Abstract
Purpose :
Color vision plays a large role in the daily life of primates, spanning social and ecological interactions. The sex-linked color vision polymorphism found among species of monkeys in the Americas is of long-standing interest. Variation is driven by single nucleotide polymorphisms (SNPs) in opsin genes at up to three tuning sites. Although Sanger sequencing and melting curve analysis of PCR amplicons have been used to determine color vision genotypes, they have several limitations. Here, we evaluate the efficiency of droplet digital PCR (ddPCR) as an exceptionally sensitive and high-throughput method for color vision assessment from feces of white-faced capuchins (Cebus imitator), and report the frequency of different alleles and genotypes in a wild population.
Methods :
DNA was extracted from fecal samples collected from 41 wild capuchins (58.5% females) in Sector Santa Rosa, Costa Rica. Two duplex ddPCR assays using probes targeting the variation at exon 3 site 180 and exon 5 site 285 were designed using FAM and HEX channels to target different SNPs. We analyzed 82 samples (2 samples from each animal). For comparison, a subset of 18 individuals (at least 2 samples from each individual) were also examined using traditional sanger sequencing.
Results :
Our results from Sanger and ddPCR were highly similar, except in one case where a female was found heterozygous by ddPCR and homozygous with Sanger sequencing. We report the former. The distribution of genotypes was: Females (N=24, two X-linked alleles each): green/red (37.5%), red/red (29.2%), green/yellow (16.7%), and yellow/red (16.7%); Males (N=17, one x-linked allele each): red (70.6%), yellow (17.6%), and green (11.8%).
Conclusions :
ddPCR was a reliable method for evaluating color vision type noninvasively in wild capuchins with the advantage of excellent sensitivity and very high throughput. In addition, many fecal samples of capuchins fail with traditional Sanger sequencing due to PCR inhibitors in feces. ddPCR is highly robust to inhibitors and we had 100% success in generating results with ddPCR. The most frequent phenotypes were red, and green/red. ddPCR can be potentially used to identify other disease-related SNP mutations noninvasively in wild animals.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.