Detection of DNA polymorphisms associated with agronomic traits in common wheat by high resolution melt analysis

LOMBARDO L.; VANZETTI L.; HELGUERA M.

San Petesburgo. Rusia

Conferencia; 8 International Wheat Conference; 2010

The complex genetics of common wheat (Triticum aestivum L 6x AABBDD) is a conditioning factor in the development of molecular markers that permit the precise detection of DNA polymorphisms defining superior alleles in genes/loci associated with agronomic traits allowing their use in breeding programs. Homeoalleles coamplification is a frequent scenario in common wheat, and, in these cases allele-specific polymorphisms can be detected by digestion with restriction enzymes followed by DNA electrophoresis (SNPs and/or InDels) or by differential migration of DNA fragments in agarose or polyacrylamide gels (InDels). An alternative strategy allowing the identification of different types of DNA polymorphisms relies on the detection of changes in the dissociation (melting) behavior of a double-stranded DNA fragment associated with polymorphisms as small as a single SNP. In a high resolution melt (HRM) analysis a DNA fragment is gradually heated from low (70-80°C) to high (90-95°C) temperatures in presence of the dsDNA-specific dye EvaGreen®, being the sample light excited with a green channel at 460nm. In the melt process a lightscaner will record the fluorescence of the sample until its loss of signal associated with dsDNA dissociation. In this study we used HRM to detect SNPs and/or InDels using a set of PCR markers associated with important agronomic traits in common wheat like leaf rust resistance (Lr47), vernalization (Vrn-A1), photoperiod response (Ppd-A1), plant height (Rht-8) and grain texture (pinb-D1) genes. Even though differences as small as 1 SNP in 84 bp were detected (pinb-D1), best results were observed when discriminating alleles carrying a higher ammount of SNPs or InDels differences (Ppd-A1, Lr47). In some cases (Ppd-A1), nested PCR was a useful strategy to improve homeoallele discrimination. Our data positionate HRM as a simple and efficient tool for screening of DNA polymorphisms in common wheat populations for different purposes like breeding, mapping, ECO TILLING, TILLING and others.