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

Characterization of two new alleles at the goat CSN1S2 locus.

Two novel alleles at the goat CSN1S2 locus have been identified: CSN1S2(F) and CSN1S2(D). Sequence analyses revealed that the CSN1S2(F) allele is characterized by a G --> A transition at the 13th nucleotide in exon 3 changing the seventh amino acid of the mature protein from Val to Ile. The CSN1S2(D) allele, apparently associated with a decreased synthesis of alpha s2-casein, is characterized by a 106-bp deletion, involving the last 11 bp of the exon 11 and the first 95 bp of the following intron. Methods (PCR-RFLP and PCR) for identification of carriers of these alleles have been developed

Italian Mediterranean river buffalo CSN2 gene structure and promoter analysis

The nucleotide sequence of the whole buffalo β-casein encoding gene (CSN2) plus 1,476 nt at the 5' flanking region and 51 nt at the 3' flanking region was determined. The gene is spread over 10.2 kb and consists of 9 exons varying in length from 24 (exon 5) to 498 bp (exon 7) and 8 introns from 92 bp (intron 5) to 22 59 bp (intron 1). Furthermore, highly conserved sequences, mainly located in the 5' flanking region, were found between this gene and the β-casein encoding genes of other species. The comparison between the obtained promoter and exonic regions and buffalo sequences present in EMBL evidenced different polymorphic sites. Finally, 5 interspersed repeated elements (4 in the bovine CSN2 gene) were also identified at 3 different locations of the sequenced region: 5' untranscribed region, intron 1, and intron 4

Use of multivariate factor analysis to characterize the fatty acid profile of buffalo milk

The suitability of multivariate factor analysis (MFA) to extract a small number of latent variables able to explain the correlation pattern among fatty acids (FA) in buffalo milk was evaluated. FA profile of milk samples from 214 Italian water buffaloes was analysed by gas chromatography. MFA, performed on the correlation matrix of 52 FA, was able to extract 10 latent factors with specific biological meaning related to a common metabolic origin for FA associated with the same factor. Scores of the factors were treated as new quantitative phenotypes to evaluate the effect of age, month of calving and lactation stage. MFA approach was effective in describing the FA profile of buffalo milk by using a low number of new latent variables that clustered FA having similar metabolic origin and function. The new variables were also useful to test the effect of environmental and individual animal factors on milk FA composition

Spontaneous rate of sister chromatid exchanges (SCEs) in mitotic chromosomes of sheep (Ovis aries L.) and comparison with cattle (Bos taurus L.), goat (Capra hircus L.) and river buffalo (Bubalus bubalis L.).

The spontaneous level of sister chromatid exchanges (SCEs) in the sheep, estimated by exposing peripheral blood lymphocytes in 0.1 microgram/ml of 5'-bromodeoxyuridine (BrdU), was 4.08 +/- 2.47 SCE/cell, 2.04 SCE/cell cycle, 0.038 SCE/chromosome. The dose-response relationships, observed by exposing the cells to 0.1, 0.25, 0.5, 1.0, 2.5, and 5.0 micrograms/ml of BrdU, rose rapidly from 0.1 to 0.25 microgram/ml, and less rapidly at higher concentrations, thus reaching a saturation level. The analysis of variance, performed on the square root transformed data at 0.1 and 5 micrograms/ml of BrdU, indicated significant differences (P < 0.001) among the four donors tested. The distribution of the SCE/cell frequencies in the cell population of the four donors followed the Poisson 'mixture' probability function, thus confirming previous findings. The spontaneous rate of SCE/cell of sheep is compared with those previously reported for cattle, goat and river buffalo. The theoretical and practical implications of the spontaneous sister chromatid exchanges are discussed in relation to their possible use in animal production for (a) better genetic evaluation of the breeding animals under selection, (b) more precise monitoring of the genotoxic effects of environmental pollutants

Genetic variability detected at the lactoferrin locus (LTF) in the Italian Mediterranean river buffalo

Lactoferrin (LTF) is multi-functional protein belonging to the whey protein fractions of the milk. The gene LTF encoding for such protein is considered a potential candidate for body measurement, milk composition and yield. This study reports on the genetic variability at LTF locus in the Italian Mediterranean river buffalo and its possible association with milk yield. Eleven polymorphic sites were found in the DNA fragment spanning the exons 15-16. In particular, the intron 15 was extremely polymorphic with 9 SNPs detected, whereas the remaining 2 SNPs were exonic mutations (g.88G>A at the exon 15 and g.1351G>A at the exon 16) and both synonymous. The genotyping of the informative samples evidenced 3 haplotypes, whose frequencies were 0.6; 0.3 and 0.1 respectively, whereas the analysis of the exonic SNPs showed a perfect condition of linkage disequilibrium (g.88A/g.1351G and g.88G/g.1351A). The association study carried out by using the SNP g.88G>A showed that buffalo LTF gene has no statistically significant influence on daily milk yield. This study adds knowledge to the genetic variability of a species less investigated than the other ruminant species, that may serve as a useful tool for large-scale screening of buffalo populations

Design and validation of a 90K SNP genotyping assay for the water buffalo (Bubalus bubalis)

Background: The availability of the bovine genome sequence and SNP panels has improved various genomic analyses, from exploring genetic diversity to aiding genetic selection. However, few of the SNP on the bovine chips are polymorphic in buffalo, therefore a panel of single nucleotide DNA markers exclusive for buffalo was necessary for molecular genetic analyses and to develop genomic selection approaches for water buffalo. The creation of a 90K SNP panel for river buffalo and testing in a genome wide association study for milk production is described here. Methods: The genomes of 73 buffaloes of 4 different breeds were sequenced and aligned against the bovine genome, which facilitated the identification of 22 million of sequence variants among the buffalo genomes. Based on frequencies of variants within and among buffalo breeds, and their distribution across the genome, inferred from the bovine genome sequence, 90,000 putative single nucleotide polymorphisms were selected to create an Axiom® Buffalo Genotyping Array 90K. Results: This 90K "SNP-Chip" was tested in several river buffalo populations and found to have ∼70% high quality and polymorphic SNPs. Of the 90K SNPs about 24K were also found to be polymorphic in swamp buffalo. The SNP chip was used to investigate the structure of buffalo populations, and could distinguish buffalo from different farms. A Genome Wide Association Study identified genomic regions on 5 chromosomes putatively involved in milk production. Conclusion: The 90K buffalo SNP chip described here is suitable for the analysis of the genomes of river buffalo breeds, and could be used for genetic diversity studies and potentially as a starting point for genome-assisted selection programmes. This SNP Chip could also be used to analyse swamp buffalo, but many loci are not informative and creation of a revised SNP set specific for swamp buffalo would be advised.Daniela Iamartino, Ezequiel L. Nicolazzi, Curtis P. Van Tassell, James M. Reecy, Eric R. Fritz-Waters, James E. Koltes, Stefano Biffani, Tad S. Sonstegard, Steven G. Schroeder, Paolo Ajmone-Marsan, Riccardo Negrini, Rolando Pasquariello, Paola Ramelli, Angelo Coletta, José F. Garcia, Ahmad Ali, Luigi Ramunno, Gianfranco Cosenza, Denise A.A. de Oliveira, Marcela G. Drummond, Eduardo Bastianetto, Alessandro Davassi, Ali Pirani, Fiona Brew, John L. William