黄瓜棒孢叶斑病抗性基因cca-2的遗传分析与定位
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摘要
黄瓜棒孢叶斑病（cucumber target leaf spot），由多主棒孢霉[Corynesporacassiicola(Berk.&Curt.)Wei]引起，是我国近几年流行的一种新兴黄瓜叶部病害。目前国内外对该病的研究主要着重于识别与防治、生物学特性等方面，对其抗性基因的遗传规律及分子标记的研究很少，而且国内外报道的黄瓜棒孢叶斑病抗性基因的遗传规律还不一致。本研究以黄瓜野生变种PI183967(Cucumis sativus var. hardwickii)和新泰密刺选系931为亲本，对亲本及其后代群体F_1、F2、BC_1P_1、BC_1P_2于苗期进行悬浮液喷雾接种，调查统计分析黄瓜棒孢叶斑病抗性遗传规律；以F2为定位群体，利用SSR标记技术，通过集团分离分析法（Bulked SegregationAnalysis，BSA）和遗传图谱法进行基因定位，并对定位区间进行基因预测和验证，以便为黄瓜棒孢叶斑病的分子辅助选择（MAS）育种、抗性基因的精细定位和克隆提供理论依据。具体研究结果如下：
（1）通过对P_1、P_2、F_1、F_2、BC_1P_1、BC_1P_2的抗病性接种鉴定和抗性遗传分析，表明黄瓜棒孢叶斑病抗性基因由单隐性核基因控制。
（2）通过BSA法筛选得到64对SSR和1对EST-SSR引物，构建了一个连锁群，包含39个SSR标记和1个EST-SSR标记，将黄瓜棒孢叶斑病抗性基因cca-2定位于黄瓜第六染色体上，位于标记SSR10954和SSR16890之间，遗传距离分别是0.6cM和1cM。
（3）通过遗传图法，构建了一张遗传图谱，该图谱包含7个连锁群、99个SSR标记，覆盖基因组长度769.2cM，平均图距7.8cM。在第六染色体SSR17624处检测到一个QTL位点，贡献率10.9%，与BSA法定位的cca-2位置距离8.5cM。
（4）定位区间SSR21318和SSR18780之间的物理距离为1254.7kb，包含180个预测基因，其中21个为抗病相关基因。
（5）cca-2侧翼标记SSR10954和SSR16890在21份黄瓜材料中验证的正确率均为85.7%，为分子标记辅助筛选（MAS）提供了依据。
Cucumber target leaf spot, caused by Corynespora cassiicola(B.&C.)have resulted in increasinglosses in cucumber production in recent years．Thus far, research about cucumber target leaf spot isprimarily focused on identification, controlling methods and biological characteristics. Study oninheritance and molecular makers related to this resistant gene is much less. Furthermore, inheritancereported is inconsistent. In this study, wide cross PI183967×931and their progenys F_1、F_2、BC_1P_1、BC_1P_2are used for genetic analysis by inoculating conidia suspension in seedling stage. Simplesequence repeat（SSR）techonology, Bulked segregation analysis（BSA）and genetic map are employedto mapping cca-2gene of cucmber in F_2population. Moreover, candidate genes are predicted accordingthe cucumber genome sequence, and verification is conducted in21cucumber lines. The results arelisted as follow.
(1) The genetic analysis showed resistance to cucumber target leaf spot is controlled by a singlerecessive nuclear gene.
(2)64SSR and1ESR-SSR primers are obtained by screening from the BSA pools. A linkagegroup corresponding to chromosome6of cucumber is constructed, containning39SSR,1EST-SSRand gene cca-2. cca-2is mapped between SSR10954and SSR16890, with genetic distance0.6cM and1cM respectively.
(3) A cucumber genetic map is constructed, with99SSR markers in seven linkage groups spanning829.7cM, the average genetic distance is7.8cM. A QTL with10.9%variance explained is detected onsite SSR17624in chromosome6of cucumber.
(4)180candidate genes are predicted during1254.7kb cucumber genome sequence betweenSSR21318and SSR18780, of which21candidate genes are related to plant disease resistance.
(5) The accuracy rate of SSR10954and SSR16890verified in21cucumber lines are both85.7%，which facilitate marker-assisted selection(MAS) for cucumber breeding.
（1）通过对P_1、P_2、F_1、F_2、BC_1P_1、BC_1P_2的抗病性接种鉴定和抗性遗传分析，表明黄瓜棒孢叶斑病抗性基因由单隐性核基因控制。
（2）通过BSA法筛选得到64对SSR和1对EST-SSR引物，构建了一个连锁群，包含39个SSR标记和1个EST-SSR标记，将黄瓜棒孢叶斑病抗性基因cca-2定位于黄瓜第六染色体上，位于标记SSR10954和SSR16890之间，遗传距离分别是0.6cM和1cM。
（3）通过遗传图法，构建了一张遗传图谱，该图谱包含7个连锁群、99个SSR标记，覆盖基因组长度769.2cM，平均图距7.8cM。在第六染色体SSR17624处检测到一个QTL位点，贡献率10.9%，与BSA法定位的cca-2位置距离8.5cM。
（4）定位区间SSR21318和SSR18780之间的物理距离为1254.7kb，包含180个预测基因，其中21个为抗病相关基因。
（5）cca-2侧翼标记SSR10954和SSR16890在21份黄瓜材料中验证的正确率均为85.7%，为分子标记辅助筛选（MAS）提供了依据。
Cucumber target leaf spot, caused by Corynespora cassiicola(B.&C.)have resulted in increasinglosses in cucumber production in recent years．Thus far, research about cucumber target leaf spot isprimarily focused on identification, controlling methods and biological characteristics. Study oninheritance and molecular makers related to this resistant gene is much less. Furthermore, inheritancereported is inconsistent. In this study, wide cross PI183967×931and their progenys F_1、F_2、BC_1P_1、BC_1P_2are used for genetic analysis by inoculating conidia suspension in seedling stage. Simplesequence repeat（SSR）techonology, Bulked segregation analysis（BSA）and genetic map are employedto mapping cca-2gene of cucmber in F_2population. Moreover, candidate genes are predicted accordingthe cucumber genome sequence, and verification is conducted in21cucumber lines. The results arelisted as follow.
(1) The genetic analysis showed resistance to cucumber target leaf spot is controlled by a singlerecessive nuclear gene.
(2)64SSR and1ESR-SSR primers are obtained by screening from the BSA pools. A linkagegroup corresponding to chromosome6of cucumber is constructed, containning39SSR,1EST-SSRand gene cca-2. cca-2is mapped between SSR10954and SSR16890, with genetic distance0.6cM and1cM respectively.
(3) A cucumber genetic map is constructed, with99SSR markers in seven linkage groups spanning829.7cM, the average genetic distance is7.8cM. A QTL with10.9%variance explained is detected onsite SSR17624in chromosome6of cucumber.
(4)180candidate genes are predicted during1254.7kb cucumber genome sequence betweenSSR21318and SSR18780, of which21candidate genes are related to plant disease resistance.
(5) The accuracy rate of SSR10954and SSR16890verified in21cucumber lines are both85.7%，which facilitate marker-assisted selection(MAS) for cucumber breeding.
引文
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