Methylation-specific PCR analysis in Col8A1 promoter in Creole cattle carrier of rob(1; 29)

Robertsonian translocation (rob(1;29)) is the most frequent structural chromosomal abnormality in cattle. Heterozygous carriers have a normal phenotype but show a 3-5 percent decrease in fertility. Chromatin decondensation was evaluated similar to the inactive X chromosome when submitted to demethylating agent. Based on this result, and the concept that imprinted genes are essential in embryonic development, we decided to query genes located on BTA1 and BTA29 that could undergo genome imprinting. The collagen typeVIII- alpha 1 (Col8A1) acted on extracellular matrix structural proteins. DNA bisulfite conversion and sequentiation methods were used to compare its differential methylation patterns. It was performed on eight Creole cattle DNA blood samples from normal and rob(1;29) carriers. An in silico screening for CpG islands in its promoter uncovered a single region of 454 bp prone to methylation. BiQ-Analizer software was used to show the selective conversion of unmethylated cytosines to uracils obtaining the following results: unmethylated CpGs: 0.000 (0 cases), methylated CpGs: 0.802 (77 cases) and CpGs not present: 0.198 (19 cases). No differences between samples were observed in this highly methylated region. This technique was successfully applied so it is a straightforward methodology that can be utilized to evaluate different tissue associated to specific gene expression.

Analysis of the population structure of Uruguayan Creole cattle as inferred from milk major gene polymorphisms

The ancestors of Uruguayan Creole cattle were introduced by the Spanish conquerors in the XVII century, following which the population grew extensively and became semi-feral before the introduction of selected breeds. Today the Uruguayan Creole cattle genetic reserve consists of 575 animals. We used the tetra primer amplification refractory mutation system polymerase chain reaction (ARMS-PCR) to analyze the kappa-casein, beta-casein, alphaS1-casein and alpha-lactoalbumin gene polymorphisms and restriction fragment length polymorphism PCR (RFLP-PCR) for the beta-lactoglobulin and the acylCoA:diacyl glycerol acyltransferase 1 (DGAT1) genes. The kappa-casein and beta-lactoglobulin genes presented very similar A and B allele frequencies, while the alphas1-casein and alpha-lactoalbumin gene B alleles showed much higher frequencies than the corresponding A alleles. The beta-casein B allele was not found in the population sampled. There was a very high frequency of the DGAT1 gene A allele which is associated with low milk fat content and high milk yield. All loci were in Hardy-Weinberg equilibrium and the level of heterozygosity agreed with the high genetic diversity observed in a previous analysis of this population. Preservation of the allelic richness observed in the Uruguayan Creole cattle should be considered for future dairy management and livestock genetic improvement. The results also emphasize the value of the tetra primers ARMS-PCR technique as a rapid, easy and economical way of genotyping cattle breeds for milk gene single nucleotide polymorphisms.

Microsatellite analysis of a sample of Uruguayan Creole bulls (Bos taurus)

The Uruguayan Creole cattle genetic reserve consists of a herd of about 600 animals (bulls, cows and calves) located in an indigenous habitat of 650 hectares. In a previous study, a random sample from this herd showed high heterozygosity and a Hardy-Weinberg equilibrium for markers of major genes related to milk production. To study its genetic diversity we genotyped a sample of bulls (N = 19 out of 23 for the whole herd) using the PCR reaction with a set of 17 microsatellite markers. Between two and seven different alleles were identified per microsatellite in a total of 73 alleles. The expected mean heterozygosity (He) per locus was between 0.465 and 0.801, except for microsatellite HEL13 which gave a He value of 0.288. The expected mean heterozygosity was 0.623 and the polymorphic information content (PIC) was between 0.266 for HEL13 and 0.794 for CSSM66. The genetic diversity found in polymorphic markers in the breeding bulls of this Creole cattle population supports previous genetic analyses using major production genes and indicate that further studies should be carried out on this population to provide data of interest to cattle production.