PCR primers were designed according to Harry et al. (1998) and Temesgen et al.(2000). Briefly, all primer sets were chosen to operate under a single thermal cycling regime which included a hot start and touchdown stages. In many cases, the “melting profile” of the amplification product predicted by the EST sequence was observed using MacMelt software (BioRad). This step allowed the effects of both primer position and GC clamping on melting profile to be examined and was used to optimize amplfication products for denaturing gradient gel electrophoresis.