Identification Of SRAP Marker And Cloning Of Protease Inhibitor Ⅰ Gene Segment Associated With Bacterial Wilt Resistance In Diploid Potato

Bacterial wilt, caused by the soil-borne bacterial pathogen Rolstonia solanacearum, is one of the most devastating diseases and occurs mainly in tropical and subtropical areas, but also epidemics in warm and even in cool temperature regions. It affects more than 300 plant species distributed in about 44 families including many economically important crops such as potato, tomato, eggplant, and tobacco. At present, there are no resistant genes found in tetraploid potato cultivars, but some wild species or their cultivated relatives are known to be resistant to bacterial wilt and thus are potential sources for the resistance. Solanum phureja displays high degree of resistance to R. solanacearum and has been used as a major source of bacterial wilt resistance in potato breeding. However, the precise inheritance model of resistance remains unknown because of strong interactions among host, environment and the pathogen races. This reduces the efficiency of screening for resistance in field and slows down the development of new resistant cultivars. The development of molecular markers linked to the resistance gene would facilitate its introgression in the offsprings and direct selection of disease resistance without arduous field-testing. The previous research in our laboratory found a RAPD marker, S27, which is liked to bacterial wilt resistance locus and locates in about 1500bp upstream of TATA-box of a protease inhibitorⅠgene implying that the proteinase inhibitor may play an important role in potato defense against bacterial wilt.In present research, bacterial wilt resistance segregation analysis was performed by field disease resistance testing of the two diploid potato segregating populations, CE and ED, and the bulked segregant analysis was used to identify sequence related amplified polymorphism (SRAP) markers linked to bacterial wilt resistance. There were 88 primer combinations screened, of these, M32 was found to be linked to bacterial wilt resistance.In the ED population, the map distance between the marker and the resistance locus was 10.2cM while it was 17.3cM in the CE population. It was observed that the accuracy of the selection by M32 was 89.86% in the ED population and 83.33% in the CE population with average of 87.62 % in the two populations. Based on the known information of protease inhibitorⅠgene, we designed specific primers and clong this gene by specific PCR and TAIL-PCR methods. A gDNA segment of 2214bp with two extrons was obtained. Amino acid prediction indicated that this segment may encode 139 amino acids and its two ends of the sequence were highly homologous to two wild tomato and one potato protease inhibitors but there was no homology detected in the region between 45～80 amino acid residue, indicating the cloned segment is a new potato protease inhibitorⅠgene.