Skip to main content
Log in

Primate genotyping via high resolution melt analysis: rapid and reliable identification of color vision status in wild lemurs

  • Original Article
  • Published:
Primates Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

References

  • Bowmaker JK, Dartnall HJA, Mollon JD (1980) Micro-spectrophotometric demonstration of 4 classes of photoreceptor in an Old World primate, Macaca fascicularis. J Physiol Lond 298:131–143

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bunce JA, Isbell LA, Neitz M, Bonci D, Surridge AK, Jacobs GH, Smith DG (2011) Characterization of opsin gene alleles affecting color vision in a wild population of titi monkeys (Callicebus brunneus). Am J Primatol 73:189–196

    Article  CAS  PubMed  Google Scholar 

  • Cho MH, Ciulla D, Klanderman BJ, Raby BA, Silverman EK (2008) High resolution melting curve analysis of genomic and whole genome amplified DNA. Clin Chem 54:2055–2058

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dacey DM (2000) Parallel pathways for spectral coding in primate retina. Annu Rev Neurosci 23:743–775

    Article  CAS  PubMed  Google Scholar 

  • Doktycz MJ (2002) Nucleic acids: thermal stability and denaturation. Wiley, Chichester. http://www.els.net. doi:10.1038/npg.els.0003123

  • Dulai KS, von Dornum M, Mollon JD, Hunt DM (1999) The evolution of trichromatic color vision by opsin gene duplication in New World and Old World primates. Genome Res 9:629–638

    CAS  PubMed  Google Scholar 

  • Dwight Z, Palais R, Wittwer CT (2011) uMELT: prediction of high-resolution melting curves and dynamic melting profiles of PCR products in a rich web application. Bioinformatics 27:1019–1020

    Article  CAS  PubMed  Google Scholar 

  • Fedigan L, Melin AD, Addicott J, Kawamura S (2014) The heterozygote superiority hypothesis for polymorphic color vision is not supported by long-term fitness data from wild Neotropical monkeys. PLoS One 9:e84872

    Article  PubMed  PubMed Central  Google Scholar 

  • Hiramatsu C, Tsutsui T, Matsumoto Y, Aureli F, Fedigan LM, Kawamura S (2005) Color vision polymorphism in wild capuchins (Cebus capucinus) and spider monkeys (Ateles geoffroyi) in Costa Rica. Am J Primatol 67:447–461

    Article  CAS  PubMed  Google Scholar 

  • Hiramatsu C, Melin AD, Aureli F, Schaffner CM, Vorobyev M, Matsumoto Y, Kawamura S (2008) Importance of achromatic contrast in short-range fruit foraging in primates. PLoS One 3:e3356

    Article  PubMed  PubMed Central  Google Scholar 

  • Hiwatashi T, Okabe Y, Tsutsui T, Hiramatsu C, Melin AD, Oota H, Schaffner CM, Aureli F, Fedigan LM, Innan H, Kawamura S (2010) An explicit signature of balancing selection for color vision variation in New World monkeys. Mol Biol Evol 27:453–464

    Article  CAS  PubMed  Google Scholar 

  • Jacobs GH (1984) Within species variations in visual capacity among squirrel monkeys (Saimiri sciureus) color vision. Vision Res 24:1267–1277

    Article  CAS  PubMed  Google Scholar 

  • Jacobs GH (1993) The distribution and nature of color vision among the mammals. Biol Rev Camb Philos 68:413–471

    Article  CAS  Google Scholar 

  • Jacobs GH (1995) Variations in primate color vision: mechanisms and utility. Evol Anthropol 3:196–205

    Article  Google Scholar 

  • Jacobs GH (1998) A perspective on color vision in platyrrhine monkeys. Vis Res 38:3307–3313

    Article  CAS  PubMed  Google Scholar 

  • Jacobs RL, Bradley BJ (2016) Considering the influence of nonadaptive evolution on primate color vision. PLoS One 11:e0149664. doi:10.1371/journal.pone.0149664

