Molecular genetic characterization of ovine beta-lactoglobulin C allele and detection by PCR-RFLP

Introduction β-Lactoglobulin (LGB), the major whey protein in sheep, consists of 162 amino acids and forms stable dimers in milk. Apart from its ability to bind and transport small hydrophobic molecules in milk, e.g. retinol and small fatty acids, its biological function is still unclear (Godovac-Zimmermann et al. 1985; MacLeod et al. 1996). A positive effect on the digestion of milk lipids has been suggested (Perez et al. 1992). In nonruminant species two different forms, LGB I and II, are found, but in the milk of cattle, sheep and goat only one LGB is expressed. An LGB-pseudogene, lacking an active promotor was characterized by Passey and Mackinlay (1995). The deduced amino acid sequence is closely related to LGB II found in cat and horse, while the expressed LGB corresponds to LGB I. The LGB-pseudogene was mapped close to the LGB genes in cattle, goat and sheep (Passey and Mackinlay 1995; Folch et al. 1996). Three allelic variants (A, B and C) of ovine LGB can be discriminated using different electrophoretic techniques. Variants A and B are present in all breeds and differ by a Tyr/His substitution in position 20 (Kolde and Braunitzer, 1983) corresponding to a single nucleotide substitution in the LGB gene (Ali et al. 1990). A molecular genetic test for the differentiation of the A and B alleles by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (RsaI) was developed (Schlee et al. 1993). The C variant was described first in 1989 in German Merinoland sheep at a frequency of 0.175 and in Hungarian Merino crosses (Erhardt 1989). In Spanish Merinos the C allele was found at low frequencies (0.013), too (Recio et al. 1995). There is little information available concerning the correlations of different LGB genetic variants with milk yield, protein content, cheese-making ability and other economic traits in sheep (Bolla et al. 1988; Garzon and Martinez 1992; Recio et al. 1995), but significant effects of LGB genotypes on whey and the resulting total protein content (Ng-Kwai-Hang et al. 1990; van den Berg et al. 1992; Hill 1993) and milk fat content (Aleandri et al. 1990; Falaki et al. 1997) were found in cattle. As the Merino breeds are one of the most widespread breed groups in the world, the inclusion of the LGB*C allele in further investigation of correlations of LGB genotypes with quantitative or economic traits in sheep is highly desirable. In addition, the occurrence of rare variants might help to clarify breed relationships. Examination of the LGB*C primary structure revealed a single amino acid substitution (Arg/Gln) in position 148 compared to the most common A allele (Erhardt et al. 1989). The test described by Schlee et al. (1993) does not discriminate LGB*C from LGB*A and leads to misidentification in animals with LGB*C. According to Ali et al. (1990) and Harris et al. (1993) amino acids 117–150 are encoded in exon V of the gene. Therefore this region was examined in the present study. Parts of exon V to VI of the gene were PCR-amplified, cloned and sequenced. Based on the sequencing results a PCR-RFLP typing procedure for identification of LGB*C was developed.