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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。
In this study, we further developed molecular markers linked to Tyr1, the major nematode resistance locus, using the NBS-LRR candidate resistance gene cloning sequences Pt8a and Pt9a obtained from the BAC library. Using Pt8a and Pt9a as probes, more than 200 positive clones were screened from the BAC library by high-density cloning hybridization. Primers were designed based on the positive cloned insertions, and the PCR products of citrus nematode and nematode were amplified by PCR Based on the cluster analysis, some cloned sequences were found to be closely linked to Tyr1, the major locus of Citrus nematode resistance. Three complete clones (7A4, 4L17 and 29F20) were obtained from 3 clones (7A4, 4L17 and 29F20) NBS-LRR candidate disease resistance gene sequence. From these sequences, more specific molecular markers were developed, and based on the original molecular markers, a higher density genetic map of citrus nematode resistant hybrid progeny (9145family) was constructed. At the same time, newly developed molecules (9401family) to detect the genetic distance between Tyr1, a major gene locus in Citrus nematode resistance, and Ctv, a Citrus recessive disease resistance gene. In this study, we further developed molecular markers linked to Tyr1, the major nematode resistance locus, using the NBS-LRR candidate resistance gene cloning sequences Pt8a and Pt9a obtained from the BAC library. Using Pt8a and Pt9a as probes, more than 200 positive clones were screened from the BAC library by high-density cloning hybridization. Primers were designed based on the positive cloned insertions, and the PCR products of citrus nematode and nematode were amplified by PCR Based on the cluster analysis, some cloned sequences were found to be closely linked to Tyr1, the major locus of Citrus nematode resistance. Three complete clones (7A4, 4L17 and 29F20) were obtained from 3 clones (7A4, 4L17 and 29F20) NBS-LRR candidate disease resistance gene sequence. From these sequences, more specific molecular markers were developed, and based on the original molecular markers, a higher density genetic map of citrus nematode resistant hybrid progeny (9145family) was constructed. At the same time, newly developed molecules (9401family) to detect the genetic distance between Tyr1, a major gene locus in Citrus nematode resistance, and Ctv, a Citrus recessive disease resistance gene.