    Article  PubMed  PubMed Central  Google Scholar 

  • Jacobs GH, Deegan JF (2003) Photopigment polymorphism in prosimians and the origins of primate trichromacy. In: Mollon JD, Pokorny J, Knoblauch K (eds) Normal and defective colour vision. Oxford University Press, Oxford, pp 14–20

    Chapter  Google Scholar 

  • Leonhardt SD, Tung J, Camden JB, Leal M, Drea CM (2009) Seeing red: behavioral evidence of trichromatic color vision in strepsirrhine primates. Behav Ecol 20:1–12

    Article  Google Scholar 

  • Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, Wittwer C (2004) Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem 50:1156–1164

    Article  CAS  PubMed  Google Scholar 

  • McIntosh A, Bennett C, Dickson D, Anestis SF, Watts DP, Webster TH, Fontenot MB, Bradley BJ (2012) The apolipoprotein E (APOE) gene appears functionally monomorphic in chimpanzees (Pan troglodytes). PLoS One 7:e47760

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Melin AD, Fedigan LM, Hiramatsu C, Sendall CL, Kawamura S (2007) Effects of colour vision phenotype on insect capture by a free-ranging population of white-faced capuchins, Cebus capucinus. Anim Behav 73:205–214

    Article  Google Scholar 

  • Melin AD, Fedigan LM, Hiramatsu C, Kawamura S (2008) Polymorphic color vision in white-faced capuchins (Cebus capucinus): is there foraging niche divergence among phenotypes? Behav Ecol Sociobiol 62:659–670

    Article  Google Scholar 

  • Mundy NI, Morningstar NC, Baden AL, Fernandez-Duque E, Bradley BJ (2016) Can colour vision re-evolve? Variation in the X-linked opsin locus of cathemeral Azara’s owl monkeys (Aotus a. azarae). Front Zool 13:9. doi:10.1186/s12983-016-0139-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nathans J (1999) The evolution and physiology of human color vision: insights from molecular genetic studies of visual pigments. Neuron 24:299–312

    Article  CAS  PubMed  Google Scholar 

  • Nathans J, Thomas D, Hogness DS (1986) Molecular genetics of human color vision: the genes encoding blue, green, and red pigments. Science 232:193–202

    Article  CAS  PubMed  Google Scholar 

  • Neitz M, Neitz J, Jacobs GH (1991) Spectral tuning of pigments underlying red-green color vision. Science 252:971–974

    Article  CAS  PubMed  Google Scholar 

  • Parant JM, George SA, Pryor R, Wittwer CT, Yost HJ (2009) A rapid and efficient method of genotyping zebrafish mutants. Dev Dyn 238:3168–3174

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Payne MS, Tabone T, Kemp MW, Keelan JA, Spiller OB, Newnham JP (2014) High-resolution melt PCR analysis for genotyping of Ureaplasma parvum isolates directly from clinical samples. J Clin Microbiol 52:599–606

    Article  PubMed  PubMed Central  Google Scholar 

  • Ramón-Laca A, Gleeson D, Yockney I, Perry M, Nugent G, Forsyth DM (2014) Reliable discrimination of 10 ungulate species using high resolution melting analysis of faecal DNA. PLoS One 9:e92043

    Article  PubMed  PubMed Central  Google Scholar 

  • Shyue SK, Boissinot S, Schneider H, Sampaio I, Schneider MP, Abee CR, Williams L, Hewett-Emmett D, Sperling HG, Cowing JA, Dulai KS, Hunt DM, Li W-H (1998) Molecular genetics of spectral tuning in New World monkey color vision. J Mol Evol 46:697–702

    Article  CAS  PubMed  Google Scholar 

  • Smith BL, Lu CP, Bremer JRA (2010) High-resolution melting analysis (HRMA): a highly sensitive inexpensive genotyping alternative for population studies. Mol Ecol Resour 10:193–196

    Article  CAS  PubMed  Google Scholar 

  • Smith AC, Surridge AK, Prescott MJ, Osorio D, Mundy NI, Buchanan-Smith HM (2012) The effect of colour vision status on insect prey capture efficiency by captive and wild tamarins (Saguinus spp.). Anim Behav 83:479–486

