兰考906抗白粉病新基因的分子标记筛选
摘要
小麦兰考906系列品系是我省培育的抗白粉病新种质。遗传分析表明,兰考906携带1个小种专化的隐性抗白粉病新基因,暂标记为PmLK906。为了深入研究和有效利用兰考906的抗白粉病基因,用SSR分子标记技术和基因芯片技术筛选了与PmLK906紧密连锁的分子标记。通过中国春缺体-四体系和缺失系将该基因定位在了小麦染色体2AL上。研究结果可直接用于分子标记辅助育种,并为通过图位克隆技术克隆抗病基因PmLK906奠定了基础。本研究的具体研究内容及结果如下:
1、以感病品种中国春、豫麦21、濮9等为母本,以兰考906为父本配制了抗、感杂交组合。用离体叶段培养法对F_1、F_2代分离群体、F_(2:3)家系分离群体进行了苗期抗病性鉴定,结果表明兰考906品系携带1对小种专化的隐性抗白粉病基因。
2、以中国春/兰考906杂交F_(2:3)家系为材料,用“集团分离分析法(BSA)”建立抗病池和感病池,筛选与抗病基因连锁的SSR标记。共筛选了150对SSR引物,其中2对引物gwm265和gdm93在抗、感亲本和抗、感DNA池之间具有稳定的多态性。
3、将2对引物在由129个家系组成的中国春/兰考906杂交F_(2:3)家系分离群体中进行连锁分析验证,结果表明gwm265和gdm93的扩增产物与PmLK906基因连锁,遗传距离分别为3.72和6.15 cM,该标记被记为Xgwm265和Xgdm93。
4、利用中国春缺体-四体系和缺失系将标记Xgwm265定位在了小麦染色体2AL上,从而将PmLK906定位于2AL上。
5、以中国春/兰考906杂交F_(2:3)家系为材料,用BSA法建立抗病和感病cDNA池。以抗、感cDNA池为探针筛选小麦基因芯片(Affymetrix产品),获得了与PmLK906相关的ESTs。在此基础上克隆了一个与粗山羊草csAtPR5类似的基因,命名为TaAetPR5。
6、在中国春/兰考906杂交F_(2:3)家系分离群体中进行连锁分析验证,结果表明TaAetPR5基因与PmLK906基因连锁,遗传距离7.62 cM。在Chancellor/CI 14124杂交分离群体中进行连锁分析证明,TaAetPR5基因与Pm4a基因紧密连锁,遗传距离为0 cM。
Wheat Lankao906 lines are new powdery mildew resistance germplasms selected in Henan province. Genetic analysis showed that Lankao906 carry a recessive powdery mildew resistance gene temporarily named PmLK906. To further study and use PmLK906, SSR technique and gene chip hybridization combined with bulked segregant analysis (BSA) was used to develop molecular markers linked to PmLK906. PmLK906 was located in chromosome 2AL by Chinese Spring nulli-tetrasomic and 2AL-deletion lines. The results can be directly used in molecular marker assistant selection, and provide a base for map based cloning of PmLK906. The detail studies are listed below:
1. Crosses of powdery mildew resistant and susceptible wheat lines were made between Chinese Spring, Yumai21,20086 and Lankao906. Seedling powdery mildew assessments of the F_1, F_2 and F_(2:3) lines were carried out by detached leaf culture method. The results showed that Lankao906 carry a recessive powdery mildew resistance gene.
2. The powdery mildew resistant and susceptible DNA bulks were constructed by BSA with the F_(2:3) families of the cross Chinese Spring / Lankao906. Total 150 pairs of SSR primers were screened, and 2 pairs primers gwm265 and gdm93 showed stable polymorphisms between the two parents and the contrasting DNA bulks.
3. Linkage analysis was carried out in a segregation population including 129 F_(2:3) lines of the cross of Chinese Spring / Lankao906. The result showed that the products of gwm265 and gdm93 were linked to PmLK906 with genetic distances of 3.72 and 6.15 cM respectively. The two SSR markers were named Xgwm265 and Xgdm93.
4. Xgwm265 was located in wheat chromosome 2AL by Chinese Spring nulli-tetrasomic and 2AL-deletion lines. Consequently PmLK906 was located in chromosome 2AL too.
5. Powdery mildew resistant and susceptible cDNA bulks were constructed by BSA with the F_(2:3) lines of the cross Chinese Spring / Lankao906. The Affymetrix? wheat genome array was hybridized using cDNAs from resistant and susceptible bulks as the probes. Several tens of ESTs related to PmLK906 were obtained. Later, a new wheat gene similar to csAtPR5 was isolated from Lankao906, and named TaAetPR5.
