P0178 LightScannerŪ High Resolution DNA Melting Analysis is a Novel Technology to Genotype Molecular Markers Conferring Disease Resistance in Commercially Valuable Crops

Matthew Poulson , Idaho Technology Inc., Salt Lake City, UT
Sukhi Pannu , California Seed & Plant Lab., Inc., Elverta, CA
Cameron Gundry , Idaho Technology, Salt Lake City, UT
High resolution melting (HRM) is set of widely-used genetic screening tools enabling investigators to rapidly screen or scan samples for the detection of for any sequence variants before proceeding to sequencing. In addition, HRM provides straightforward genotyping of known mutations such as SNPs at a medium to high throughput scale. LunaProbes® (unlabeled probe) assays were designed to genotype a variety of SNPs in crops:  the Tm2 locus for tomato mosaic virus resistance, the Ve1 locus for verticilium wilt disease resistance, the Mi locus for nematode resistance in Solanum lycopersicum (tomato), and the cor locus for corky root resistance in Lactuca sativa (lettuce). LCGreen® Plus-containing LightScanner Master Mix was used in this study. After PCR the 96 or 384 well plate was transferred without sample manipulation to the LightScanner for a simple 5 minute melt protocol, preventing contamination in subsequent reactions. Each Lunaprobe assay was challenged by testing at least 50 samples of DNA isolated from resistant, susceptible and heterozygous samples which had previously been genotyped using agarose gel electrophoresis.  In each case all assays could differentiate the resistant, susceptible, and heterozygous samples and showed 100% concordance with the current genotyping practices.  An advantage of HRM over agarose gel electrophoresis for mutation genotyping is that HRM does not require post PCR manipulation of the sample. In this study HRM proved to be a fast, accurate, and cost effective genotyping method that can be used to identify disease resistance and cultivar identification.