To enable the efficient analysis of a highly polymorphic swine major histocompatibility complex (MHC) class II gene, swine leukocyte antigen (SLA)-DQB1, we developed a simple and comprehensive high-resolution genotyping protocol. To obtain sufficient sequence information to design a set of common genotyping primers for SLA-DQB1, we cloned SLA-DQB1 introns 1 and 2 from 11 alleles with official four-digit allelic designations and sequenced the regions directly surrounding the SLA-DQB1 exon 2. Significant intronic nucleotide variations, including several deletions, were identified. Based on 733-bp assembled genomic sequences including introns 1 and 2 and exon 2 from 11 different alleles, a primer set was identified that allowed the ubiquitous amplification and analysis of the complete SLA-DQB1 exon 2 sequence. We then developed a method to directly sequence the amplified polymerase chain reaction (PCR) products without further experimental steps. We especially focused on avoiding superimposed peaks, which arose from the presence of allelic deletions, in the sequencing electropherogram of SLA-DQB1 heterozygous animals. The genotyping accuracy was evaluated by comparing the results of genomic sequence-based typing (GSBT) with those of other available methods, including cDNA sequence-based typing (SBT), low-resolution PCR typing with sequence-specific primers, allelic segregation analysis, and heterozygote simulation typing. In all cases, the results were consistent between SLA-DQB1 GSBT and previously reported methods or expected results. We applied it to genotype 350 animals from seven pig breeds. The observed level of heterozygosity from our genotyping was ∼51%, reflecting that a large portion of the animals were inbred miniature pigs. Among the seven pig breeds tested, the allelic diversity of SLA-DQB1 was highest in Berkshire pigs. In conclusion, we have developed a simple and effective SLA-DQB1 GSBT method by combining simple genomic DNA PCR and direct sequencing. Our new method may aid in the study of SLA diversity and disease resistance and susceptibility.

Sequences from the clones of full-length enriched cDNA libraries serve as valuable resources for functional genomic studies. We have analysed 1970 high-quality chromatograms (Phred value >or= 30) that were obtained from sequencing the 5' ends of brainstem, liver, neocortex and spleen clones derived from full-length enriched cDNA libraries from Korean native pigs. In addition, 50,000 pig expressed sequence tag (EST) sequence trace files were obtained from Genbank and combined with our sequencing information to facilitate SNP identification in silico. The process generated 8118 contigs, of which 239 included minimum one sequence from Korean native pig and contained 678 putative coding single nucleotide polymorphisms (cSNPs). Of these, 33 putative cSNPs were randomly selected for confirmatory analysis and validated using 20 pigs from four different breeds (Duroc, Landrace, Yorkshire, Korean native pig). Of the 33 putative cSNPs, 20 were confirmed (61%), which was similar to the frequency reported in other studies. We also identified 15 new cSNPs from the validation process, which were not detected by our in silico analysis. Our study shows that analysing genetically diverse pig breeds including the Korean native pig could serve as a useful strategy for generating a large number of cSNPs.

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A novel human leukocyte antigen, A*24 (HLA-A*24), was identified in the Korean population. HLA-A*2474 allele shows one nucleotide difference from A*24020101 in exon 2 at nucleotide position 186 (C --> A), resulting in an amino acid change, Ser38Arg.