High-throughput microsatellite isolation through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries
DNA from 12 individuals, one male and one female from six different regions in the Iberian Peninsula was pooled to increase the detection of polymorphism. A total of 1 μg was used to develop a microsatellite library through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries, as described in Malausa et al. (2011). Briefly, total DNA was enriched for AG, AC, AAC, AAG, AGG, ACG, ACAT, and ATCT repeat motifs and subsequently amplified. PCR products were purified, quantified, and GS-FLX libraries were then constructed following the manufacturer’s protocols (Roche Diagnostics) and sequenced in a quarter of a run on a GsFLX-PTP