稻瘟菌无毒基因簇的RAPD标记及其SCAR转化

为克隆稻瘟菌无毒基因簇Avr-Pi1、Avr-Pi2和Avr-Pi4a,以RAPD方法对稻瘟菌(Magnaporthe grisea）菌株FJ81278ZB15、GUY11及其F1后代群体进行扩增，得到2个与该无毒基因簇连锁的分子标记P1414700和P1389420 。对2个特异片段进行克隆和测序，并根据序列分别设计2对特异引物，对菌株FJ81278ZB15、GUY11及其F1后代群体进行扩增，扩增结果P1414700 成功转化为SCAR标记SC1414，而P1389420未能成功转化。对分子标记SC1414、P1389420和报道的RPF1.2与无毒基因簇Avr-Pi1、Avr-Pi2和Avr-Pi4a遗传连锁关系进行了分析，结果SC1414、RPF1.2、P1389420与Avr-Pi2的遗传距离分别为5.8、2.2 和4.4 cM；与Avr-Pi1的遗传距离分别为15.9、7.9和5.7 cM；与Avr-Pi4a的遗传距离分别为20.7、12.7 和10.5 cM。

SCAR Conversion of Two RAPD Markers Linked to An Avirulence Gene Cluster in Magnaporthe grisea

To clone and characterize the avirulence gene cluster of Avr-Pi1, Avr-Pi2 and Avr-Pi4a, genomic DNAs of Magnaporthe grisea strains FJ81278ZB15, GUY11 and their F1 progenies were isolated and used as templates to screen for tightly linked random amplified polymorphic DNA(RAPD) markers . Two RAPD markers, P1414700 and P1389420 were identified using bulk segregant analysis, then cloned and sequenced. To overcome difficulty in reproducibility of RAPD markers, conversion of sequence characterised amplified region(SCAR) was further conducted. Two pairs of primers were designed based on sequences of P1414700 and P1389420 and their amplification polymorphism was validated with templates from FJ81278ZB15, GUY11 and their F1 progenies. The PCR results indicated that P1414700 was converted into a SCAR marker SC1414 successfully, but not for P1389420 . The genetic distances of molecular markers, including SC1414, P1389420, RPF1.2 and the avirulence gene cluster of Avr-Pi1,Avr-Pi2 and Avr-Pi4a, were then calculated by using program Mapmaker. The genetic distances were 5.8, 2.2 and 4.4 cM respectively from SC1414, RPF1.2 and P1389420 to Avr-Pi2; 15.9,7.9 and 5.7 cM to Avr-Pi1; 20.7, 12.7 and 10.5 cM to Avr-Pi4a. These markers will facilitate the map-based cloning of the avirulence gene cluster.