6. Linkage analysis in the segregation population of the cross of Chinese Spring / Lankao906 showed that TaAetPR5 linked to PmLK906 with a genetic distance of 7.62 cM. Linkage analysis in the segregation population of the cross of Chancellor / CI 14124 showed that TaAetPR5 linked to Pm4a with a genetic distance of 0 cM.
1、以感病品种中国春、豫麦21、濮9等为母本,以兰考906为父本配制了抗、感杂交组合。用离体叶段培养法对F_1、F_2代分离群体、F_(2:3)家系分离群体进行了苗期抗病性鉴定,结果表明兰考906品系携带1对小种专化的隐性抗白粉病基因。
2、以中国春/兰考906杂交F_(2:3)家系为材料,用“集团分离分析法(BSA)”建立抗病池和感病池,筛选与抗病基因连锁的SSR标记。共筛选了150对SSR引物,其中2对引物gwm265和gdm93在抗、感亲本和抗、感DNA池之间具有稳定的多态性。
3、将2对引物在由129个家系组成的中国春/兰考906杂交F_(2:3)家系分离群体中进行连锁分析验证,结果表明gwm265和gdm93的扩增产物与PmLK906基因连锁,遗传距离分别为3.72和6.15 cM,该标记被记为Xgwm265和Xgdm93。
4、利用中国春缺体-四体系和缺失系将标记Xgwm265定位在了小麦染色体2AL上,从而将PmLK906定位于2AL上。
5、以中国春/兰考906杂交F_(2:3)家系为材料,用BSA法建立抗病和感病cDNA池。以抗、感cDNA池为探针筛选小麦基因芯片(Affymetrix产品),获得了与PmLK906相关的ESTs。在此基础上克隆了一个与粗山羊草csAtPR5类似的基因,命名为TaAetPR5。
6、在中国春/兰考906杂交F_(2:3)家系分离群体中进行连锁分析验证,结果表明TaAetPR5基因与PmLK906基因连锁,遗传距离7.62 cM。在Chancellor/CI 14124杂交分离群体中进行连锁分析证明,TaAetPR5基因与Pm4a基因紧密连锁,遗传距离为0 cM。
Wheat Lankao906 lines are new powdery mildew resistance germplasms selected in Henan province. Genetic analysis showed that Lankao906 carry a recessive powdery mildew resistance gene temporarily named PmLK906. To further study and use PmLK906, SSR technique and gene chip hybridization combined with bulked segregant analysis (BSA) was used to develop molecular markers linked to PmLK906. PmLK906 was located in chromosome 2AL by Chinese Spring nulli-tetrasomic and 2AL-deletion lines. The results can be directly used in molecular marker assistant selection, and provide a base for map based cloning of PmLK906. The detail studies are listed below:
1. Crosses of powdery mildew resistant and susceptible wheat lines were made between Chinese Spring, Yumai21,20086 and Lankao906. Seedling powdery mildew assessments of the F_1, F_2 and F_(2:3) lines were carried out by detached leaf culture method. The results showed that Lankao906 carry a recessive powdery mildew resistance gene.
2. The powdery mildew resistant and susceptible DNA bulks were constructed by BSA with the F_(2:3) families of the cross Chinese Spring / Lankao906. Total 150 pairs of SSR primers were screened, and 2 pairs primers gwm265 and gdm93 showed stable polymorphisms between the two parents and the contrasting DNA bulks.
3. Linkage analysis was carried out in a segregation population including 129 F_(2:3) lines of the cross of Chinese Spring / Lankao906. The result showed that the products of gwm265 and gdm93 were linked to PmLK906 with genetic distances of 3.72 and 6.15 cM respectively. The two SSR markers were named Xgwm265 and Xgdm93.
4. Xgwm265 was located in wheat chromosome 2AL by Chinese Spring nulli-tetrasomic and 2AL-deletion lines. Consequently PmLK906 was located in chromosome 2AL too.
5. Powdery mildew resistant and susceptible cDNA bulks were constructed by BSA with the F_(2:3) lines of the cross Chinese Spring / Lankao906. The Affymetrix? wheat genome array was hybridized using cDNAs from resistant and susceptible bulks as the probes. Several tens of ESTs related to PmLK906 were obtained. Later, a new wheat gene similar to csAtPR5 was isolated from Lankao906, and named TaAetPR5.
6. Linkage analysis in the segregation population of the cross of Chinese Spring / Lankao906 showed that TaAetPR5 linked to PmLK906 with a genetic distance of 7.62 cM. Linkage analysis in the segregation population of the cross of Chancellor / CI 14124 showed that TaAetPR5 linked to Pm4a with a genetic distance of 0 cM.
引文
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