Major Histocompatibility Complex Polymorphism And Its Association With Disease Resistance In "Whole Red" Color Pattern Of Cyprinus Carpio Var. Color

The major histocompatibility complex (MHC) is a group of closely linked loci inhetro-dimer gene which encoding glycoprotein in vertebrates. It plays a crucial role inthe adaptive immune system for its function of presenting pathogen peptides to T cells.And a lot of evidences have proved the association between different MHC alleles andresistance or susceptibility to pathogens. This research studied the cloning,polymorphism and the associations between some genotypes and resistance ability ofMHC after infected Aeromonas hydrophila artificially from important fresh wateraquiculture fish in our country, Oujiang color common carp. Our aim was to searchsome MHC alleles associated with resistance to Aeromonas hydrophila in order torealize application of molecular markers in fish breeding. The results are shown asfollows:1．Major histocompatibility complex DAB allele polymorphism and its associationwith resistance/susceptibility to Aeromonas hydrophila in＂whole red＂colorpattern of Cyprinus carpio var. colorA total of76different nucleotide acid sequences of MHC gene, which classified as20alleles, were identified in185clones from16resistant and21susceptible “wholered” color pattern of Cyprinus carpio var. color individuals using the specific primerpair of DABF and DABR. The624bp-long nucleotide fragment consisted of the exon1,exon2, exon3and exon4, which encoding signal peptide, β1domain, β2domain and part transmembrane, respectively. There were144nucleotide (52.17%) and70aminoacid (76.07%) variable sites in the276bp-long β1domain, whereas the nucleotide andamino acid variable sites were98(34.75%) and50(53.19%) in the282bp-long β2domain. It was obviously found that β1domain had more variable sites than β2domain.Meanwhile,23variable sites were observed in total24peptide binding region (PBR) ofβ1domain. The ω values (ω=dN/dS) were1.367,0.886, and0.754for PBR andnon-PBR of the β1domain, and for β2domain, respectively, implying positiveselection pressure conducted on the MHC-DAB gene (especially for β1domain) of C.carpio var. color. The percentage (13.75%) of Cyca-DAB3*15was significant higher inthe resistant stock than that (3.81%) in the susceptible stock (P<0.05). Interestingly, theCyca-DAB3*09and Cyca-DAB3*10were only found in the resistant stock, withpercentage of3.75%(P<0.05), whereas the Cyca-DAB3*02and Cyca-DAB3*14wereonly observed in the susceptible stock, with the percentage of7.62%(P<0.05) and8.57%(P<0.01), respectively.2. Major histocompatibility complex class II α allele polymorphism and itsassociation with resistance/susceptibility to Aeromonas hydrophila in＂wholered＂color pattern of Cyprinus carpio var. colorTotally,85different encoding sequences of MHC II α gene were identified within180clones from16resistant and21susceptible individuals of “Whole Red” colorpattern of Cyprinus carpio var. color using the specific primer pair of DXAF andDXAR which based on the known common carp MHC class II α sequence. All thesequences were classfied as10alleles. The624bp-long nucleotide fragment consisted ofthe33bp-exon1,252bp-exon2,285bp-exon3and54bp-exon4, which encoding signalpeptide, α1domain, α2domain and part transmembrane, respectively. It was obviouslyfound that α1domain had more variable sites than α2domain. There were105nucleotide (41.67%) and49amino acid (58.33%) variable sites in α1domain, whereasthe nucleotide and amino acid variable sites were only84(29.47%) and43(45.26%) in the α2domain. Meanwhile,15variable sites were observed in total21peptide bindingregion (PBR) of α1domain, The ω values (ω=dN/dS) were3.982for PBR,0.725fornon-PBR of the α1domain, and0.500for α2domain, implying positive selectionpressure mainly conducted on the α1domain of MHC class II α gene of “Whole Red”color pattern of Cyprinus carpio var. color. The gene Cyca-DXA7was only observed inthe resistant individuals, with percentage of9.09%(P<0.01).3. The cloning and polymorphism of MHC class I gene from＂whole red＂colorpattern of Cyprinus carpio var. colorIn order to explore the polymorphism of MHC class I gene in “Whole Red” colorpattern of Cyprinus carpio var. color, we extracted cDNA from30fish, and MHC classI gene were successfully cloned in18samples. Then seventy five clones weresequenced and seventy one972bp~993bp-long sequences were obtained. Thesenucleotide fragments encoded324~331amino acid, which including signal peptide(SP), α1, α2, α3domains, connecting peptide (CP), transmembrane (TM) and partcytoplasmic (CY). We detected49different amino acid sequences, which could beclassified into11alleles. The total percentages of polymorphic sites in nucleotide andamino acid were45.68%and60.84%, respectively. Meanwhile, the range ofnucleotide/amino acid sequences variability were0%~57.7%and0%~75.9%,respectively. All these revealed high polymorphism of MHC class I gene in “WholeRed” color pattern of Cyprinus carpio var. color. The percentages of variable sites ofnucleotide/amino acid in α1and α2domains were65.90%/79.31%and56.38%/72.34%, respectively, significantly higher than that in α3domain (30.24%/46.39%).The ratio of nonsynonymous substitution (dN) and synonymous substitution (dS) in thepeptide binding region (PBR) of α1and α2domains were1.219and1.639, respectively,both larger than1. At the same time, the positive selection amino acid sites were mostlylocated in PBR except for loci23A and130F. These results provide evidence thatpositive selection is one of the main mechanisms in maintaining MHC class I gene polymorphism of “whole red” color pattern of Cyprinus carpio var. color.