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1 December 2017 Duplication and Variation in the Major Histocompatibility Complex Genes in Blakiston's Fish Owl, Bubo blakistoni
Keita Omote, Tetsuo I. Kohyama, Chizuko Nishida, Keisuke Saito, Satoshi Fujimoto, Ryuichi Masuda
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Abstract

The major histocompatibility complex (MHC) includes many genes that are essential for the adaptive immune system, and variation in the antigen binding site (ABS) is related to resistance against pathogens. In the present study, quantitative real-time PCR indicated a larger number of MHC gene copies in the endangered population of Blakiston's fish owl (Bubo blakistoni) than in five other owl species, and massively parallel pyrosequencing detected more MHC class IIβ per individual alleles in B. blakistoni than in the other species. A chromosomal fluorescence in situ hybridization (FISH) analysis showed that the MHC class I and class IIβ loci are closely linked on a single pair of microchromosomes, indicating that the MHC genes were tandemly duplicated in a limited chromosomal region. Because B. blakistoni has twice as many MHC genes as its sister species, the tawny fish owl (Bubo flavipes), the duplication of MHC genes occurred after these species diverged by speciation. A Bayesian molecular phylogenetic analysis showed that the DAB1 and DAB2 lineages of MHC class IIβ alleles from various strigid species each formed a separate clade, indicating that the two allelic lineages preceded the radiation of Strigidae and evolved as paralogs. By contrast, the ABS sequences did not form distinct clades between DAB1 and DAB2 alleles but were intermixed, presumably due to gene conversion. Despite the low diversity of alleles per locus, B. blakistoni had many lineages of MHC class IIβ alleles. Gene duplication increases variation in the MHC genes in this species, and could have facilitated adaptation in small populations.

INTRODUCTION

The major histocompatibility complex (MHC) is a polymorphic genomic region that contains many genes and plays a role in the adaptive immune system of jawed vertebrates. In humans, the MHC is large and has a complex genomic structure, consisting of over 200 genes that include class I, class II, and class III gene families (MHC sequencing consortium, 1999). The MHC class I (MHCI) genes encode cell-surface proteins that present antigen peptides from pathogens in the cytoplasm or nucleus. MHC class II (MHCII) molecules, which consist of α and β chains, are expressed on subsets of cells such as B cells and macrophages to present antigen peptides from pathogens in intracellular vesicles and extracellular spaces.

Among birds, MHC organization is best characterized in the chicken (Gallus gallus). The chicken MHC has a highly streamlined organization that contains only 19 genes, including two class I and two class IIβ genes (Kaufman et al., 1999), all located on a single microchromosome. By contrast, while the Japanese quail (Coturix japonica) MHC is similar in overall organization to that in G. gallus, it is more diverse, having seven class I and 10 class IIβ genes (Shiina et al., 2004). More complex MHC organizations, with highly duplicated genes, have been found in several songbirds, which are the most diverse group of birds and are included in a clade of landbirds, along with a variety of other groups such as parrots, falcons, raptors, woodpeckers, and owls. In the great reed warbler (Acrocephalus arundinaceus), Westerdahl et al. (2000) found seven different sequences for exon 3 of MHCI, and seven divergent sequences for the antigen-binding site (ABS) of MHCIIβ per individual, and suggested that the class I and class IIβ restriction fragments were linked. The zebra finch (Taeniopygia guttata) has highly duplicated MHCI and MHCIIβ genes that are dis persed among four chromosomes (Balakrishnan et al., 2010). This indicates that gene duplications and chromosome rearrangements have caused the evolution of complex MHC organization in birds.

Blakiston's fish owl (Bubo blakistoni) is an endangered fish-eating owl endemic to northeastern Asia. The population of B. blakistoni on Hokkaido Island, Japan, decreased in the 20th century, resulting in low genetic diversity at present (Omote et al., 2015). Recently, Kohyama et al. (2015) reported up to 16 different MHCIIβ alleles per individual in B. blakistoni based on the next-generation sequencing approach. This indicates that the B. blakistoni genome contains at least eight copies of MHCIIβ genes, the highest number of gene copies reported in a non-passerine bird. We speculate that the large number of MHCIIβ genes in B. blakistoni arose through recent gene duplication events, but the sequencing-based approach is insufficient to reveal accurately the number of gene copies and the gene organization at the chromosomal level.

The aims of the present study were fourfold: (1) estimate the copy numbers of MHCI and MHCIIβ genes in B. blakistoni, (2) map the locations of the loci on chromosomes, (3) reconstruct the phylogenetic relationships among alleles, and (4) discuss evolutionary patterns and variation in the MHC genes in B. blakistoni and closely related species.

MATERIALS AND METHODS

Sampling, and RNA and DNA ext