2.5 Primer design
Primers were designed using Primer3 (Sequences are summarized in Table
S1). Briefly, we used the 801 bp sequence and designed the primers
asymmetrically around the target SNP, i.e., that approximately one third
(or two thirds) of the amplicon is 5’ of the target SNP to avoid that
the fragments have the same size after digestion. If the previous
analysis indicated additional restriction sites in the 801 bp sequence
we confirmed that the restriction sites are either outside of the
amplicon, or at least do not complicate the detection of the RFLP (e.g.,
for the Lake Managua marker Chr3:33,260,056 the alleles resulted in 402,
106, and 50 bps or 448 and 106 bps long fragments).