Haplotype determination of the upstream regulatory region and the second exon of the BoLA-DRB3 gene in Holstein cattle.

Polymorphisms of the BoLA-DRB3 gene are located primarily in the second exon [antigen binding site (ABS)] and, to a lesser extent, in the upstream regulatory region (URR). It can be hypothesised that exon 2 and the URR are under different types of natural selection. The aim of this work was to determine the URR-exon 2 haplotypes; 34 Holstein samples were genotyped by direct sequencing. A total of 7 URR alleles and 23 exon 2 alleles were detected, and 3 of the URR alleles were novel. Our results may suggest that no relationship exists between the URR and exon 2 of the BoLA-DRB3 gene (linkage disequilibrium P value > 0.05), most likely due to recombination over time. Our results also suggest that both regions of class II genes may be included in the development of new genotyping methods based on next-generation DNA sequencing technologies.

Association between BoLA-DRB3 and somatic cell count in Holstein cattle from Argentina.

Different studies have proved that the resistance/susceptibility to mastitis is genetically determined. The major histocompatibility complex in cows is known as bovine lymphocyte antigen (BoLA). Genes from the BoLA have been associated with the occurrence of infectious diseases such as mastitis and leukosis, especially the BoLA-DRB gene. The object of the present study was to detect associations between BoLA-DRB3 alleles and somatic cell count (SCC), as an indicator of resistance/susceptibility to mastitis in Holstein cattle (N = 123) from La Pampa, Argentina. Fisher's exact test and Woolf-Haldane odds ratio were applied to study the association between SCC and BoLA-DRB3 allele frequencies. Significant association was noted between BoLA-DRB3.2*23 and *27 alleles (p < 0.05) and protective or susceptibility effects, respectively. In addition, alleles BoLA-DRB3.2*20 and *25 exhibit suggestive association with high SCC (p < 0.1). These results were partially in agreement with data reported from Japanese Holstein cattle, but differed from those published by other authors. A possible explanation for the contrasting results could be that the mastitis is a multifactor disease caused by different pathogens. Moreover, most of the studies were carried out using PCR-RFLP method, which has less resolution than PCR-SBT because PCR-RFLP defined alleles included more than one sequenced alleles.

Analysis of a polymorphism in the DGAT1 gene in 14 cattle breeds through PCR-SSCP methods.

The diacylglycerol O-acyltransferase (DGAT1) is a microsomal enzyme that catalyzes the final step of triglyceride synthesis. Recent work have evidenced a significant association between lysine at amino acid position 232 with elevated milk fat content, while an alanine at this position is associated with lowered milk fat content. The aim of the present work was to develop a simple and inexpensive PCR-SSCP assay in order to discriminate the CG/AA alleles in exon 8 of the DGAT1 gene. In addition, this method was used to analyze the polymorphism of the DGAT1 through PCR-SSCP methods in 14 populations of cattle from Argentine, Bolivia and Uruguay. The PCR primers were designed from GenBank reported sequences. In this study, we found three PCR-SSCP variants, which were denominated from "A" to "C". However, DNA sequencing analysis showed that "A" variant corresponded with the A allele, while both "B" and "C" observed pattern have the motif AA at positions 10,433-10,434 (K allele), being two alternative conformations of the same DNA sequence. Both variants were detected within each breed with the exception of Hereford, and the heterozygosity varied between 0.000 and 0.524. The gene frequency analysis evidenced significant differences among the studied breeds (F(ST) = 0.325, p = 0.000). European Bos taurus breeds, with the exception of Jersey breed, showed the lowest frequency of the K allele, while highest K allele frequencies were harboured by Bos indicus type cattle. In addition, unselected South American Creole cattle breeds and the synthetic Brangus breed had intermediate allele frequencies.

Effectiveness of a 95 SNP panel for the screening of breed label fraud in the Chinese meat market.

