Application of PCR-RF-SSCP to study major histocompatibility class II B polymorphism in common carp (Cyprinus carpio L.) Krzysztof L. Rakus a, *, Geert F. Wiegertjes b , Mikolaj Adamek a,c , Vitaliy Bekh a,d , Rene´ J.M. Stet e,1 , Ilgiz Irnazarow a a Polish Academy of Sciences, Institute of Ichthyobiology and Aquaculture in Golysz, Zaborze, ul. Kalinowa 2, 43-520 Chybie, Poland b Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, Marijkeweg 40, 6709 PG Wageningen, The Netherlands c Department of Ichthyobiology and Fisheries, University of Agriculture, 30-199 Krako´w-Mydlniki, Poland d Institute of Fisheries, Ukrainian Academy of Agrarian Sciences, Obukhovska Street 135, Kyiv, 03164, Ukraine e Scottish Fish Immunology Research Centre, University of Aberdeen, Zoology Building, Tillydrone Avenue, Aberdeen, AB24 2TZ, Scotland, UK Received 1 August 2007; revised 19 November 2007; accepted 19 November 2007 Available online 5 December 2007 KEYWORDS MHC; Polymorphism; Mutation detection techniques; SSCP; Common carp Abstract A variety of methods have been applied for the characterization of major histocom- patibility (MH) polymorphism in fish. We optimized a technique designated polymerase chain reaction-restriction fragments-single strand conformation polymorphism (PCR-RF-SSCP) for screening a large number of individuals for the Cyca-DAB1 and Cyca-DAB2 genes polymorphism in common carp. The advantages of this technique are simplicity, high sensitivity and low costs. PCR-RF-SSCP analysis revealed different genotypes consisting of unique combinations of the Cyca-DAB1 and Cyca-DAB2 sequences with the number of SSCP bands clearly correlating with the degree of heterozygosity of the Cyca-DAB1 and Cyca-DAB2 genes. We found four alleles for Cyca-DAB1 (*02-*05) gene but only one allele for Cyca-DAB2 (*02) and noted that the Cyca-DAB2 gene was either homozygous or absent. PCR-RF-SSCP analysis of n Z 79 carp in- dividuals challenged with Aeromonas hydrophila indicated that individuals bearing no Cyca- DAB2 gene showed higher cumulative mortality and lower bacterial agglutination titers during the experiment. We suggest that our PCR-RF-SSCP method can be used to study correlations of different MH class II B genotypes/alleles with resistance of common carp to specific pathogens. ª 2007 Elsevier Ltd. All rights reserved. * Corresponding author. Tel.: þ48 33 8561551; fax: þ48 33 8589292. E-mail address: krzysztof.rakus@fish.edu.pl (K.L. Rakus). 1 In memory of Prof. Rene ´ J.M. Stet e deceased on September 12, 2007. 1050-4648/$ - see front matter ª 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2007.11.015 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/fsi Fish & Shellfish Immunology (2008) 24, 734e744