EcoTILLING candidate genes for drought tolerance in rice

摘要

EcoTILLING of diverse germplasm allows the discovery of SNPs and the delineation of haplotypes at loci of interest (Comai et al., 2004). This technique relies on the enzymatic cleavage of heteroduplex molecules formed between reference and query linesby an SI type single-strand endonuclease from celery, CELL Dual-labeling of the PCR amplicons by different fluorescent tags allows the detection of the cleavage products on denaturing PAGE with automated genotyping. The appearance of new bands against the background products allows the detection of single nucleotide polymorphisms. Furthermore, the banding patterns across a range of diverse germplasm can be grouped into haplotypes. This combination of haplotyping and identifying SNP loci reduces the cost associated with SNP discovery so that only those germplasm carrying SNP differences need be sequenced in order to establish the identity of the nucleotide difference. As such, EcoTILLING is a powerful, inexpensive tool for the detection of natural variation.