Discussion
From a set of 269 microsatellite loci widely applied in catarrhine primates, we selected a total of 45 loci that can be universally applied to genotype catarrhine primates. Due to the relatively small amplicon sizes, even low-quality DNA could be genotyped and since the selected loci were evenly distributed throughout the genome (at least according to the human genome), the risk of linkage was significantly reduced. Moreover, our panel could be multiplexed to a great extent. The testing of different multiplex settings revealed that a 5-pool approach produced the best result, but that a 3-pool approach containing one pool of 18 and two of 12 loci is the best compromise between locus amplification efficiency and laboratory effort and costs.
We tested the panel with high-quality DNA samples from all major lineages of catarrhines in multiplex settings and revealed successful amplification rates of 33 to 41 (average 38) loci per species (Table 2). We additionally showed the applicability of the 3-pool approach to degraded DNA samples such as fecal samples, which is a common material in many non-invasive wildlife studies (Carroll et al., 2018; Waits & Paetkau, 2005). The results for fecal samples were similar to the results of the high-quality samples (Table 2). All loci, besides D7s503 and D13s1291, were in accordance with Mendelian inheritance, demonstrating the suitability of the new microsatellite panel for kinship and relatedness analyses.
Through multiplexed GBS, cryptic alleles can be detected (Barbian et al., 2018; Sarhanova, Pfanzelt, Brandt, Himmelbach, & Blattner, 2018; Vartia et al., 2016), and even in our test panel of only ten catarrhine species with one individual each, we found cryptic alleles at nine loci (Tables S7-S10). As more individuals per species get tested, this number will most likely increase and provide further accuracy and a higher statistical power of our panel.
Another advantage of GBS is that the resulting genetic data, in form of allele sequences, is independent of the used sequencing platform. Thus, data produced by different laboratories can be easily shared and compared. By applying validated bioinformatics pipelines, such as the CHIIMP pipeline (Barbian et al., 2018), one can further ensure that the resulting data are reproducible and less prone to arbitrary allele calling by different researchers while still allowing the customization of e.g. filtering parameters to fit different datasets.
Although we recommend the 3-pool approach, the amplification success of individual loci can be improved, for example, by amplifying all loci in individual reactions and then pooling before or after the indexing PCR. However, this would largely increase workload in the laboratory and costs. It is also important to check which loci are polymorphic in the species of interest, so that monomorphic loci can be excluded from large-scale population genetic investigations. Likewise, as several species exhibit mismatches in primer binding sites (0 - 12 loci with mismatches per species), primer design for a given species can be adjusted and optimized, which becomes easier to do with an increasing number of sequenced catarrhine genomes.
In summary, with our microsatellite panel, we provide a tool to universally genotype catarrhine primates via GBS from samples of varying DNA quality in a cost- and time-efficient way; with higher resolution, better comparability among laboratories, and largely mitigated problems of traditional FLA.