2.3 Screen for RFLP alleles
To screen for RFLP alleles, we extracted 801 bp flanking the target
variants (focal marker ± 400 bp) using samtools faidx from the A.
citrinellus reference genome (Kautt et al., 2020). Genotypes for all
loci were extracted from an in-house filtered statistically-phased
variant call format (vcf) file (Kautt et al., 2020) using tabix.
Nucleotide sequences and genotypes of variants (using vcfR (Knaus
et al., 2020)) were loaded into R (R Development Core Team, 2019). For
each locus, we generated a sequence with the alternative allele
(alternative sequence) – for this initial analysis we did not use
phased haplotypes. Next, we generated a list of the recognition sites of
237 commercially available restriction enzymes and performed in
silico restriction digests of reference and alternative sequences for
each locus. For each locus, we identified restriction enzymes cutting
one of the alleles but not the other one and generated a list of all
enzymes, including information on how many restriction sites could be
found within the 801 bp reference sequence. For every locus, we then
sorted the restriction sites by number of cutting sites within the 801
bp sequence.