ABSTRACT
The major histocompatibility complex (MHC) is central to the vertebrate immune system and its highly polymorphic genes are considered to influence several life-history traits of individuals. To characterize the MHC in a natural population of blue tits (Cyanistes caeruleus) we investigated the class I exon 3 diversity of more than 900 individuals. We designed two pairs of motif-specific primers that reliably amplify independent subsets of MHC alleles. Applying denaturing gradient gel electrophoresis (DGGE) we obtained 48 independently inherited units of unique band patterns (DGGE-haplogroups), which were validated in a segregation analysis within 105 families. In a second approach, we extensively sequenced 6 unrelated individuals to confirm that DGGE-haplogroup composition reflects individual allelic variation. The highest number of different DGGE-haplogroups in a single individual corresponded in 19 MHC exon 3 sequences, suggesting a minimum of 10 amplified MHC class I loci in the blue tit. In total, we identified 50 unique functional and 3 non-functional sequences. Functional sequences showed high levels of recombination and strong positive selection in the antigen binding region, whereas nucleotide diversity was comparatively low in the range of all passerine species. Finally, in a phylogenetic comparison of passerine MHC class I exon 3 sequences we discuss conflicting evolutionary signals possibly due to recent gene duplication, recombination events and concerted evolution. Our results indicate that the described method is suitable to effectively explore the MHC diversity and its ecological impacts in blue tits in future studies.