Systematic analysis of swine leukocyte antigen-DRB1 nucleotide polymorphisms using genomic DNA-based high-resolution genotyping and identification of new alleles.

In an attempt to enable comprehensive high-resolution genotyping of the swine leukocyte antigen (SLA) gene, we performed a systemic analysis of nucleotide polymorphisms at introns 1 and 2 and exon 2 from diverse alleles of SLA-DRB1 and DRB1 pseudogenes. We amplified and cloned 16 partial sequences of SLA-DRB1 and DRB2 introns 1 and 2 from different alleles, and analyzed them together with sequences of four reported SLA-DRB pseudogenes, DRB2, 3, 4, and 5. The results showed the presence of extreme nucleotide variations within introns 1 and 2 of SLA-DRB-related genes including substitutions and deletions. On the basis of these results, we developed a comprehensive genotyping method for SLA-DRB1 by genomic polymerase chain reaction (PCR) and subsequent direct sequencing. A total of 415 animals were genotyped and 67 allelic combinations from 18 DRB1 alleles were identified. Among them, two alleles, SLA-DRB1*kn04 and *kn05, were previously unreported. SLA-DRB1 genotyping results from this study combined with those of SLA-DQB1 from our previous study presented 10 SLA class II haplotypes, three of which were previously unreported. Population analysis using seven different pig breeds showed differences in the allele frequency of SLA-DRB1 among breeds. Our results should benefit biological experiments requiring sequence-level genotyping results of SLA-DRB1 and further study of the complete genetic diversity of SLA-DRB1 using field samples.