title: The use of reference strand-mediated conformational analysis for the study of cheetah (Acinonyx jubatus) feline leucocyte antigen class II DRB polymorphisms.
abstract: There is now considerable evidence to suggest the cheetah (Acinonyx jubatus) has limited genetic diversity. However, the extent of this and its significance to the fitness of the cheetah population, both in the wild and captivity, is the subject of some debate. This reflects the difficulty associated with establishing a direct link between low variability at biologically significant loci and deleterious aspects of phenotype in this, and other, species. Attempts to study one such region, the feline leucocyte antigen (FLA), are hampered by a general reliance on cloning and sequencing which is expensive, labour-intensive, subject to PCR artefact and always likely to underestimate true variability. In this study we have applied reference strand-mediated conformational analysis (RSCA) to determine the FLA-DRB phenotypes of 25 cheetahs. This technique was rapid, repeatable and less prone to polymerase chain reaction (PCR)-induced sequence artefacts associated with cloning. Individual cheetahs were shown to have up to three FLA-DRB genes. A total of five alleles were identified (DRB*ha14-17 and DRB*gd01) distributed among four genotypes. Fifteen cheetahs were DRB*ha14/ha15/ha16/ha17, three were DRB*ha15/ha16/ha17, six were DRB*ha14/ha16/ha17 and one was DRB*ha14/ha15/ha16/ha17/gd01. Sequence analysis of DRB*gd01 suggested it was a recombinant of DRB*ha16 and DRB*ha17. Generation of new alleles is difficult to document, and the clear demonstration of such an event is unusual. This study confirms further the limited genetic variability of the cheetah at a biologically significant region. RSCA will facilitate large-scale studies that will be needed to correlate genetic diversity at such loci with population fitness in the cheetah and other species.
authors.affiliation: University of Liverpool, Veterinary Teaching Hospital, Leahurst, Chester High Road, Neston, CH64 7TE, UK.
authors.forename: G J C
authors.initials: GJ
authors.lastname: Drake
authors.forename: L J
authors.initials: LJ
authors.lastname: Kennedy
authors.forename: H K
authors.initials: HK
authors.lastname: Auty
authors.forename: R
authors.initials: R
authors.lastname: Ryvar
authors.forename: W E R
authors.initials: WE
authors.lastname: Ollier
authors.forename: A C
authors.initials: AC
authors.lastname: Kitchener
authors.forename: A R
authors.initials: AR
authors.lastname: Freeman
authors.forename: A D
authors.initials: AD
authors.lastname: Radford
mesh_terms: D002612:Acinonyx / Q000235:genetics*; D000483:Alleles; D000818:Animals; D001483:Base Sequence; D016000:Cluster Analysis; D017931:DNA Primers; D015395:Histocompatibility Antigens Class I / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D015395:Histocompatibility Antigens Class I; C059600:feline leukocyte antigen