Evolutionary history of black grouse major histocompatibility complex class IIB genes revealed through single locus sequence-based genotyping

Abstract

Background: Gene duplications are frequently observed in the Major Histocompatibility Complex (MHC) of many species, and as a consequence loci belonging to the same MHC class are often too similar to tell apart. In birds, single locus genotyping of MHC genes has proven difficult due to concerted evolution homogenizing sequences at different loci. But studies on evolutionary history, mode of selection and heterozygosity correlations on the MHC cannot be performed before it is possible to analyse duplicated genes separately. In this study we investigate the architecture and evolution of the MHC class IIB genes in black grouse. We developed a sequence-based genotyping method for separate amplification of the two black grouse MHC class IIB genes BLB1 and BLB2. Based on this approach we are able to study differences in structure and selection between the two genes in black grouse and relate these results to the chicken MHC structure and organization.

Results: Sequences were obtained from 12 individuals and separated into alleles using the software PHASE. We compared nucleotide diversity measures and employed selection tests for BLB1 and BLB2 to explore their modes of selection. Both BLB1 and BLB2 are transcribed and display classic characteristics of balancing selection as predicted for expressed MHC class IIB genes. We found evidence for both intra- and interlocus recombination or gene conversion, as well as indication for positive but differential selection at both loci. Moreover, the two loci appear to be linked. Phylogenetic analyses revealed orthology of the black grouse MHC class IIB genes to the respective BLB loci in chicken.

Conclusions: The results indicate that the duplication of the BLB gene occurred before the species divergence into black grouse, chicken and pheasant. Further, we conclude that BLB1 and BLB2 in black grouse are subjected to homogenizing concerted evolution due to interlocus genetic exchange after species divergence. The loci are in linkage disequilibrium, which is in line with the theory of tightly coevolving genes within the MHC under the minimal essential MHC hypothesis. Our results support the conclusion that MHC form and function in birds derived from studies on the domesticated chicken are not artefacts of the domestication process.

Figure 1

Schematic figure of the genomic focus region of the MHC. Genes are illustrated by white arrows indicating their orientation, with the names above the drawing. The exon-intron structure is given for BLB1 and BLB2 to show the target region and primer positions. The positions and amplification directions of all primers used in this study are indicated by small one-headed arrows. Below the upper part of the drawing, the primers for the long-range PCR amplifications of BLB1 and BLB2 (C275, NBG262 and preBLB2F, BLBex3R) are given. Further down, the target region Exon 2 is enlarged, with the positions of the nested PCR primers (BLB1: RNAR1a, RNAF1a, NBG262; BLB2: Vorinex, RNAF1a, RNAR1a, Postex) given below. Note that BLB1 and BLB2 are orientated in opposite directions.

Figure 2

Alignment of amino acid sequences. PBR positions from Tong et al. [73] are marked with a +. The shaded amino acids are the PAML derived positively selected codon positions, blue for BLB2 and pink for BLB1.

Figure 3

Sliding window Tajimas’ D, for the 125 bp exon 2 fragments of the black grouse BLB1 and BLB2 (window size 11 bp, step size 1 bp). The threshold for P < 0.05 is shown by the dotted line.

Figure 4

Neighbour joining tree for the 3′UTR. BLB1 and BLB2 sequences derived from black grouse (fosmid individual JHGO 213 [48], chicken [GenBank AB268588] and pheasant [GenBank AJ224349]. The scale bar represents substitutions per site.

Figure 5

Neighbour network for BLB1 and BLB2. Sequences of the black grouse (pink and blue), chicken (dark and light green) [GenBank AB426144, AB426150-51, AJ248577, M29763] and red jungle fowl (brown and orange) [GenBank AM489767 - AM489776] for the a) 125 nucleotide sequence of exon 2 and b) only 3^rd codon positions in the 125 nucleotide sequence of exon 2. Long-eared Owl Asot-DAB1*02 [GenBank EF641225] was used as an outgroup.