Breed assignment has proved to be useful to control meat trade and protect the value of special productions. Meat-related frauds have been detected in China; therefore, 95 SNPs selected from the ISAG core panel were evaluated to develop an automated and technologically updated tool to screen breed label fraud in the Chinese meat market. A total of 271 animals from four Chinese yellow cattle (CYC) populations, six Bos taurus breeds, two Bos indicus and one composite were used. The allocation test distinguished European, Japanese and Zebu breeds, and two Chinese genetic components. It correctly allocated Japanese Black, Zebu and British breeds in 100, 90 and 89% of samples, respectively. CYC evidenced the Zebu, Holstein and Limousin introgression. The test did not detect CYC components in any of the 25 samples from Argentinean butchers. The method could be useful to certify Angus, Hereford and Japanese Black meat, but a modification in the panel would be needed to differentiate other breeds.

Growth, carcass and meat quality traits in beef from Angus, Hereford and cross-breed grazing steers, and their association with SNPs in genes related to fat deposition metabolism.

Grazing steers from Angus and Hereford breeds, their cross-breeds and a three-way cross-breed (Limousin × Angus-Hereford) were measured for growth, carcass and meat quality traits. Breed effects were studied, and the association of SNPs with fat deposition and fatty acid (FA) composition (leptin, melanocortin-4 receptor, stearoyl-CoA desaturase, FA synthase and thyroglobulin) was tested. Limousin cross-breed showed the greatest final body weight, ultrasound rib eye area, dressing percentage, carcass and leg length, and the lowest backfat thickness and intramuscular fat content. Genetic groups had similar pH, shear force, cooking loss, L* and b* and n-6:n-3 ratio. Meat from 1/2-Angus presented greater a* than Limousin cross-breed. Whereas Angus had the highest total SFA content, Hereford had the lowest total SFA and the highest total MUFA. Limousin cross-breed had greater content of several individual PUFAs, total PUFA, n-6 and n-3 FA than Angus and 1/2-Angus. Leptin and FA synthase were associated with some FAs, supporting their influence over fat metabolism for grazing animals.

Polymorphism in the bovine BOLA-DRB3 upstream regulatory regions detected through PCR-SSCP and DNA sequencing.

In the present work, we describe through polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing the polymorphism within the URR-BoLA-DRB3 in 15 cattle breeds. In total, seven PCR-SSCP defined alleles were detected. The alignment of studied sequences showed six polymorphic sites (four transitions, one transversion and one deletion) in the interconsensus regions of the BoLA-DRB3 upstream regulatory region (URR), while the consensus boxes were invariant. Five out of six detected polymorphic sites were of one nucleotide substitution in the interconsensus regions. It is expected that these mutations do not affect significantly the level of expression. In contrast, the deletion observed in the sequence between CCAAT and TATA boxes could have some effect on affinity interactions between the promoter region and the transcription factors. The URR-BoLA-DRB3 DNA analyzed sequences showed moderate level of nucleotide diversity, high level of identity among them and were grouped in the same clade in the phylogenetic tree. In addition, the phylogenetic tree, the similarity analysis and the sequence structure confirmed that the fragment analyzed in this study corresponds to the URR-BoLA-DRB3. The functional role of the observed polymorphic sites among the regulatory motifs in bovine needs to be analyzed and confirmed by means of gene expression assays.

Incidence of 1/29 translocation in Bolivian Creole and Brahman Yacumeño cattle.

In Bolivia, four different Creole cattle breeds can be found, as well as other European and Zebu breeds adapted to local environments. The relationship between the occurrence of the 1/29 translocation and subfertility is well known, and analysis of Y chromosome morphology is useful to determine a possible introgression with Bos indicus. The incidence of the 1/29 translocation was analyzed in four Bolivian Creole cattle breeds and the Brahman Yacumeño population, as well as on four farms with phenotypical Creole-type cattle. In 259 (164 dams and 95 sires) Bolivian Creole cattle, 10.42% of the individuals demonstrated the 1/29 translocation, with a variation from 0 to 28.20% between the breeds. In contrast, 43 (19 dams and 24 sires) Yacumeño Brahman and the Creole-type cattle did not show the centric fusion. The highly significant differences between Creole cattle breeds in relation to the incidence of 1/29 translocation could be a consequence of factors such as founder group, genetic drift, and selection. The low frequency observed in the Saavedreñio Creole dairy cattle might be explained by its breeding under a more intensive system, and selection according to milk yield and fertility traits. Finally, no relation between acrocentric Y chromosomes and 1/29 translocation was observed.

Patagonian Argentine Creole cattle polymorphism: comparison with North-West populations of this breed.

