Simple and comprehensive SLA-DQB1 genotyping using genomic PCR and direct sequencing

Area of Science:

• Immunogenetics
• Molecular Biology
• Animal Genetics

Background:

• The swine major histocompatibility complex (MHC) class II gene, swine leukocyte antigen (SLA)-DQB1, is highly polymorphic.
• Efficient high-resolution genotyping is crucial for analyzing SLA-DQB1 diversity and its association with disease resistance.

Purpose of the Study:

• To develop a simple, comprehensive, and high-resolution genotyping protocol for the SLA-DQB1 gene.
• To enable efficient analysis of SLA-DQB1 allelic variations in pigs.

Main Methods:

• Cloned and sequenced introns 1 and 2 and surrounding regions of SLA-DQB1 exon 2 from 11 alleles.
• Designed a primer set for ubiquitous amplification of the complete SLA-DQB1 exon 2.
• Developed a direct sequencing method for PCR products, addressing challenges with allelic deletions in heterozygotes.

Main Results:

• Identified significant nucleotide variations and deletions in SLA-DQB1 introns.
• Established a primer set enabling amplification and analysis of the complete SLA-DQB1 exon 2.
• Validated the genomic sequence-based typing (GSBT) method against other typing techniques, showing consistent results.
• Genotyped 350 pigs across seven breeds, revealing high heterozygosity and the greatest SLA-DQB1 allelic diversity in Berkshire pigs.

Conclusions:

• A simple and effective SLA-DQB1 GSBT method combining genomic DNA PCR and direct sequencing was developed.
• This method provides accurate, high-resolution genotyping for SLA-DQB1.
• The developed protocol can facilitate studies on SLA diversity, disease resistance, and susceptibility in swine populations.