Primate genotyping via high resolution melt analysis: rapid and reliable identification of color vision status in wild lemurs.
Analyses of genetic polymorphisms can aid our understanding of intra- and interspecific variation in primate sociality, ecology, and behavior. Studies of primate opsin genes are prime examples of this, as single nucleotide variants (SNVs) in the X-linked opsin gene underlie variation in color vision. For primate species with polymorphic trichromacy, genotyping opsin SNVs can generally indicate whether individual primates are red-green color-blind (denoted homozygous M or homozygous L) or have full trichromatic color vision (heterozygous ML). Given the potential influence of color vision on behavior and fitness, characterizing the color vision status of study subjects is becoming commonplace for many primate field projects. Such studies traditionally involve a multi-step sequencing-based method that can be costly and time-consuming. Here we present a new reliable, rapid, and relatively inexpensive method for characterizing color vision in primate populations using high resolution melt analysis (HRMA). Using lemurs as a case study, we characterized variation at exons 3 and/or 5 of the X-linked opsin gene for 87 individuals representing nine species. We scored opsin genotypes and color vision status using both traditional sequencing-based methods as well as our novel melting-curve based HRMA protocol. For each species, the melting curves of varying genotypes (homozygous M, homozygous L, heterozygous ML) differed in melting temperature and/or shape. Melting curves for each sample were consistent across replicates, and genotype-specific melting curves were consistent across DNA sources (blood vs. feces). We show that opsin genotypes can be quickly and reliably scored using HRMA once lab-specific reference curves have been developed based on known genotypes. Although the protocol presented here focuses on genotyping lemur opsin loci, we also consider the larger potential for applying this approach to various types of genetic studies of primate populations.
Department of Anthropology, Center for the Advanced Study of Human Paleobiology, The George Washington University, Washington, DC, USA. rachel_jacobs@gwu.edu.|Interdepartmental Doctoral Program in Anthropological Sciences, Stony Brook University, Stony Brook, USA. rachel_jacobs@gwu.edu.|Centre ValBio Research Station, Ranomafana, Fianarantsoa, Madagascar. rachel_jacobs@gwu.edu.
Rachel L
RL
Jacobs
Department of Anthropology, Center for the Advanced Study of Human Paleobiology, The George Washington University, Washington, DC, USA.|Department of Anthropology, University at Albany-SUNY, Albany, USA.
Amanda N
AN
Spriggs
Department of Zoology, University of Cambridge, Cambridge, UK.
Tammie S
TS
MacFie
Centre ValBio Research Station, Ranomafana, Fianarantsoa, Madagascar.|Department of Anthropology, Hunter College of City University of New York, New York, USA.|Departments of Anthropology and Biology, The Graduate Center of City University of New York, New York, USA.|The New York Consortium in Evolutionary Primatology, New York, USA.
Andrea L
AL
Baden
Department of Anthropology, Northern Illinois University, DeKalb, USA.
Mitchell T
MT
Irwin
Centre ValBio Research Station, Ranomafana, Fianarantsoa, Madagascar.|Department of Anthropology, Stony Brook University, Stony Brook, USA.
Patricia C
PC
Wright
Conservation Genetics Department, Omaha's Henry Doorly Zoo and Aquarium, Omaha, USA.
Edward E
EE
Louis
Department of Sociology and Anthropology, James Madison University, Harrisonburg, USA.
Richard R
RR
Lawler
Department of Zoology, University of Cambridge, Cambridge, UK.
Nicholas I
NI
Mundy
Department of Anthropology, Center for the Advanced Study of Human Paleobiology, The George Washington University, Washington, DC, USA. bradleyjbrenda@gwu.edu.
Brenda J
http://orcid.org/0000-0002-7084-0385
BJ
Bradley
D000818:Animals; D055253:Color Vision; D060005:Genotyping Techniques / Q000379:methods*; D007901:Lemuridae / Q000235:genetics / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D011443:Strepsirhini / Q000235:genetics / Q000502:physiology*
Opsin; Sensory ecology; Single nucleotide variant genotyping; Strepsirrhines; Technique