    Article  Google Scholar 

  • Surridge AK, Osorio D, Mundy NI (2003) Evolution and selection of trichromatic vision in primates. Trends Ecol Evol 18:198–205

    Article  Google Scholar 

  • Surridge AK, Suárez SS, Buchanan-Smith HM, Mundy NI (2005) Non-random association of opsin alleles in wild groups of red-bellied tamarins (Saguinus labiatus) and maintenance of the colour vision polymorphism. Biol Lett 1:465–468

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tan Y, Li WH (1999) Vision—trichromatic vision in prosimians. Nature 402:36

    Article  CAS  PubMed  Google Scholar 

  • Thomsen N, Ali RG, Ahmed JN, Arkell RM (2012) High-resolution melt analysis (HRMA); a viable alternative to agarose gel electrophoresis for mouse genotyping. PLoS One 7:e45252

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tovee MJ (1994) The molecular genetics and evolution of primate color vision. Trends Neurosci 17:30–37

    Article  CAS  PubMed  Google Scholar 

  • Tung J, Primus A, Bouley AJ, Severson TF, Alberts SC, Wray GA (2009) Evolution of a malaria resistance gene in wild primates. Nature 460:388–391

    CAS  PubMed  Google Scholar 

  • Valenta K, Edwards M, Rafaliarison RR, Johnson SE, Holmes SM, Brown KA, Dominy NJ, Lehman SM, Parra EJ, Melin AD (2015) Visual ecology of true lemurs suggests a cathemeral origin for the primate cone opsin polymorphism. Funct Ecol. doi:10.1111/1365-2435.12575

    Google Scholar 

  • Veilleux CC, Bolnick DA (2009) Opsin gene polymorphism predicts trichromacy in a cathemeral lemur. Am J Primatol 71:86–90

    Article  CAS  PubMed  Google Scholar 

  • Veilleux CC, Jacobs RL, Cummings ME, Louis EE, Bolnick DA (2014) Opsin genes and visual ecology in a nocturnal folivorous lemur. Int J Primatol 35:88–107

    Article  Google Scholar 

  • Vogel ER, Neitz M, Dominy NJ (2007) Effect of color vision phenotype on the foraging of wild white-faced capuchins, Cebus capucinus. Behav Ecol 18:292–297

    Article  Google Scholar 

  • Vossen RHAM, Aten E, Roos A, den Dunnen JT (2009) High-resolution melting analysis (HRMA): more than just sequence variant screening. Hum Mutat 30:860–2866

    Article  CAS  PubMed  Google Scholar 

  • Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ (2003) High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49:853–860

    Article  CAS  PubMed  Google Scholar 

  • Wooding S, Bufe B, Grassi C, Howard MT, Stone AC, Vazquez M, Dunn DM, Meyerhof W, Weiss RB, Bamshad MJ (2006) Independent evolution of bitter-taste sensitivity in humans and chimpanzees. Nature 440:930–934

    Article  CAS  PubMed  Google Scholar 

  • Yokoyama S, Radlwimmer FB (1998) The “five-sites” rule and the evolution of red and green color vision in mammals. Mol Biol Evol 15:560–567

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Shoji Kawamura, Amanda Melin, and Michael James Montague for helpful comments and suggestions, and we thank Gary Aronsen for logistical support in the Yale Molecular Anthropology Lab. Funding was provided by the National Science Foundation (FSML 1227143), the UK Natural Environment Research Council, Yale University, and the George Washington University. All applicable international, national, and institutional guidelines for the care and use of animals were followed. The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Rachel L. Jacobs or Brenda J. Bradley.

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jacobs, R.L., Spriggs, A.N., MacFie, T.S. et al. Primate genotyping via high resolution melt analysis: rapid and reliable identification of color vision status in wild lemurs. Primates 57, 541–547 (2016). https://doi.org/10.1007/s10329-016-0546-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10329-016-0546-y

Keywords

Navigation