454a sequencing tag

Plant DNA was isolated from samples using DNeasy Plant Mini Kits (QIAGEN), with modifications to the manufacturer's protocol as described by Pettigrew et al. [12 ]. The microsatellite loci Ad01, Ad02, Ad06, Ad08, Ad09, Ad12, Ad13, Ad14, Ad15 and Ad18 [47 ] were amplified following the method of James et al. [48 ], modified for multiplexing polymerase chain reactions (PCRs) using the Type-It Microsatellite PCR Kit (QIAGEN). Amplification reactions contained a final concentration of 1x PCR Master Mix (QIAGEN), 0.075 μM each multiplexed forward primer appended to the 454A sequencing tag (Applied Biosystems, Foster City, CA, USA), 0.25 μM each reverse primer, 0.1 μM per multiplexed locus of 454A sequencing tag labelled with either 6-FAM, NED, HEX or PET (Applied Biosystems). Thermal cycling followed the instructions provided with the Type-It Kit. Specifically, initial heat activation of 5 min at 95°C was followed by 28 cycles of denaturation for 30 s at 95°C, annealing for 90 s at 60°C, and extension for 30 s at 72°C, with a final extension of 30 min at 60°C. Following PCR, amplifications using compatible dye types were diluted to equal concentrations and combined, then separated on an ABI 3730XL sequencer with a GS500LIZ size standard (Applied Biosystems) at Macrogen Inc. (Seoul, Korea). Allele sizes were scored using the Geneious microsatellite plugin v. 1.0.0 (Biomatters Ltd).