Microsatellite Genotyping of Miscanthus

Root genomic DNA was also used for host plant genetic analysis. Sixteen previously described microsatellite loci [28 (link), 29 (link)] allowed observations of polymorphisms in M. sinensis and M. floridulus (Table S3). DNA was amplified by PCR cycling with an initial denaturation of 5 min at 95 °C, followed by 35 cycles of 1 min at 95 °C (denaturation), 1 min at a primer-specific annealing temperature, and 1 min at 72 °C (extension), with a final extension at 72 °C for 10 min. The reaction mixture (10 µl) contained 10 × reaction buffer (New England Biolabs, Beverly, MA), 2 mM MgSO₄, 0.125 µM dNTPs, 0.25 µM of each primer 0.5 U of Taq DNA polymerase (New England Biolabs) and 40 ng template DNA. Forward primers were then fluorescently labeled so that they could be used for automated genotyping. The PCR products were treated with poly(A) at 65 °C for 30 min, then diluted in ddH₂O if too concentrated and sized using the LIZ500 internal sizing standard on an ABI 3130xl automated DNA sequencer with GENEMAPPER V4.0 software (Applied Biosystems, Foster City, CA).