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.

Low MHC DRB class II diversity in the mountain goat: past bottlenecks and possible role of pathogens and parasites

“…The number of MHC DRB class II alleles in the three populations of mountain goats studied is low and comparable to those reported amongst species known to have gone through population bottlenecks (Hedrick et al 1999(Hedrick et al , 2000aSommer et al 2002;Blankenburg et al 2003;Drake et al 2004;Babik et al 2005;Wan et al 2006). The low genetic diversity in mountain goats could be largely attributable to Pleistocene glaciations as has been suggested in other ungulates (Mikko et al 1999;Loehr et al 2006), where depletion of the gene pool would have occurred via the combined actions of inbreeding and genetic drift.…”

Analysis of Pigtail Macaque Major Histocompatibility Complex Class I Molecules Presenting Immunodominant Simian Immunodeficiency Virus Epitopes

“…While a rigorous but low-throughput cloning and sequencing strategy was required for the initial characterization of alleles, the RSCA technology permitted sensitive and rapid screening of MHC class I alleles in multiple pigtail macaques. Throughput could potentially be increased by using liquid-phase capillarybased technologies now utilized for high-throughput sequencing (23,59). Using a capillary sequencer, multiple reference alleles labeled with different fluorescent dyes can be used, eliminating the need to run multiple nondenaturing gels with different reference alleles.…”

Genotyping of black grouse MHC class II B using reference Strand-Mediated Conformational Analysis (RSCA)

“…RSCA has also been optimised to various Capillary Electrophoresis (CE) instruments, firstly to the one capillary instrument ABI PRISM 310 (Applied Biosystems) [47,48]. Recently, besides CE instruments, also Capillary Array Electrophoresis (CAE) instruments, with several capillaries for faster genotyping, are used in RSCA typing, four capillaries in CAE ABI 3130 [28,49] and 16 capillaries in CAE ABI 3100 [21,28,36,50,51]. To our knowledge, RSCA genotyping has not previously been optimised for a MegaBACE™ instrument (GE Healthcare).…”