Development of MHC-Linked Microsatellite Markers in the Domestic Cat and Their Use to Evaluate MHC Diversity in Domestic Cats, Cheetahs, and Gir Lions

Abstract

Diversity within the major histocompatibility complex (MHC) reflects the immunological fitness of a population. MHC-linked microsatellite markers provide a simple and an inexpensive method for studying MHC diversity in large-scale studies. We have developed 6 MHC-linked microsatellite markers in the domestic cat and used these, in conjunction with 5 neutral microsatellites, to assess MHC diversity in domestic mixed breed (n = 129) and purebred Burmese (n = 61) cat populations in Australia. The MHC of outbred Australian cats is polymorphic (average allelic richness = 8.52), whereas the Burmese population has significantly lower MHC diversity (average allelic richness = 6.81; P < 0.01). The MHC-linked microsatellites along with MHC cloning and sequencing demonstrated moderate MHC diversity in cheetahs (n = 13) and extremely low diversity in Gir lions (n = 13). Our MHC-linked microsatellite markers have potential future use in diversity and disease studies in other populations and breeds of cats as well as in wild felid species.

Keywords: major histocompatibility complex; Acinonyx jubatus; Felis catus; Panthera leo.

Figure 1.

Diagram of domestic cat MHC class II and class I classical regions showing position of MHC genes and microsatellite markers.

Figure 2.

Amino acid alignment of MHC class I alpha domain alleles in the cheetah. Dots indicate identity to the top sequence. Included are previously characterized MHC alleles AJUMHCAJUI1, AJUMHCAJUI3 (Yuhki and O’Brien 1994), and Acju-MHCI*02,04,05, 07,12 (Castro-Prieto et al. 2011) (only those unique at the alpha 1 domain are shown). Predicted classical alleles, nonclassical alleles, and pseudogenes are indicated by red, blue, and green dots, respectively. Asterisks above the sequence indicate putative peptide-binding sites based on alignment with human MHC I.

Figure 3.

Phylogenetic analysis of 43 MHC class I alpha I domain nucleotide sequences, including cheetah and Gir lion alleles from this study, previously characterized cheetah alleles (Yuhki and O’Brien 1994; Castro-Prieto et al. 2011), and domestic cat MHC class I alleles (Yuhki et al. 2008). FLAI-E, FLAI-H, and FLAI-K are domestic cat classical class I alleles, whereas FLAI-C, FLAI-F FLAI-J, FLAI-M, FLAI-O, and FLAI-Q are nonclassical alleles. The phylogenetic relationship was inferred using the neighbor-joining method (Saitou and Nei 1987). The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (1000 replicates) are displayed next to the branches, indicating the level of reliability of the phylogeny (Felsenstein 1985). Predicted classical alleles, nonclassical alleles, and pseudogenes are indicated by red, blue, and green dots, respectively. Domestic cat, cheetah, and lion alleles are indicated by black squares, stars, and diamonds, respectively.

Figure 4.

Amino acid alignment of partial MHC class I alpha domain alleles in the Gir lion. Dots indicate identity to the top sequence. Predicted classical alleles, nonclassical alleles, and pseudogenes are indicated by red, blue, and green dots, respectively. Asterisks above the sequence indicate putative peptide-binding sites based on alignment with human MHC I.