Authors: Chitra Raghavan Ma Elizabeth B Naredo Hehe Wang Genelou Atienza Bin Liu Fulin Qiu Kenneth L McNally Hei Leung
Publish Date: 2006/11/17
Volume: 19, Issue: 2, Pages: 87-101
Abstract
TILLING Targeting Induced Local Lesions IN Genomes exploits the fact that CEL I endonuclease cleaves heteroduplexes at positions of single nucleotide or small indel mismatches To detect single nucleotide polymorphisms SNPs across a population DNA pools are created and a target locus under query is PCRamplified and subjected to heteroduplex formation followed by CEL I cleavage Currently the common method used to detect cleaved products is by polyacrylamide gel electrophoresis using a highthroughput genotyping platform Exact SNPs are then determined by sequencing We sought to simplify the detection of CEL Icleaved products on conventional agarose gels to make the technique more accessible to collaborating partners in developing countries where access to instrumentation could be limiting Here we used a panel of stressrelated genes to evaluate SNP detection across 48 rice genotypes by contrasting them individually against IR64 and Nipponbare SNP detection calls corresponded perfectly with those obtained from the LiCor genotypers We were able to detect SNPs in pools of eight DNA templates suggesting that the agarose gel system could be used to screen for SNPs with comparable throughput as that of the LiCor genotypers and showed that the throughput can be increased by analyzing larger amplicons ∼3 kb The agarose method offers a significant advantage by alleviating the need for labeled primers We further demonstrated that the agarose method can be effectively used in gene mapping an application particularly useful for parental lines with low levels of polymorphism The lower cost and simplicity of the technique make it possible for broader applications of SNPbased markers for germplasm characterization and mapping studiesThis work was supported in part by grants from the Generation Challenge Program Rockefeller Foundation Bundesministerium für wirschaftliche Zusammenarbeit und Entwicklung BMZ Swiss Agency for Development and Cooperation and USDANRI We thank L Comai University of Washington and S Henikoff Fred Hutchinson Cancer Research Center for their help in establishing the TILLING/EcoTILLING procedures at IRRI
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