Genotyping and Mutation Scanning by High Resolution Melting (HRM) Analysis of Citrus EST-SNPs and SSRs

Citrus taxonomy is very complex mainly due to specific aspects of its reproductive biology. A number of studies have been performed using various molecular markers in order to evaluate the level of genetic variability in citrus. SNP markers were used for genetic diversity assessment using different methods; recently, the availability of EST database and whole genome sequences increased the information on sequence variability in citrus leading to wider employment of these markers so that reliable SNP detection methods are needed. In the present study, for the first time we applied high resolution melting curve analysis (HRM) in citrus to detect the presence of SNPs polymorphisms and their involvement in functional and structural genomics. In citrus, the possibility of characterizing genetic diversity was drastically increased using this method since it allowed analyzing more polymorphisms in a single PCR fragment. Furthermore, we identified SNPs associated with gene coding regions which revealed some aspects of their functionality in biochemical variations. The results suggested that HRM is an efficient, cost-effective and accurate method to detect sequence variations. In fact, HRM analysis provided a panel of primers for HRM genotyping in citrus representing also a useful tool for bridging functional and structural genomics.