Polymorphism And Expression Analysis Of MHS Class â…¡ Gene And Its Association With Resistance/Susceptibility To Aeromonas Hydrophila In Cyprinus Carpio Var. Singuonensis And Cyprinus Carpio Var. Wuyuanensis

Red common carp is one kind of very important fish breeding materials in ourcountry, which economic value of Xingguo red common carp (Cyprinus carpio var.singuonensis, XG) and purse red common carp (Cyprinus carpio var. wuyuanensis,PR) are bigger, playing an important role in breeding production. Aeromonashydrophila is one of the main pathogen in breeding process, but there are no effectiveways against outbreak of the bacteria. The basic approach to resolve the problem is toculture strains of fish with enhanced resistance to A. hydrophila using molecularmarker-assisted selective breeding. Major histocompatibility complex (MHC) hasbeen thought candidate for molecular markers association between polymorphism andresistance/susceptibility to diseases. The relationship between polymorphism of theMHC II genes and the disease resistance of XG and PR was analyzed in the presentstudy. Real-time quantitative PCR was used to detect the expression levels in varioustissues. This study has identified potential candidate MHC genes for further researchinto the breeding of disease-resistant strains of red common carp. The main results areas follows:1. Polymorphism and expression analysis of MHC class IIα gene and itsassociation with resistance/susceptibility to A.hydrophila in XGA total of171sequences of MHC class IIα gene were determined from41XGsusceptible and22resistant XG individuals. These sequences were collapsed into26 alleles, thirteen of which were novel (Cyca-DXA24－Cyca-DXA36). The total lengthof the sequence was624bp, consisting a33bp exon1, a252bp exon2, a285bpexon3, and a54bp exon4. These exons encoded a partial signal peptide (SP), a α1domain, a α2domain and a connecting peptide (CP)/partial transmembrane (TM),respectively. The nucleotide or amino acid variable sites in α1domain (55.16%,79.76%) were significantly higher than those in α2domain (45.96%,68.42%). The ωvalues (ω=dN/dS) were5.742for peptide-binding region (PBR),0.755for non-PBR inthe α1domain and0.592for the α2domain, implying that positive selection pressuremaybe had occurred in the PBR region of the α1domain. Cyca-DXA24(P<0.01) wassignificantly correlated with the carp’s resistance to A.hydrophila, while Cyca-DXA3(P<0.05), Cyca-DXA4(P<0.01), Cyca-DXA6(P<0.05) and Cyca-DXA33(P<0.05)alleles were significantly correlated with the fish’s susceptibility to the disease.Real-time quantitative PCR demonstrated that MHC IIα genes were ubiquitouslyexpressed in ten tissues, but up-regulated in kidney, down-regulated in liver andspleen during infection.2. Polymorphism and expression analysis of MHC class II β gene and itsassociation with resistance/susceptibility to A.hydrophila in XGThe MHC classⅡβ gene fragment was amplified from41XG susceptible and22resistant XG individuals within291clones, using primer pairs of DABF and DABR.The length of the PCR product was624bp. The sequences were classified into33alleles, and Cyca-DAB1*17－Cyca-DAB1*21and Cyca-DAB3*24－Cyca-DAB3*34represented the16novel alleles. The nucleotide fragment encoded apartial SP, a β1domain, a β2domain, and a CP/partial TM, respectively. There were189nucleotide (68.48%) and80amino acid (86.96%) variable sites in the β1domain,whereas nucleotide and amino acid variable sites were145(51.42%) and72(76.60%)in the β2domain, indicating the β1domain possessed more variable sites than the β2domain. The ω value was1.796for PBR,0.956for non-PBR in the β1domain, and0.740for the β2domain, respectively. So, it was implied that positive selectionpressure maybe was exerted on the β1domain. There was a higher percentage of Cyca-DAB1*17in the resistant individuals (12.87%) than in the susceptibleindividuals (4.74%, P<0.05). Real-time quantitative PCR demonstrated that MHCclass IIβ genes were ubiquitously expressed in the ten normal tissues but changedduring the infection process in kidney, liver and spleen tissues.3. Polymorphism and expression analysis of MHC class IIα gene and itsassociation with resistance/susceptibility to A.hydrophila in PROne hundred and eleven sequences from24PR susceptible individuals and85sequences from17PR resistant individuals were analysed. A total of114differentencoding sequences were classified into15alleles and Cyca-DXA18－Cyca-DXA23were represented the six novel alleles. The624bp nucleotide fragment encoded apartial SP, a α1domain, a α2domain, a CP/TM. The relative numbers of nucleotideand amino acid variable sites in the a1domain were49.21%and77.38%, respectively.This was markedly higher than in the α2domain (34.04%,53.68%). These may becorrelated with highly polymorphic PBR in a1domain. The ω values were7.167forPBR and0.925for non-PBR in the α1domain and0.397for the α2domain, implyingthat positive selection pressure was exerted on α1domain. Cyca-DXA4(P<0.01) andCyca-DXA5(P<0.01) alleles were closely associated with susceptibility to A.hydrophila, and Cyca-DXA17(P<0.01), Cyca-DXA18(P<0.01), and Cyca-DXA19(P<0.05) were closely associated with resistance. Real-time quantitative PCRdemonstrated that the expression of MHC IIα genes in healthy PR and bacteriachallenged PR were in accordance with the trend in XG.4. Polymorphism and expression analysis of MHC class Ⅱβ gene and itsassociation with resistance/susceptibility to A.hydrophila in PRBased on cDNA templates from24PR susceptible and17PR resistantindividuals,110different encoding sequences of the MHC classⅡβ gene wereidentified by PCR amplification and clone. The sequences were classified into17alleles, and Cyca-DAB1*14－Cyca-DAB1*16and Cyca-DAB3*23represented the4novel alleles. The624bp nucleotide fragment encoded a partial SP, a β1domain, a β2 domain, and a CP/TM, respectively. The percentages of nucleotide or amino acidvariable sites in β1domain were63.04%/81.52%, significantly higher than that in β2domain (47.52%/71.28%). The ω values were2.525for PBR,0.927for non-PBR inthe β1domain, and0.735for the β2domain, respectively. Allele Cyca-DAB1*07wassignificantly more prevalent in individuals from the susceptible stock than inindividuals from the resistant stock:18.26%and5.00%, P<0.05. In contrast,Cyca-DAB3*23was significantly more prevalent in resistant individuals than insusceptible individuals:13.75%and5.22%, P<0.05. Real-time quantitative PCRshowed that the expression of MHC IIβ genes in healthy PR and bacteria challengedPR were also in accordance with the trend in XG.