The relict Patagonian Argentine Creole cattle population consist of a small feral population (Los Glaciares population) that is geographically isolated in the South-West of Patagonia. In order to determine the level of genetic variability of this population, the polymorphism of eight structural genes and two microsatellites loci were studied using the polymerase chain reaction (PCR). In addition, genetic characterisation was used to compare Los Glaciares population and the ACc breed of cattle. Results obtained in this study show that the value of average heterozygosity of the studied loci for the Los Glaciares were not significantly different from the ACc. Furthermore, the data of this report were consistent with the hypothesis that Los Glaciares originated from ACc brought to the area by colonialists in the last century. Such data may be useful in formulating management plans for Feral Patagonian Creole cattle populations.

DNA typing in a cattle stealing case.

DNA profiling was used as probative evidence in a cattle stealing case. The carcasses of the dead animals were found from a report and a farmer recognized the remains as those corresponding to the stolen animals by the farm mark on the coat. Those remains were collected as reference samples. Meat pieces were sequestered from a butchery and then sent to our Laboratory by the Justice Department of Buenos Aires (Argentine) to perform a DNA comparative analysis with the reference. Matches were found between the evidences and the references, supporting the hypothesis that the meat pieces had been obtained from the stolen animals. The butcher was suspected of stealing animals but no direct incrimination had been made yet.

Foreign meat identification by DNA breed assignment for the Chinese market.

Methods for individual identification are usually employed for traceability, whereas breed identification is useful to detect commercial frauds. In this study, Chinese Yellow Cattle (CYC) samples plus data from six Bos taurus breeds, two Bos indicus breeds, and one composite breed were used to develop an allocation test based on 22 microsatellites. The test allowed discriminating all foreign breeds from the CYC, although some CYC individuals were wrongly allocated as Limousin or Holstein, probably due to the recent introduction of these breeds into China. In addition, CYC evidenced a previously reported Zebu cline (south-north) and a possible structure within the B. taurus component that should be confirmed. An independent test performed with meat samples of unknown breed origin from Argentina allocated 92% of them to either Angus, Hereford, or their crossbreed, but none was identified as CYC. We conclude that the test is a suitable tool to certify meat of foreign breed origin and to detect adulterations of CYC beef labeled as imported meat.

Malformación Vascular Linfática en un Recién Nacido / Vascular Lymphatic Malformation in a Newborn

Las malformaciones vasculares son enfermedades poco frecuentes que representan errores en el desarrollo de los vasos sanguíneos. Las malformaciones vasculares linfáticas (MVL) consisten en lesiones congénitas del sistema linfático compuestas por canales y cavidades de diferente tamaño que resultan en la acumulación de linfa. Las malformaciones macroquísticas se manifiestan como tumoraciones palpables, de consistencia blanda pero no compresible. La localización más frecuente es en la cabeza y el cuello Se presenta el caso de un neonato con una malformación vascular linfática localizada en zona postero-lateral de cuello. Se arribó al diagnóstico en base a la clínica y las características ecográficas. El tratamiento fue muy satisfactorio luego de dos procedimientos de escleroterapias, presentando una excelente evolución

Vascular malformations are rare conditions associated to anomalies in blood vessel development. Lymphatic vascular malformations are congenital lesions consisting of channels and sacs of different size caused by lymph fluid acumulation. Macrocystic vascular malformations present as non-compresible soft tissue masses. They are generally found withinthe head and neck (70%-80%). We present the case of a newborn with a lymphatic vascular malformation in the posterior cervical área. The diagnosis was made on the basis of clinical and ultrasound findings. Sclerotherapy was succesful wihout signs of recurrence

South American camelid illegal traffic detection by means of molecular markers.

South American camelids comprise the wild species guanaco and vicuña and their respective domestic relatives llama and alpaca. The aim of the present study was to determine by DNA analysis to which of these species belong a herd of camelids confiscated from a llama breeder but alleged to be alpacas by the prosecution, and to evaluate the usefulness of mitochondrial and autosomal DNA markers to solve judicial cases involving camelid taxa. Cytochrome b and cytochrome oxidase I mitochondrial genes and 7 STR were analyzed in 25 confiscated samples. Mitochondrial results were inconclusive because 18 of the sequestered samples presented haplotypes that corresponded to the guanaco haplogroup and the remaining seven belonged to a vicuña linage. Microsatellite data of casework samples and llama reference samples revealed different genetic profiles by the presence of private alleles at two microsatellites suggesting that the confiscated animals could be alpaca, or at least alpaca hybrids instead of pure llama.

