离。

The NBS-LRR class resistance-gene candidate sequences （Pt8a and Pt9a） were used to develop new specific markers to the citrus nematode resistance gene locus Tyrl. By high-density colony screening, over 200 positive clones were pulled out from the BAC library. A few of the clones were found to be closely linked with the Tyrl region, because the primers from these clones insert sequence produced polymorphism which matched up with the phenotype after bulked segregant analysis. By primer walking approach, three integrate NBS-LRR class resistance-gene sequences were tagged and identified separately in three clones （7A4, 4L17 and 29F20）. More specific markers were developed from these tagged sequences and relatively high-density genetic maps were constructed by incorporating the newly developed markers and previously developed markers in the ＇9145 family＇. New markers were applied in ＇ 9401 family＇ trying to estimate roughly the genetic distance between the Cry and TyrI region.