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.