Validation of variants using cost effective highresolution melting (HRM) analysis predicted from target re-sequencing in Eucalyptus

The advent of next-generation sequencing has facilitated large-scale discovery and mapping of genomic variants for high-throughput genotyping. Several research groups working in tree species are presently employing next generation sequencing (NGS) platforms for marker discovery, since it is a cost effective and time saving strategy. However, most trees lack a chromosome level genome map and validation of variants for downstream application becomes obligatory. The cost associated with identifying potential variants from the enormous amount of sequence data is a major limitation. In the present study, high resolution melting (HRM) analysis was optimized for rapid validation of single nucleotide polymorphisms (SNPs), insertions or deletions (InDels) and simple sequence repeats (SSRs) predicted from exome sequencing of parents and hybrids of Eucalyptus tereticornis Sm. ? Eucalyptus grandis Hill ex Maiden generated from controlled hybridization. The cost per data point was less than 0.5 USD, providing great flexibility in terms of cost and sensitivity, when compared to other validation methods. The sensitivity of this technology in variant detection can be extended to other applications including Bar-HRM for species authentication and TILLING for detection of mutants.