Rapid and Sensitive Detection of Low-Level Mutations in a Mutagenized Petunia hybrida Population Using High-Resolution Melting

Genetic variation for plant improvement can come from a number of places; elite germplasm, gene banks and closely related species.  Variation can also be introduced through chemical mutagenesis creating alleles that may not be available through other sources.  Reverse genetics is the process of identifying a gene of interest and then identifying a plant within a mutagenized population for mutations in that gene.  A successful reverse genetics platform has three pillars; a densely mutagenzied population, methodology to identify genes and regions susceptible to mutagenesis, and an effective method of finding mutants in the population.  Here we describe using DNA High-Resolution Melting (HRM) to identify single base mutations in a pooled population of Petunia hybrida.  Arrayed in a 384-well format, we were able to interrogate over 2 million bases from 11,000 M2 plants, representing 2200 M2 families, in a single day at a cost of pennies per plant. The presence of mutations, either homozygous or heterozygous, was easily detected by HRM analysis.  Our results demonstrate that HRM is a fast, affordable and sensitive technique amenable to screening pooled mutagenized plant populations.