In silico selection of microsatellite loci
We screened 269 human microsatellite loci widely used in catarrhine
population genetic studies. We extracted the human (GRCh38/hg38)
sequence of each locus with 500 bp flanking regions from GenBank
(https://www.ncbi.nlm.nih.gov/genbank/) and performed BLAT
searches against the 16 available (status: 5 December 2018) non-human
Catarrhini reference genomes (Table S1) using the UCSC
(genome.ucsc.edu) or Ensembl
(ensembl.org) genome browsers with
standard settings. In addition, we checked the human sequence for
repetitive elements (SINEs, LINES, etc.) in flanking regions using the
RepeatMasker Web Server (http://www.repeatmasker.org/) with
standard settings. We generated alignments for each locus containing the
16 non-human catarrhine species, the human and the human repeat-masked
sequences with Muscle 3.8.31 (Edgar, 2004) in SeaView 4 (Gouy, Guindon,
& Gascuel, 2010) and added published primer sequences to the
alignments.
Loci were selected for further analysis if they fulfilled the following
criteria: (1) primer binding sites are not in repetitive elements thus
increasing locus-specific amplifiability and reducing the risk of
off-target PCR products particularly in multiplex PCR reactions; (2)
primer binding sites are conserved among catarrhines so that loci can be
universally amplified in this taxonomic group with >180
species (Mittermeier, Wilson, & Rylands, 2013); (3) the microsatellite
motif is relatively short (max. 150 bp) to allow small amplicon size and
secure locus amplification from degraded DNA samples, such as faecal
samples; and (4) loci are evenly (1-3 loci per chromosome) distributed
throughout the genome (using the human genome as a reference) to avoid
potential linkage problems.
For loci which passed the selection criteria, we designed new primers
using Primer-Blast
(http://www.ncbi.nlm.nih.gov/tools/primer-blast/). To allow for
multiplexing, primers were designed to have similar annealing
temperatures. Locus specificity of primers was checked by BLAT search
against the 17 available catarrhine genomes. As primer-binding sites
were not always fully conserved among the 17 catarrhines, primers of 21
loci were designed with wobble positions. To simplify library
preparation for GBS, we added adapter nucleotide sequences to the 5’ end
of the locus-specific primers (5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’
to forward primers, 5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’ to reverse
primers; locus-specific primers are provided in Table S2).