During second visit (9.11.-5.12.2014) at Max-Plank Institute of Evolutionary Biology in Ploen, Oleksiy Yanchukov focused on obtaining the CNV (gene copy-number variation) genotypes for 252 individual samples of the house mouse DNA. I used the fluorescent probe assays to simultaneously estimate the amount of template DNA for a single-copy reference gene, Tert, and the target gene with unknown copy number. The genotypes for three genes, Smok2a, Cma1 and Retnlb, were scored for the whole set of samples. A few additional experiments were performed using the indiscriminate DNA dye, Eva-Green, to directly compare with the probe-based results. I found that using Eva-Green method might be suitable for the initial optimisation of the digital PCR for specific genes, but for the bulk processing of hundreds of samples, the probe-based method is the most optimal method.
The CNV results for the three genes were quite meaningful: the Smok2a and Cma1 genes show clear stepped pattern of geographical variation across the hybrid zone, with populations of Mus m. musculus demonstrating higher copy number on average. The Retnlb gene is uniformly diploid (copy number =2) in both subspecies, but the samples close to the center of the hybrid zone show elevated copy number (~3-4). A third visit to Ploen is planned in 2015, to obtain the CNV data for several more candidate genes --- we expect to publish the combined results from 2014-15 towards the end of 2015.