THE PCR TYPING OF MHC-DRB GENES IN THE SHEEP USING PRIMERS FOR AN INTRONIC MICROSATELLITE - APPLICATION TO NEMATODE PARASITE RESISTANCE

The strong association between polymorphisms in an intronic microsatel lite and the coding sequences for (BoLA)-DRB3 genes, previously descri bed for demonstrating alleles of class II major histocompatibility com plex (MHC) in the cow, was examined in sheep to see if similar polymor phisms could be demonstrated in the DRB region of the MHC. The bovine primers LA53 and LA54, previously used to amplify the bovine DRB3 micr osatellites, were used with DNA from Australian sheep, eight DRB allel es were identified by length polymorphisms of polymerase chain reactio n (PCR) products amplified from the DRB microsatellite region. Incompl ete amplification of both alleles was sometimes found for sheep DNA sa mples using bovine primers, so a modified primer (LA53b) was used, and found to amplify the microsatellite next to intron 2 of the MHC more reliably than the LA53 primer. Two additional primers (LA31 and LA32), used in amplification of the exon 2 region of bovine DRB3, were used in the sheep, and the PCR products were analysed by single-stranded co nformation polymorphism (SSCP). These primers successfully amplified t he variable region of the ovine DRB region coded by exon 2, and the SS CP technique demonstrated polymorphisms with sheep DNA. Family studies demonstrated the segregation of alleles, by amplification both of int ronic microsatellites and of the exon 2 variable region. Close corresp ondence was found between the two regions for several alleles, suggest ing that the intronic microsatellites were closely linked to DRB-varia ble region alleles. Three families of Merino sheep with different anti body responses to intestinal nematode parasites were examined. The sir e group with the highest antibody levels possessed two microsatellite alleles of closely similar length (alleles 3 and 4) inherited from the sire and present in high frequency in the lambs. In contrast, the oth er two sires did not possess these two alleles and the alleles were in low frequency in their progeny. Further studies are required in unrel ated sheep to confirm whether these two alleles are associated with re sistance to nematode parasites.