Identification of carriers of the Welsh CASA1 variant using an allele-specific polymerase chain reaction method

In sheep, an electrophoretic CASA1 variant (called Welsh) was first observed by King.1 This variant has subsequently been found in various breeds with an extremely low frequency. Only in a flock of Sarda breed did this variant show a relatively high frequency (0·21). The Welsh variant is characterized by the substitution SerP-68-Asn-68.3 The aim of the present study was to develop a DNA-based method for identifying, directly and at birth, carriers of the Welsh CASA1 variant. The research was carried out on individual milk and DNA samples obtained from 180 ewes belonging to the Sarda breed. Individual milk samples were analysed by polyacrylamide gel electrophoresis at alkaline pH (UREA–PAGE). Primers were designed according to the nucleotide sequence of the ovine CASA1C cDNA located in the EMBL Database (acc. no. X03237). Allele-specific primers are part of the 9th exon and differ in the mutation (transition A-G) that is responsible for the SerP-68-Asn-68 amino acid substitution. The common primer sequence is part of the 10th exon. The amplified fragment has a length of about 800 bp and spans part of the 9th exon, the 9th intron and part of the 10th exon. The asl-Cn genotype determined by ASPCR in 180 ewes (158 homozygotes for the ‘non-Welsh’ allele, two homozygotes and 20 heterozygotes for the ‘Welsh’ allele) agreed in all cases with the phenotype determined by milk electrophoresis. Typing of the two homozygotes for the ‘Welsh’ allele has been accomplished four times with identical results.