There were dropouts from the initial genotyping, so fresh aliquots of DNA from those individuals were analyzed using a different primer pair, HE3236/HE3244 (Table 1), which yielded a 336 bp band (without indel) or a 1166 bp band with the indel; we reasoned that reducing the size of the fragments should make the assay more robust to potential DNA degradation. The final reaction contained 1× R-Taq Master Mix (Bulleye R-Taq DNA pol 2.0× Mix, MidWest Scientific, St Louis, MO, USA), 0.375 µM primer in 20 µl total volume with 15 ng genomic DNA. PCR reactions were carried out in GeneAmp PCR system 9700 (Applied Biosystems) using the following conditions: 95°C for 3 min for initial DNA denaturation, then 35 cycles of 94°C for 20 s, 62°C for 15 s, 72°C for 1 min 30 s, final extension at 72°C for 7 min. Aliquots (6 µl) of PCR product were separated on 2% agarose gels (E-Gel 96, Invitrogen) and independently read by three individuals. Only unambiguous data were used. Results of the genotyping were tested for Mendelian inheritance using the program PEDCHECK (75). Marker allele frequency and heterozygosity were computed using the program USERM13 (76).
