IDENTIFICATION OF HOLSTEIN COWS CARRIERS OF COMPLEX VERTEBRAL MALFORMATION BY HIGH RESOLUTION MELTING CURVES (HRM)

The objective of this study was the optimization and implementation of a reliable and economical molecular screening method for the detection of the mutant allele of CVM (complex vertebral malformation, c.559G>T, SLC35A3) by HRM analysis, as well as analyzing its existence in a representative sample of Holstein cows from the Milk Genomic DNA Bank of Uruguay. The optimization of the HRM methodology in the RotorGene™ 6000 equipment (Corbett Life Science, Australia) by amplification of the 79 bp PCR products clearly differentiated the two genotypes: homozygous, wild type: GG; and heterozygous, carrier for the mutation CVM: GT (c.559G>T; SLC35A3). In the analyzed sample, the frequency of the mutant allele (T) for CVM was high (q = 0.032), with a prevalence of carrier cows of 6.45%. It is concluded that the PCR-HRM analysis is a fast, easily interpretable, low cost, and highly accurate technique for the detection of this mutation in Holstein cattle, which may be implemented in genetic selection programs

Estudios preliminares de la estructura genética del perro cimarrón uruguayo usando microsatélites

Analizar la estructura poblacional en una muestra de perros “Cimarrón Uruguayo” usando marcadores moleculares tipo microsatélites. Materiales y métodos. Se analizaron treinta caninos de diferentes zonas de Uruguay con un set de nueve marcadores moleculares microsatélites empleando PCR. La estructura poblacional se analizó con el software de distribución libre “Structure”. Resultados. Según nuestros datos, los resultados preliminares muestran que no es posible establecer una subdivisión entre los animales de la muestra. Conclusiones. El estudio realizado apoya la hipótesis de que los perros que existen en la actualidad derivan del núcleo fundador que se refugió en la región noreste del país. La distribución de la raza entre las distintas áreas de Uruguay continúa hoy en día, no existe aislamiento entre los diferentes grupos de animales y el intercambio es constante

Conociendo al perro cimarrón uruguayo

La publicación de este libro fue realizada con el apoyo de la Comisión Sectorial de Investigación Científica (CSIC) de la Universidad de la República. El trabajo que se presenta fue seleccionado por el Comité de Referato de Publicaciones de la Facultad de Veterinaria integrado por Luis Barros, José Luis Repetto y Celia Tasende.Capítulo 1. Primeros estudios de genética molecular en el perro cimarrón uruguayo / Silvia Llambí Dellacasa -- Capítulo 2. Estructura poblacional en el perro cimarrón uruguayo / Rosa Gagliardi, Silvia Llambí -- Capítulo 3. Caracterización racial del perro cimarrón / Gabriel E. Fernández de Sierra, Beatriz E. Mernies Falcone -- Capítulo 4. Estudios genealógicos en la raza canina cimarrón uruguayo / Mónica Martínez, Eileen Armstrong -- Capítulo 5. Etología clínica canina, agresividad y el cimarrón uruguayo / Juan Pablo Damián, María Belino, Ruben Rijo, Paul Ruiz -- Capítulo 6. Estacionalidad y carácterísticas reproductivas en perras cimarronas, Danilo Fila -- Capítulo 7. Diagnóstico genético de las anomalías del desarrollo sexual en caninos / Rody Artigas, María Montenegro, Silvia Llambí -- Capítulo 8. Estudio ecocardiográfico en la raza cimarrón / Benech, Pisón, Sehabiaga, Jiménez, Rossi -- Capítulo 9. Estudios farmacogenéticos en el perro cimarrón uruguayo / Rosa Gagliardi, Silvia Llambí -- Capítulo 10. Razas caninas españolas / M. Victoria Arruga Laviña, José Ignacio Bonafonte Zaragozano

HIGH- RESOLUTION MELTING (HRM) CURVE ANALYSIS: NEW APPROACH USED TO DETECT BLAD AND DUMPS IN URUGUAYAN HOLSTEIN BREED

The widespread use of artificial insemination has allowed the expansion of genetic progress. However, it also brought consequences such as the expansion of lethal hereditary diseases and the increase in inbreeding. The object of this study was to establish a fast and sensitive molecular assay to detect bovine leukocyte adhesion deficiency (BLAD) and deficiency of uridine monophosphate synthase (DUMPS) carriers in Uruguayan Holstein cattle by means of high resolution melting (HRM) curve analysis. By testing previously confirmed carrier and non-carrier animals, we set up a rapid, simple, and inexpensive diagnostic test using PCR followed by HRM curve analysis. The PCR-HRM genotyping method was effective for the discrimination of BLAD and DUMPS homozygous genotypes, and the BLAD heterozygous genotype. We conclude that the PCR-HRM assay is a robust, reliable, and economical tool for the detection of these mutations in the Holstein breed, which may be implemented in genetic selection programs

