Rapid identification of maternal lineages in common carp (Cyprinus carpio L.) using real-time PCR and high resolution melt-curve analysis

This study presents a protocol for using real-time PCR and high-resolution melt-curve (HRMC) analysis to score polymorphisms in the mitochondrial DNA control region of common carp. This is the first time HRMC analysis has been used in an aquacultural species. The technique is accurate, robust and rapid to apply. It has a number of advantages over other existing techniques for rapidly scoring DNA polymorphisms, namely it is rapid, taking less than 3 h from start to finish; all procedures take place in closed PCR tubes, reducing the risk of contamination and human error; cycling conditions in the Rotorgene 6000 PCR machine used in the methodology are more homogenous than in traditional block-based PCR machines; and the progress and success of each individual PCR is monitored real-time. The primers were designed to score a greater number of polymorphic sites than in previous studies, and specifically target a section of the control region that is polymorphic amongst European carp races, which otherwise have very little mitochondrial DNA variation. The technique was used to accurately identify three common carp and one goldfish haplotype, with no haplotypes incorrectly identified. Although the method outlined here is optimised for scoring common carp mitochondrial haplotypes using the Rotorgene 6000 machine, real-time PCR and HRMC analysis can be applied in a similar way to almost any species and/or loci, with a number of different real-time PCR machines available for scoring genetic differences.