MHC Haplotype Screening Of HBK-SPF Duck And Bioinformatics Analysis Of TAP Gene

HBK-SPF duck has been developed from the indigenous Shaoxing duck breed at the HarbinVeterinary Research Institution, CAAS. There are two lines of HBK-Q and HBK-B, both at their eighthgenerations. TAP molecules are part of the MHC class I antigen-processing pathway, through which theendogenous antigen is transported into the lumen of endoplasmic reticulum (ER), followed by itspresentation to MHC class I molecules and subsequently binding to the surface of CD8+T cells.In this study, the genetic backgrounds of HBK-SPF duck lines at their F5and F6generations werefurther investigated using16microsatellite DNA markers. A total of71alleles were identified at the16loci across all birds while the average effective number of alleles was4.14per locus. Compared to theF2and F3generations, the numbers of observed alleles have been declining at most of loci overgenerations, the numbers of loci deviated from Hardy-Weinberg equilibrium have been reduced to twoin HBK-SPF-Q and four in HBK-SPF-B lines of both F5and F6generations, and the numbers of lociwith polymorphic information contents (PICs) higher than0.5have also been decreased with averagePICs as of0.511in HBK-SPF-Q and0.512in HBK-SPF-B of F5generation as well as0.480inHBK-SPF-Q and0.505in HBK-SPF-B of F6generation, however, both observed and expectedheterozygosities remained at similar levels across the generations, an indication of the rapid loss of ‘rarealleles’ in small populations following genetic bottlenecks leading to inbreeding. Nevertheless, it wasevident that the changes in allelic number and combination of predominant and minor alleles at majorityof loci followed an expected pattern across generations of HBK-SPF-Q line with relatively stablepopulation sizes while the number of alleles was reduced in F3generation but increased slightlyafterwards in HBK-SPF-B line due to their fluctuated population sizes.Four putative short tandem repetitive DNA sequences (A-D) were predicted in the37kb longMHC I region partial DNA sequence deposited in the GenBank using SSR Hunter software. One pair ofprimers was then designed for A and D loci each while five pairs of primers were synthesized for B andC loci each. The PCR products were resolved based on SSCP analyses and subsequent direct DNAsequencing using relevant PCR primers. The results showed that only A locus was polymorphic in bothSSCP gels and DNA sequences and four homozygous fragments defined by a number of SNPs presentin108bp upstream and127bp downstream sequences flanking the (GT)n tandem repeat were identifiedin close linkage to different numbers of the (GT)n motifs. They were named as B1, B2, B3and B4haplotypes following the nomenclature system of chicken MHC B complex. The different homozygousindividuals were screened using DNA sequencing data and then selected for homogamic mating toestablish homozygous duck MHC lines from F0generation maintained in isolates with positive pressure.Samples and birds of F1generation were employed for further investigation of the duck MHC structureand function.TAP1and TAP2genes were completely sequenced in the four duck MHC haplotypes at around8kb. Compared to the reference sequence from GenBank, their nucleotide homologies were around97.0%. The length of TAP1gene was4061-4187bp and97.0%of nucleotide identity was found among the four haplotypes and reference sequence while their nucleotide homologies were at66.0%and67.0%~68.0%against chicken and quail, respectively. The length of coding sequence was1218bp,coding for406amino acids with eight variable residues based on the alignment against the referencesequence. The predicted duck TAP1molecule has four trans-membrane domains (TMDs), locatedtowards its N terminal with highly conserved sequences.The length of TAP2genomic DNA sequencewas around3013~3045bp among the four haplotypes with97.0%of nucleotide identity amongthemselves and the reference sequence. It shared77.0%of nucleotide identity with both chicken andquail. The length of coding sequence was2103bp, coding for701amino acids with24variable residuesamong the four haplotypes as well as27and26variable residues compared to the reference TAP2A andTAP2B protein sequences, suggesting its higher polymorphism at protein level than the TAP1molecule.Duck TAP2molecule was predicted to have seven TMDs, located at its N terminal. There were7variable residues in its peptide-binding domains, seven amino acids which determining peptidespecificity, there is a mutation form F to G at263sites for B2hapoltype, which were probablyassociated to its peptide presentation specificity.The result of RA toxicity attack test revealed no significant difference in the mortality among theduck B1, B2, B3homozygous lines and heterozygosis lines. However, the DHV-I toxicity attack testrevealed a significantly lower mortality in B haplotype (36%) than the other three haplotypes(73%~93%), an strong indication of specific TAP functions in responding to different pathogeninfections warranted for further investigation.