EcoTILLING stress tolerance genes in cultivated and wild rice

EcoTILLING is the application of TILLING (Targeted Local Lesions IN genomes) for the identification of variation in natural populations. At IRRI, the authors used EcoTILLING to characterize diverse cultivated and wild germplasm at candidate genes involved in stress tolerance. Target loci include drought response binding protein 1 (DREB 1), trehalose6-phosphatase (TPP) for drought tolerance and 14-3-3 protein (CG18) for blast tolerance. Locus-specific primers were designed for upstream regularity and coding sequence regions using the Nipponbare genomic sequence. The loci were tested on a panel of cultivated Oryza sativa or wild Oryza species, contrasted against two reference lines, Nipponbare (japonica type) and IR64 (indica type). Screening 96 O. sativa accessions for the coding region of the TPP locus on CH2 and 109 CM (RGP map) revealed 2 putative SNPs in Kunmim Tsieh Hunan and Mudgo by either IR64 or Nipponbare. For the DREB1 locus at 16.1 cM, mismatches for indica/japonica contrasts were detected in both coding and upstream regions. For the DREB1 locus at 136.6 cM, 20 putative mismatches were detected among 400 varieties. For the wild species, amplicons were generally obtained at all test loci in the AA genome species. In addition, amplicons were also obtained from the genomes BBCC and CCDD using the TPP coding primers; from BB, CC, BBCC, and CCDD at the DREB 1 locus at 136.6 cM; and in all genomes except FF and GG at the cg18 locus. Contrasts at these amplicons to either IR64 or Nipponbare revealed a number of mismatches. A sampling of these and other putative SNPs are being sequenced to confirm the mismatches and establish the identity of the nucleotide difference(s). These results indicate the utility of EcoTILLING for identification of SNPs/haplotypes across both cultivated and wild Oryza, demonstrating the use of EcoTILLING to detect inter-specific differences.