Simultaneous detection of a sex-specific sequence and the Ryr1 point mutation in porcine genomic DNA

The advantages are becoming increasingly apparent of designing livestock breeding programmes around the detection of specific sequences in genomic DNA using amplification by the polymerase chain reaction (PCR). Furthermore, by subjecting the products of such reactions to restriction enzyme digestion, important information conveyed by single-base substitutions can be retrieved and used in marker-assisted selection. The potential for the rapid diagnosis of several DNA markers simultaneously would seem to offer particular benefits in the field of in vitro fertilisation and embryo transfer, where only a few cells constitute the source of the DNA, and where keeping the duration of the tests to a minimum is imperative. However, where the markers to be detected fall into different categories, different kinds of amplification reactions may need to be combined. The present study with porcine DNA combines a one-step multiplex PCR test for sex-determination with a specialised PCR reaction designed to diagnose the Ryrl or 'halothane' genotype. A total of seven primers have been utilised to amplify by, firstly, a control sequence related to the Zfx/y genes present in both sexes, secondly to amplify a Y chromosome sex-specific sequence related to the Sry gene and lastly, to detect either allele of the Ryr1 mutation associated with porcine stress syndrome and pale, soft exudative meat. The presence of PCR products of characteristic size on agarose gel electrophoresis gives a visual read-out of animal sex and halothane genotype. Although primarily a model system, the test may have direct applications in the context of embryo transfer, sperm separation technology and also in the characterisation of pork samples undergoing sensory evaluation by meat scientists.