Application of Simplified Ecotype Targeting Induced Local Lesions in Genomes(EcoTILLING) Technique for Genotyping and Single Nucleotide Polymorphism(SNP) Detection in Rice
Abstract：Ecotype targeting induced local lesions in genomes (EcoTILLING) is a high-throughput method of detection and analysis of single nucleotide polymorphism (SNP) variations in natural populations. However, it requires a substantial investment in sophisticated equipment and costly fluorescence labeled primers to detect CELⅠ- cut products that limit its use in laboratories with common financial resources. The technology of traditional EcoTILLING had been appropriately improved in this study. Here, we simplified extraction procedure of the CELⅠ enzyme, prepared heteroduplex DNA products for CELⅠdigestion by use of natural cooling method, and detected the CEL I-cleaved products through general silver staining. The results of activities of crude CELⅠenzyme extracts showed that the activity of CELⅠwas high enough for detection of mismatch bases. Using the simplified EcoTILLING technique, the G/T polymorphism at the first intron 5' splice site of the Wx gene among 18 rice(Oryza sativa L.) accessions were successfully genotyped. In addition, CELⅠdigested bands of a 417 bp fragment within the Wx gene of the 18 rice accessions were distinguished into several types. On comparisons with sequencing results, CELⅠcut bands could accurately reflect the SNP variations between the materials. The results suggested that the simplified EcoTILLING is an effective method for SNP discovery and genotyping in rice that will provide powerful tool for rice functional genomics research.
