High resolution melting peaks and amplicon sequences from Category 2 candidate SNPs and the corresponding amplicon sequences between M. sativa subs. sativa var 'Chilean' (red) and M. sativa subsp. falcata var 'Wisfal' (gray). SNPs predicted by 454 sequencing are shown in blue and additional SNPs identified through amplicon sequencing are shown in black. A) Primers SNP MSSNPV072 confirms the presence of a single SNP at position 37 (Chilean = A/T, Wisfal = T) in a 92 bp amplicon. B) Primers SNP MSSNPV075 confirms SNP at position 94 (Chilean = G, Wisfal = A/G), and additional SNPs at position 42 (Chilean = A/C, Wisfal = A/C), and position 48 (Chilean = C/T, Wisfal = C/T), in a 115 bp amplicon. C) Validated candidate SNP MSCWSNP032 at position 85 (Chilean = G, Wisfal = A/G), and additional SNPs at positions 26 and 27 (Chilean = C/T and A/C, and Wisfal = C/T and A/C, respectively), and position 61 (Chilean = A/T and Wisfal = A), in a 130 bp amplicon). D) Non-validated candidate SNP MSCWSNP058 (Chilean = A and Wisfal = A) in a 169 bp amplicon.