A point mutation in the splice donor site of intron 7 in the as2-casein encoding gene of the Mediterranean River buffalo results in an allele-specific exon skipping

The CSN1S2 cDNA of 10 unrelated Mediterranean River Buffaloes reared in Southern Italy was amplified by RT-PCR, while the region from the 6th to the 8th exon of the CSN1S2 gene was amplified from genomic template. cDNA sequence comparisons showed that five individuals had a normal transcript only (named CSN1S2A), one had a deleted transcript only (named CSN1S2B), because of the splicing out of the 27-bp of exon 7, and the remaining four had a heterozygous pattern. Analysis of the genomic sequences revealed a FM865620: g.773G>C transversion that caused inactivation of the intron 7 splice donor site and, consequently, the allele-specific exon skipping characteristic of the CSN1S2B allele. The g.773G>C mutation creates a new AluI restriction site enabling a PCR– RFLP rapid genotyping assay. The cDNA sequences showed three additional exonic mutations forming an extended haplotype with the g.773G>C polymorphism: FM865618: c.459C>T, c.484A>T and c.568A>G homozygous and heterozygous respectively in the CSN1S2BB and CSN1S2AB buffaloes. The first is silent, while the remaining two are non-conservative (p.Ile162Phe and p.Thp200Ala respectively). The genotype frequencies (37 CSN1S2A/A, 15 CSN1S2A/B and one CSN1S2B/B) are in agreement with Hardy–Weinberg equilibrium, with the frequency of the deleted B allele being 0.16. The predicted bubaline as2B protein is 198 aa long instead of 207 aa and would also be characterized by the presence of Phe at position 147 and Ala at 185

Genotyping at the CSN1S1 locus by PCR-RFLP and AS-PCR in a Neapolitan Goat Population

The goat CSN1S1 gene has for many years been an excellent model for demonstrating that most of the variability observed in the as1-casein content in goat’s milk is due to the presence of autosomal alleles at a single structural locus. Until now, about 17 alleles associated to at least four levels of as1-casein expression in milk have been described at the CSN1S1 locus in the domestic goat (Capra hircus). The great importance of goat as1-casein polymorphism is due to its qualitative as well as quantitative implications. In the present work five PCR protocols (PCR-RFLPs, AS-PCR) were set up for rapid genotyping of B1, B2*, B3, B4 and C CSN1S1 alleles, until nowdetectable only by milk electrophoresis. Application of these protocols, together with previously described methods to identify CSN1S1 01, E, M, F, N and A* (CSN1S1 A, G, I, H) alleles, allow us to define, at DNA level, the genetic structure of the autochthonous goat reared in the province of Naples for the highest number of possible alleles at this locus. Monitoring of CSN1S1 variability in the Neapolitan goat population indicates a high frequency of low (F, 0.368) and null (N, 0.227) alleles

Preliminary analysis of Stearoyl Co-A Desaturase gene transcripts in River buffalo

Stearoyl-CoA desaturase (SCD) is a key enzyme in the biosynthesis of monounsaturated fatty acids (MUFAs). In cattle, SCD gene extends over a DNA segment of ~17.0 Kb, and it is organized in 6 exons and 5 introns. The SCD gene has been indicated as the candidate gene to change the saturated/unsaturated FAs ratio and hence it has been suggested as the gene influencing the fat quality. In cattle, eight SNPs have been identified and one of them, (T→C) at 231st nt of 5th exon, is responsible for the Val→Ala amino acid change. The C allele has been associated with higher content of MUFAs in carcasses, and it is positively related to a higher index of desaturation (C18:0/C18:1 and C16:0/C16:1) in the milk. In this study, we report on preliminary results of analysis of transcripts of the SCD encoding gene in river buffalo. The electrophoretic analysis of the RT-PCR products and the subsequent sequencing showed at least five different populations of mRNA. The most represented population is correctly assembled (~1300 bp), followed by the one which is deleted of ~750bp, corresponding to the 3rd, 4th and 5th exon and partially to the 2nd and 6th exon