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.