Phylogenetic Analysis Of Disease Resistance Gene Analogs And Cloning Of Late Blight Resistance Gene In Potato

Potato (Solanum tuberosum. L) is one of the most important crops in the world ranking fourth after rice, wheat and maize, and plays a significant role in economy. One of the most destructive diseases of potato is the late blight, which can lead to severe yield decrease. Traditionally, people combated the late blight of potato by fungicides and conventional breeding. With the development of Genetic Engineering, a lot of R genes in wild sources have been isolated and subsequent transformation of theses genes into potato cultivars could be a fast way to obtain disease resistance cultivars. In this study, we employ the RGA method combined with RACE technology to clone the late blight resistance gene of potato, the results showed below:(1) Isolation and Phylogenetic Analysis of Potato Resistance Gene Analogs (RGAs)We choose the classical late blight resistant species Solanum dimissum as plant material, degenerated oligonucleotide primers were designed according to the two conservative motifs: P-loop and GLPL sequences of previously reported resistance genes (R3a, R1, Rpi-blb1, Rpi-blb2, I2, I2C-1 etc.), as a result, 30 sequences with Open Reading Frame (ORF) were acquired from the cDNA of S.demissum, which includes the NBS conservative motifs. Sequences alignment and phylogenetic analysis were performed by bioinformatics softwares, the results indicated that 30 sequences with ORF were divided into 9 types based on the 90% nucleotide similarity. All of them belong to nonTIR-NBS-LRR resistance gene analogs (RGAs).(2) Identification of Candidate sequences of Late Blight resistanceNine specific primers were designed based on the specific domain of these nine RGAs, respectively, PCR and RT-PCR identification were performed in S.demissum and six domestic cultivars (Long 3, Nileke, Atlantic, Zihuabai, MC-4, Favorita), results showed that groupâ…£appeared uniquely in S.demissum, as well as groupâ…¥in S.demissum and another cultivar Atlantic, while all the other seven groups were present in all the samples tested, which indicated that the groupâ…£and groupâ…¥might highly relate to the late blight resistance (Candidate Genes).(3) Cloning of Late Blight Resistance GeneWe cloned the whole sequence of Groupâ…£RGA (PB6-22) by RACE technology, this gene was named Sdpa, with coding sequence is 3570 bp. The deducted open reading frame (ORF) of the Sdpa gene encodes a predicted polypeptide of 1189 amino acid. The gene includes the conservative motifs of R genes (P-loop, Kinase2, Kinase-3a, GLPL etc.), and at least eight LRR motifs were identified in this protein. This resistance protein shares sequence homology of 66.24% with reported potato late blight resistance protein R3a. Our future studies will focus on expressing this disease resistance gene for functional analysis.