Initial Characterization of Major Histocompatibility Complex (Mhc) Class Iib Exon 2 in an Endangered Rattlesnake, the Eastern Massasauga (Sistrurus Catenatus)

Abstract

Genes of the major histocompatibility complex (MHC) play an important role in the vertebrate immune system and exhibit remarkably high levels of polymorphism, maintained by strong balancing selection. While the conservation implications of MHC variation have been explored in a variety of vertebrates, non-avian reptiles (most notably snakes) have received less attention. To address this gap and take the first steps toward more extensive population-level analyses, we cloned and sequenced MHC IIB exon 2 in an endangered rattlesnake, the Eastern Massasauga (Sistrurus catenatus). Based on three individuals, we found evidence of at least four putatively functional loci. These sequences exhibited relatively high levels of variation and significantly higher rates of nonsynonymous to synonymous substitutions, especially within the antigen-binding sites, indicating strong positive selection. Phylogenetic analysis revealed a pattern of trans-species polymorphism, also suggesting positive selection. These results contribute to our understanding of MHC variation in non-avian reptiles and form a basis for more studies of MHC variation in snakes of conservation concern.

Microsatellite and major histocompatibility complex variation in an endangered rattlesnake, the Eastern Massasauga (Sistrurus catenatus)

“…In each population, estimates of Tajima's D based on the MHC sequences were significantly greater than zero indicating balancing selection or population contraction (Tajima 1989). This finding is consistent with previous analyses of synonymous and nonsynonymous mutation rates in the Eastern Massasauga MHC (Jaeger et al 2014). Although we found a great deal of variation, the number of MHC alleles per individual showed an increasing trend from Cook to Piatt and Clinton counties, consistent with our microsatellite results.…”

“…As predicted, the 24 putatively functional Eastern Massasauga MHC alleles were highly variable, at both the nucleotide and amino acid levels. Consistent with previous work (Jaeger et al 2014), we found an excess of nonsynonymous substitutions in the Eastern Massasauga MHC, indicating historical positive selection. Although this signature of selection was higher on the putative antigen-binding sites than the non-antigen-binding sites predicted based on the human MHC protein structure (Brown et al 1993), only three of the 14 amino acid residues predicted to bind antigens were identified as positively selected sites based on the maximum-likelihood analysis of evolutionary models.…”

“…5 0 -TAG TTG TGS CKG CAG WAS GTG TC -3 0 ) to amplify MHC IIB exon 2 from genomic DNA. This primer pair targets conserved regions of the gene and has been used successfully in several reptile species (Edwards et al 1995;Li et al 2008), including the Eastern Massasauga (Jaeger et al 2014). Amplifications were performed in 30 lL volumes containing 19 GoTaq Flexi Buffer (Promega), 1 U of GoTaq DNA Polymerase, 200 lmol/L of each dNTP, 1.5 mmol/L of magnesium chloride (MgCl 2 ), 0.5 lmol/L of each primer, and 50-100 ng of genomic DNA.…”

“…Of the 25 putative alleles, five alleles (Sica-DAB*01-*04, *07) were previously identified (Jaeger et al 2014). The 20 newly identified putative alleles were named Sica-DAB*10-29, according to the nomenclatural standards (Klein et al 1990).…”

“…At the population level, the importance of MHC variation in maintaining population viability remains poorly understood . While the relevance of MHC variation in snake conservation efforts is understood Madsen and Ujvari 2006), relatively little is known regarding snake MHC evolution Kelley et al 2005;Ujvari and Belov 2011;Jaeger et al 2014).…”