Alternative ecotilling protocol for rapid, cost-effective single-nucleotide polymorphism discovery and genotyping in rice (Oryza sativa L.)

Ecotilling is a high-throughput method of discovery and analysis of single-nucleotide polymorphism (SNP) variations in natural populations, but it requires a substantial investment in sophisticated equipment, costly reagents, and specialized software programs and implementation of several time-consuming steps that limit its use in laboratories with modest financial resources. Moreover, labeling efficiency of PCR primers with fluorescent dyes during Ecotilling can be reduced by unwanted exonuclease activity of single strand-specific nucleases. A new alternative protocol involving a simplified gel system, unlabeled primers, DNA staining after single strand-specific nuclease digestion, and standard gel data analysis was optimized to address these constraints. Using this alternative protocol, we successfully identified four new SNPs verified by sequencing in a collection of 57 diverse rice accessions along with 2 previously reported SNPs in a 922-bp DNA region from thealk gene. An SNP cluster containing a deletion within a 472-bp fragment of thewaxy gene was also characterized. In addition, 4 previously reported SNPs in thealk andwaxy genes were faithfully genotyped among the 57 accessions based on comparisons with sequencing results. Associations between the genotyped SNPs and amylose class and starch gelatinization temperature were as anticipated. These results, along with detailed time and cost comparisons between the 2 methods, suggest that alternative Ecotilling is a simple and reproducible method for SNP discovery and genotyping in rice that leads to substantial savings in equipment, reagents, software, and time compared with the standard Ecotilling procedure.