Sequence variations of the locus‐specific 5′ untranslated regions of SLA class I genes and the development of a comprehensive genomic DNA‐based high‐resolution typing method for SLA‐2

Abstract

The genetic diversity of the major histocompatibility complex (MHC) class I molecules of pigs has not been well characterized. Therefore, the influence of MHC genetic diversity on the immune-related traits of pigs, including disease resistance and other MHC-dependent traits, is not well understood. Here, we attempted to develop an efficient method for systemic analysis of the polymorphisms in the epitope-binding region of swine leukocyte antigens (SLA) class I genes. We performed a comparative analysis of the last 92 bp of the 5' untranslated region (UTR) to the beginning of exon 4 of six SLA classical class I-related genes, SLA-1, -2, -3, -4, -5, and -9, from 36 different sequences. Based on this information, we developed a genomic polymerase chain reaction (PCR) and direct sequencing-based comprehensive typing method for SLA-2. We successfully typed SLA-2 from 400 pigs and 8 cell lines, consisting of 9 different pig breeds, and identified 49 SLA-2 alleles, including 31 previously reported alleles and 18 new alleles. We observed differences in the composition of SLA-2 alleles among different breeds. Our method can be used to study other SLA class I loci and to deepen our knowledge of MHC class I genes in pigs.

SLA-1 Genetic Diversity in Pigs: Extensive Analysis of Copy Number Variation, Heterozygosity, Expression, and Breed Specificity

“…Several inbred lines were selected as homozygous for SLA in NIH miniature pigs for biomedical studies 38 . Previous and current studies showed the establishment of homozygosity in class II (observed heterozygosity, DRB1-0%; DQA1-0%; DQB1-34.72%) but not in class I genes (observed heterozygosity, SLA-1, 83% and SLA-2, 46.6%) 18,[20][21][22] for the breed. Our analysis on SLA-1 diversity in NIH miniature pigs, however, showed that the high SLA-1 (83%) heterozygosity was not due to the actual genetic diversity at the chromosomal level but to the presence of a haplotype with duplicated SLA-1 (Table 3).…”

“…SLA-2 also showed a lower allele sharing among breeds with a large number (n = 28) of breed-specific alleles 21 . The allele SLA-2*04:01 was the most shared allele for SLA-2 among the major pig breeds and belongs to the same www.nature.com/scientificreports www.nature.com/scientificreports/ haplotype as SLA-1*04:01 (Hp-4) 28 .…”

“…The preparation of genomic DNA and mRNA is described as Supplementary Information. To determine SLA-1 specific primer binding sites, 2086 bp amplicons corresponding to the region from 5′ UTR to the middle of exon 4 of SLA class I genes were produced by PCR with 0.5 μM SLA class I specific primers (SLA-CATF and SLA-R, Table 1) as described previously 21 . For genomic DNA-based typing, SLA-1 specific amplicons were generated in a 20-μL reaction containing 50-100 ng of genomic DNA, 0.5 μM specific primers (SLA1-e1F1 and SLA e4R4) ( Table 1), 200 μM dNTPs, 1 × PCR buffer, and 0.5 U of Supertherm ™ DNA polymerase (JMR Holdings, Kent, UK) using a ABI 9700 Thermocycler (Applied Biosystems).…”

“…SLA-1 exons 2 and 3 sequence information is the minimum requirement to officially assign alleles of SLA class I genes 28 . It has been proven that combining the locus-specific PCR and subsequent direct sequencing using independent primers is a successful method for the comprehensive typing of hyper polymorphic MHC genes 18,[20][21][22] . Taking the advantage of the small sizes of the introns surrounding the exons 2 and 3, we succeeded in comprehensively amplifying the 1844-bp SLA-1-specific amplicons from all the tested samples ( Fig.…”

“…However, these DNA based typing methods still require further improvement in the resolution of the typing results, comprehensiveness in allele coverage, and usability for large-scale typing. To improve these, we previously developed the genomic sequence-based high-resolution typing (GSBT) methods for SLA-2, -DQA, -DQB1, and -DRB1, and presented the results of new allele identification using a large number of field samples and population genetic analysis on diverse breeds [18][19][20][21][22] However, the precise typing of SLA-1 has been particularly difficult because of the presence of a large number of novel alleles and copy number variations (CNVs) of the locus. Thus, the development of a robust typing method for SLA-1 is necessary.…”