History and selection imprinting on genetic relationships among bovine breeds analyzed trough five genes related with marbling.

Many candidate genes have been suggested as responsible for marbling in beef cattle, for instance diacylglycerol O-acyltransferase 1, thyroglobulin, growth hormone, leptin and stearoyl CoA desaturase. The objective of the present work was to evaluate the polymorphisms of five SNPs of these candidate genes in 389 animals of 18 Bos Taurus and Bos indicus breeds. The obtained results were compared with ones previously obtained with STRs and loci related to milk production in these populations. Moreover we analyzed whether the phylogenies reconstructed using SNPs associated with marbling resulted in the known tree topology. The tree constructed with UPGMA, using genetic distance D(A), exhibit a topology partially consistent with the historical origin of breeds. The result observed in the Correspondence Analysis coincided with the topology of the UPGMA tree. This work allowed us to evaluate the five SNPs genetic diversity and to demonstrate that the grouping of the breeds may be the result of its history, selection process, or both at once.

Characterization and validation of bovine gonadotripin releasing hormone receptor (GNRHR) polymorphisms.

Gonadotropin releasing hormone and its receptor (GNRHR) play a critical role in sexual differentiation and reproduction. Available evidence shows a strong genetic component in the timing of puberty. In bovines, there are significant differences within and among beef breeds in the time when bulls reach puberty. Despite its economic importance, there are not many SNPs or genetic markers associated with this characteristic. The aims of the study were to identify DNA polymorphism in the bovine GNRHR by re-sequencing analysis, determine haplotype phases, and perform a population study in a selected tag SNP in six breeds. Eight SNPs were detected, including: one in the Upstream Regulatory Region (URR), five in the coding regions, and two in non-coding regions. This polymorphism level corresponds to one variant every 249.4bp and a global nucleotide diversity of 0.385. Two haplogroups comprising nine haplotypes and two linkage blocks were detected. Despite 5 tag SNPs were required to capture all variability, just one SNP allowed to define both haplogroups, and only two SNPs were needed to differentiate the most common haplotypes. An additional taq SNP was necessary to identify both URR variants. Allele-frequency analysis of a selected taq SNP among breeds showed a geographical cline. European Bos taurus breeds had lower frequencies of the C allele than B. indicus type cattle, while Creole cattle and Wagyu breeds had intermediate frequency. There was a significant correlation between frequency profile and timing of puberty among the studied breeds, which seems to suggest that genetic variation within bovine GNRHR gene could explain at least part of the reported variability.

Effect of consanguinity on Argentinean Angus beef DNA traceability.

Since the 1990s several authors have envisaged the use of DNA to certify meat origin. Two major parameters must be assessed before a DNA based traceability protocol can be implemented in the food chain: (i) the information content of a DNA marker set in a specific livestock breed or group of breeds; (ii) the minimum number of DNA markers needed to obtain a statistically acceptable match probability. The objective of the present work was to establish the effect of different levels of inbreeding in the matching efficiency, and the minimum number of microsatellite markers needed, in a DNA based meat traceability program, starting from an 11-microsatellite marker panel. Samples were obtained from beef production farms in South America, where animals are typically bred under pasture-based extensive conditions. Three groups of animals with different consanguinity rates were sampled. Exclusion power (Q) was higher than 0.999998 and match probability lower than 3.01E-08, for the whole set of markers within each group. Both values were affected by consanguinity. To reach a two mismatch criteria exclusion power (Q(2)) of 99.99, six markers were needed in unrelated animals whereas seven markers were needed in related animals. To reach Q(2)=99.9999, 8 and 10 microsatellite markers, respectively, were needed. In general, one or two more microsatellite markers were needed to identify consanguineous animals. This study proved the DNA marker set used to be suitable for the identification of the meat from all slaughtered animals in Argentina, per week, month, and year.

New polymorphisms for the BoLA-DRB3 upstream regulatory region.

