not covered. Forward and reverse primers incorporated the -21M13F (TGTAAAACGACGGCCAGT) or M13R (CAGGAAACAGCTATGAC) extensions, respectively, at their 5′ ends. PCR, sequencing reactions and sequence analysis procedures were carried out as described previously [47]. Briefly, PCR reactions were optimized for each individual primer pair using genomic test DNA and a temperature gradient (48–65°C). Standard PCR conditions were 15 s annealing time, 30 s extension time, and 35 cycles. A standard 10 ul (optimization) or 20 ul (sample) PCR reaction mix contained 1 mM MgSO4, 0.2 mM dNTPs, 0.5 uM of each primer, 0.0125 units Platinum Pfx Polymerase, 1× Enhancer solution, 1× Pfx amplification buffer (all from Invitrogen, CA, USA) and 10 ng genomic DNA. Primer pairs showing no product or high background amplification were re-tested at slightly different conditions (annealing time: 5 s–1 min; extension time: 10 s–1 min) or re-designed if necessary (∼5% of primers). The majority of primers worked at 60°C (±3°C). All PCR products, optimizations as well as sample PCRs, were checked on 2% agarose gels (SeaKem LE, Cambrex, ME, USA). For cycle sequencing, we used Big Dye Terminator Mix v3.1 (Applied Biosystems, Foster City, CA) at 0.33 µl of mix per reaction in a total volume of
