Identification and characterization of major histocompatibility complex class IIB alleles from three species of European ranid frogs

Document Type: Original article


1 Department of Parasitology, University of Agricultural Science and Veterinary Medicine, Mănăștur Street nr. 3-5, RO-400372 Cluj-Napoca, Romania

2 Department of Biological Sciences, Humboldt State University 95521 USA

3 Faculty of Biology and Geology, Babes-Bolyai-University Cluj-Napoca, Clinicilor street 5-7, RO-400006 Cluj-Napoca, Romania

4 Association for Bird and Nature Protection “Milvus Group”, Targu Mures, 22 Crinului Street, RO-540343

5 Molecular Biology Center, Interdisciplinary Research Institute on Bio-Nano-Sciences, Babes-Bolyai University Cluj-Napoca, 42 Treboniu Laurian Street, RO-400271 Cluj-Napoca, Romania


Immune genes of the major histocompatibility complex (MHC) are among the most polymorphic genes in the vertebrate genome. Due to their polymorphic nature, they are often used to assess the adaptive genetic variability of natural populations. This study describes the first molecular characterization of 13 partial MHC class IIB sequences from three European ranid frogs.  The utility of previously published primers was expanded by using them to successfully amplify eight exon 2 alleles from Rana arvalis. We also designed a novel primer set that successfully amplified exon 2 from Pelophylax kurtmuelleri. Pelophylax lessonae was also designed as part of this study. Results indicate the presence of one or two class IIB loci in these three species. In R. arvalis, significant evidence of positive selection acting on MHC antigen binding sites was found. Many European ranid populations are experiencing disease-related declines; the newly developed primers can, therefore, be used for further population analyses of native frogs.


1.Klein J. Natural history of the major histocompatibility complex.Wiley, New York 1986.

2.Edwards SV, Nusser J, Gasper J. Characterization and evolution of major histocompatibility complex (MHC) genes in non-model organisms, with examples from birds. In: Baker AJ (ed.): Molecular methods in ecology. Blackwell Science Ltd, Oxford, 2000;168-207.

3.Hughes AL, Yeager M. Natural selection at major histocompatibility complex loci of vertebrates. Annu Rev Genet 1998;32:415-435.

4.Berger L, Speare R, Daszak P, Green DE, Cunningham AA, Goggin CL, Slocombe R, Ragan MA, Hyatt D, McDonald KR,Hines HB, Lips KR, Marantelli G, Parkes H.Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests ofAustraliaandCentral America. Proc Natl Acad Sci USA1998;95:9031-9036.

5.Granoff A. Viruses of Amphibia: an historical perspective. In: Ahne W, Kurstak E (eds.): Viruses of Lower Vertebrates. Springer Verlag Berlin 1989;3-12.

6.Wood LR, Griffiths RA, Schley L. Amphibian chytridiomycosis inLuxembourg. Bull Soc Naturalistes Luxemb 2009;110:109-114.

7.Teacher AG, Cunningham AA, Garner TW. Assessing the long-term impact of ranavirus infection in wild common frog populations. Anim Conserv 2010;13:514-522.

8.Hauswaldt J, Stuckas SH, Pfautsch S, Tiedemann R. Molecular characterization of MHC class II in a nonmodel anuran species, the fire-bellied toad Bombina bombina. Immunogenetics 2007;59:479-491.

9.Zeisset I, Beebee JC. Molecular characterization of major histocompatibility complex class II alleles in the common frog, Rana temporaria. Mol Ecol Resour 2009;9:738-745.

10.Sommer S. The importance of immune gene variability (MHC) in evolutionary ecology and conservation. Front Zool 2005;2:16-34.

11.Palumbi SR. Nucleic acids II: the polymerase chain reaction. In: Hills DM, Moritz C, Mable B (eds.): Molecular Systematic. Sinauer & Associates Inc, Sunderland 1996;205-247.

12.Kiemnec-Tyburczy KM, Richmond JQ, Savage AE, Zamudio KR. Selection, trans-species polymorphism, and locus identification of major histocompatibility complex class IIβ alleles ofNew Worldranid frogs. Immunogenetics 2010;62:741-751.

13.Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analyses program for Windows 95/98/NT. Nucleic Acids Symp Ser1999;41:95-98.

14.Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular Evolutionary Genetics Analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011;28:2731-2739.

15.Tong J, Bramson J, Kanduc D, Chow S, Sinha AA, Ranganathan SH. Modeling the bound conformation of Pemphigus Vulgaris-associated peptides to MHC class II DR and DQ alleles. Immunome Res 2006;2:1745-7580.

16.Scheffler KD, Martin DP, Seoighe C. Robust inference of positive selection from recombining coding sequences. Bioinformatics 2006;22:2493-2499.

17.May S, Beebee T. Characterisation of major histocompatibility complex class II alleles in the Natterjack toad, Bufo calamita.Cons Genet Resour 2009;1:415-417.

18.Duffus AL, Cunningham AA. Major disease threats to European amphibians. J Herpetol 2010;20:117-127.

19.Teacher AG, Garner TW, Nichols RA. Evidence for directional selection at a novel major histocompatibility class I marker in wild common frogs (Rana temporaria) exposed to a viral pathogen (Ranavirus). PLoS One 2009;4:e4616.2009.

20.May S, Zeisset I, Beebee TJ. Larval fitness and immunogenetic diversity in chytrid-infected and uninfected natterjack toad (Bufo calamita) populations. Conserv Genet 2011;12:805-811.