Two new alleles, named BoLA-DRB3-P*06 and BoLA-DRB3-P*07, have been identified for the upstream regulatory region of the BoLA-DRB3 gene. The 228-bp nucleotide sequences of the promoter comprising the W, X, Y, CAAT and TATA regulatory boxes were analysed. The BoLA-DRB3-P*06 exhibits one insertion between the W and X boxes, and one transition between the X and Y boxes. On the other hand, the BoLA-DRB3-P*07 showed one insertion in the X box.

Indigenous domestic breeds as reservoirs of genetic diversity: the Argentinean Creole cattle.

Contrary to highly selected commercial breeds, indigenous domestic breeds are composed of semi-wild or feral populations subjected to reduced levels of artificial selection. As a consequence, many of these breeds have become locally adapted to a wide range of environments, showing high levels of phenotypic variability and increased fitness under natural conditions. Genetic analyses of three loci associated with milk production (alpha(S1)-casein, kappa-casein and prolactin) and the locus BoLA-DRB3 of the major histocompatibility complex indicated that the Argentinean Creole cattle (ACC), an indigenous breed from South America, maintains high levels of genetic diversity and population structure. In contrast to the commercial Holstein breed, the ACC showed considerable variation in heterozygosity (H(e)) and allelic diversity (A) across populations. As expected, bi-allelic markers showed extensive variation in He whereas the highly polymorphic BoLA-DRB3 showed substantial variation in A, with individual populations having 39-74% of the total number of alleles characterized for the breed. An analysis of molecular variance (AMOVA) of nine populations throughout the distribution range of the ACC revealed that 91.9-94.7% of the total observed variance was explained by differences within populations whereas 5.3-8.1% was the result of differences among populations. In addition, the ACC breed consistently showed higher levels of genetic differentiation among populations than Holstein. Results from this study emphasize the importance of population genetic structure within domestic breeds as an essential component of genetic diversity and suggest that indigenous breeds may be considered important reservoirs of genetic diversity for commercial domestic species.

Gene frequency distribution of the BoLA-DRB3 locus in Saavedreño Creole dairy cattle.

The objective of this study is to describe the gene frequency distribution of the bovine lymphocyte antigen (BoLA)-DRB3 locus in Saavedreño Creole dairy cattle and to compare it with previously reported patterns in other cattle breeds. One hundred and twenty-five Saavedreño Creole dairy cattle were genotyped for the BoLA-DRB3.2 allele by polymerase chain reaction and restriction fragment length polymorphism. Twenty-two out of 53 previously identified BoLA-DRB3.2 alleles were detected, with gene frequencies ranging from 0.4 to 16.8%. Seventy percent of the variation corresponded to the seven most frequent alleles (BoLA-DRB3.2*7, *8, *11, *16, *27, *36, and *37). The studied population exhibits a high degree of expected heterozygosity (he = 0.919). The FIS index did not show significant deviation from Hardy-Weinberg equilibrium. However, the neutrality test showed an even gene frequency distribution. This result could be better explained assuming balancing selection instead of neutral or positive selection for one or a few alleles. In conclusion, the results of this study demonstrated that BoLA-DRB3.2 is a highly polymorphic locus in Saavedreño Creole dairy cattle, with significant variation in allele frequency among cattle breeds.

Male-mediated introgression of Bos indicus genes into Argentine and Bolivian Creole cattle breeds.

The geographic distribution and frequency of Bos taurus and Bos indicus Y chromosome haplotypes amongst Argentine and Bolivian Creole cattle breeds were studied, using cytogenetic and molecular genetic techniques. A complete correspondence between Y chromosome morphology and the haplotype of the Y-linked microsatellite marker INRA 124 was found in all males examined. The taurine and indicine haplotypes were detected in 85.7 and 14.3% of the males studied, respectively, although these frequencies varied amongst the different breeds examined. The geographic distribution of this polymorphism suggests a pattern of zebu introgression in South America. The highest frequencies of the Zebu Y-chromosome are found in Brazilian populations (43-90%), in the eastern part of the continent, while it is absent in the southernmost breeds from Uruguay and Argentina. Bolivian breeds, at the centre of the continent, exhibit intermediate values (17-41%). This east/west and north/south gradient of male Zebu introgression could be explained by historical events and environmental factors.