Agarose-based ecotilling: a rapid method for SNP detection in Oryza species

Ecotilling, a derivative of Tilling Reverse genetics tool that is potentially useful in allele mining to identify multiple types of polymorphisms in natural populations. It involves pooling of a standard reference variety with the sample of interest, amplification using OCR primers of selected candidate genes, heteroduplex formation and mismatch cleavage by CELL digestion, and electrophoresis to detect digestion products. Tilling exploits the property of CEL 1 endonuclease to cleave heteroduplexes at positions of single nucleotide or small indle mismatches. The common method used to detect cleaved products is by polyacrylamide gel electrophoresis using a high throughput genotyping platform. To simplify the technique and alleviate the need for labeled markers, a modified ecotilling procedure was developed at IRRI allowing the detection of digestion products on agarose gels. The modified procedures offers the advantages of lower cost and simplicity, making the technique accessible to laboratories with limited access to instrumentation. At IRRI the authors used agarose-based ecotilling to characterize diverse germplasm for drought candidate genes. Locus-specific primers were designed for the upstream regulatory and coding sequence regions using the Nipponbare genomic sequence and tested on a mini-core collection of 1536 O. sativa accessions, a set of 190 O. glaberrima accessions, and a panel of 95 AA genome wild Oryza accessions. Samples were contrasted against two reference lines, Nipponbare (japonica type) and IR64 (indica type). The agarose-based ecotilling method proved to be a fast and robust method to detect SNPs in both cultivated and wild Oryza germplasms. Mismatch sites were confirmed by sequencing samples representing particular mismatch patterns. Among the wild AA genome Oryza species , species-specific mismatch patterns were observed, suggesting that agarose-based ecotilling can also be potentially useful as a diagnostic tool in taxonomic analysis.