8th INTERNATIONAL WHEAT CONFERENCE

deTeCTIoN of dNA PoLymoRPhISmS ASSoCIATed
WITh AgRoNomIC TRAITS IN CommoN WheAT By hIgh
ReSoLuTIoN meLT ANALySIS
Lucio Lombardo, Leonardo Vanzetti, Marcelo Helguera
INTA EEA Marcos Juárez, Ruta 12 S/N, 2580 Marcos Juárez, Córdoba, Argentina.
E-mail Address of presenting author: mhelguera@mjuarez.inta.gov.ar
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 dsDNAspecific
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.
444