DHPLC Technology As a High-Throughput Detection of Mutations in a Durum Wheat Tilling Population

Durum wheat (Triticum turgidum ssp. durum) has a tetraploid genome (AABB genome) with genes in two homoeologous copies, a high proportion of repetitive DNA regions and a low gene density. Current tools for the discovery of sequence alterations in target genes rely on reverse genetic approaches such as the TILLING technique. TILLING is based on the detection of chemical induced mutations in heteroduplexes using endonucleases (CelI enzyme) or by DNA melting temperature. In the present work we present a modified approach for SNP detection based on melting temperature relived with a  Denaturing High Performance Liquid Chromatography (DHPLC) (Transgenomic, Omaha, USA). The screening regarded more than 1,200 M₂ mutant lines derived from durum wheat MarcoAurelio cultivar and pooled in groups of four DNAs. The M₂ lines were analyzed for four carotenoid genes (Lcy-ε and Lcy-β genes in A and B genome). We identify over 57 novel alleles in the four targeted genes, revealing a high mutation density of the TILLING population (~1/79 kb). In addition we compared the screening results of the use of Surveyor nuclease (CelI enzyme) on agarose gel and the sensitivity of DHPLC approach in order to identify the most rapid, sensitive and low cost method to detect mutations. The DHPLC technology will offer an alternative tool to current strategies of SNP detection based on genotyping array.