Characterization, Polymorphism and Selection of Major Histocompatibility Complex (MHC) DAB Genes in Vulnerable Chinese Egret (Egretta eulophotes)

Abstract

The major histocompatibility complex (MHC) is an excellent molecular marker for the studies of evolutionary ecology and conservation genetics because it is a family of highly polymorphic genes that play a key role in vertebrate immune response. In this study, the functional genes of MHC Class II B (DAB) were isolated for the first time in a vulnerable species, the Chinese egret ( Egretta eulophotes ). Using a full length DNA and cDNA produced by PCR and RACE methods, four potential MHC DAB loci were characterized in the genome of this egret and all four were expressed in liver and blood. At least four copies of the MHC gene complex were similar to two copies of the minimal essential MHC complex of chicken, but are less complex than the multiple copies expressed in passerine species. In MHC polymorphism, 19 alleles of exon 2 were isolated from 48 individuals using PCR. No stop codons or frameshift mutations were found in any of the coding regions. The signatures of positive selection detected in potential peptide-binding regions by Bayesian analysis, suggesting that all of these genes were functional. These data will provide the fundamental basis for further studies to elucidate the mechanisms and significance of MHC molecular adaptation in vulnerable Chinese egret and other ardeids.

Characterization of a non-classical MHC class II gene in the vulnerable Chinese egret (Egretta eulophotes)

“…First, two new forward locus-specific primers for Egeu-DAB3 and -DAB4 were designed and designated as DAB03F1 and DAB04F1, because the previously published primers, DAB03F and DAB04F (Wang et al 2013), were not specific enough for some samples in present study. These new primers and two others of Wang et al (2013) were each combined with the reverse primer DAB2exR (Table 1) to specially amplify the four Egeu-DAB genes (836, 836, 850, and 302 bp, respectively). PCR was carried out in a final volume of 20 μL, which contained 1 μL (approximately 100 ng) gDNA, 0.7 U of Taq polymerase (TaKaRa), 1.5 mM MgCl 2 , 200 μM of each dNTP, and 0.4 μM of each primer.…”

“…Thermocycling conditions were as follows: 94°C for 3 min, 25 amplification cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 60 s, and final extension at 72°C for 10 min. To obtain suitable length fragments for SSCP genotyping, second round PCR was done on each respective sample, using the primer sets DAB2exF and DAB2exR, which could amplify the entire exon 2 (270 bp) in each locus (Wang et al 2013). PCR products diluted 40-fold from the first round were used as the template for second round PCR.…”

“…When compared with Egeu-DAB1, -DAB2, and -DAB3, the Egeu-DAB4 gene had a similar complete structure but a shorter intron 1 (280 bp for Egeu-DAB4, 543-600 bp for Egeu-DAB1-3) and low nucleotide identity in exon 2, and it was not found in all studied individuals (Wang et al 2013), implying that it was potentially a non-classical (class IIb) gene. As little is known about the non-classical MHC genes in this vulnerable Chinese egret and in other ciconiiform birds, we initiated the present study with the following specific aims:…”

“…Recently, studies on MHC class II genes have been reported for several groups of Aves, including Anseriformes (Li et al 2011a), Charadriiformes (Ekblom et al 2010), Ciconiiformes (Li et al 2011b;Wang et al 2013;Taniguchi et al 2014), Falconiformes (Alcaide et al 2007;Burri et al 2008;Bollmer et al 2011), Galliformes (Zoorob et al 1993;Promerová et al 2013), Gruiformes (Alcaide et al 2014), Procellariiformes (Silva and Edwards 2009;Strandh et al 2011), and Passeriformes (Sato et al 2001;Aguilar et al 2006;Bollmer et al 2010). Our previous studies on the Chinese egret (Ciconiiformes, Ardeidae, Egretta eulophotes), a vulnerable waterbird with an estimated global population of 2600-3400 individuals BirdLife International 2015;IUCN 2015), have isolated some MHC class II genes and established a preliminary locus-specific MHC genotyping technique (Li et al 2011b;Wang et al 2013).…”

“…Our previous studies on the Chinese egret (Ciconiiformes, Ardeidae, Egretta eulophotes), a vulnerable waterbird with an estimated global population of 2600-3400 individuals BirdLife International 2015;IUCN 2015), have isolated some MHC class II genes and established a preliminary locus-specific MHC genotyping technique (Li et al 2011b;Wang et al 2013). In this egret, four class II genes, designated as Egeu-DAB1, -DAB2, -DAB3, and -DAB4, were described as potential classical MHC genes based on a comparison of full-length complementary DNA (cDNA) between Egeu-DAB genes and other published class II genes (Wang et al 2013).…”