Acción de la 5'azacitidina en cromosomas de células linfocitarias de bovinos

La azacitidina (5–azaC) es una sustancia que actúa inhibiendo la metilación del ADN, es utilizada en experiencias de clonación, transgénesis por transferencia nuclear y estudios de fragilidad cromosómica. Se ha observado la manifestación de diversas patologías en animales domésticos nacidos de experiencias de clonación. Ellas estarían asociadas a una inexacta reprogramación nuclear relacionada con fenómenos epigenéticos de metilación del ADN. En el presente trabajo se realizó una curva de inducción con diferentes concentraciones finales de 5–azaC (0 uM, 5 uM, 10 uM, 20 uM, 50 uM y 70 uM) con el objetivo de estudiar los efectos sobre la estructura cromosómica en cultivos linfocitarios bovinos. Se utilizó la técnica de macrocultivo para análisis cromosómico a partir de sangre de un bovino macho (Bos taurus, Holstein Friesian). Se analizó un total de 50 placas metafásicas por tubo de cultivo y se encontraron diferencias significativas (p and lt;0.01) en cuanto a las fracturas del cromosoma X en el cultivo control frente al cultivo con mayor concentración de 5–azaC. El cromosoma Y no presentó fracturas. En los cultivos con concentraciones de 50 uM y 70 uM se observó descondensación cromosómica (12% y 16%) y fracturas centroméricas en autosomas (2% y 4%).Azacytidine (5–azaC) is a substance that inhibits the metilation of DNA, being used in cloning experiments, nuclear transference transgenesis and in chromosomal fragility studies. The manifestation of diverse pathologies has been observed in domestic animals born from cloning experiments. These would be associated to an incorrect nuclear reprogramation related to epigenetic metilation phenomena of the DNA. In the present work an induction curve with different final concentrations of 5–azaC (0 uM, 5 uM, 10 uM, 20 uM, 50 uM and 70 uM) were carried out, with the objective of studying the effects on chromosome structure in bovine lymphocyte cultures. The macroculture technique was used for chromosomic analysis, from a blood sample of a male bovine (Bos taurus, Holstein Friesian). A total of 50 metaphases were analyzed from each culture. Significative differences (p and lt;0.01) were found regarding the break points of chromosome X in the control culture with respect to the culture with higher concentration of 5–azaC. Chromosome Y did not show break points. The cultures with higher concentration of 5–azaC (50 uM and 70 uM) presented chromosomic decondensation (12% and 16%) and autosomic centromeric break points (2% and 4%)

Comparative analysis of PRNP 12-bp and 23-bp indels in healthy Aberdeen Angus, Aberdeen Angus x Hereford, Holstein Friesian and Uruguayan Creole cattle

ABSTRACT: Bovine spongiform encephalopathy (BSE) is a transmissible progressive neurodegenerative disease characterized by the accumulation of a pathological isoform (PrpSC) of the cellular prion protein (PrpC) in the brain of cattle. Two insertion/deletion polymorphisms in the PRNP gene (23bp in the promoter and 12bp in intron 1) have been associated with resistance or susceptibility to the disease. The aim of this study was to analyze the distribution of these polymorphisms in 214 healthy bovines belonging to four different breed groups (Aberdeen Angus, Aberdeen Angus x Hereford, Holstein Friesian and Uruguayan Creole cattle). DNA samples were amplified by end-point PCR. A high frequency of the alleles and haplotype associated with susceptibility to BSE (del12 and del23, and del12-del23, respectively) were found in the Aberdeen Angus, Aberdeen Angus x Hereford and Holstein Friesian animals. At the same time, the Uruguayan Creole cattle presented a higher frequency of the alleles and haplotype associated with resistance to BSE (ins12 and ins23, and ins12-ins23, respectively). These data could indicate a greater genetic resistance of the Uruguayan Creole cattle to BSE compared to other analyzed breeds, reinforcing its value as a zoogenetic resource

Localisation of aphidicolin-induced break points in Holstein-Friesian cattle (Bos taurus) using RBG-banding

Fragile sites (FS) seem to play a role in genome instability and may be involved in karyotype evolution and chromosome aberrations. The majority of common fragile sites are induced by aphidicolin. Aphidicolin was used at two different concentrations (0.15 and 0.30 μM) to study the occurrence of FS in the cattle karyotype. In this paper, a map of aphidicolin induced break points and fragile sites in cattle chromosomes was constructed. The statistical analysis indicated that any band with three or more breaks was significantly damaged (P < 0.05). According to this result, 30 of the 72 different break points observed were scored as fragile sites. The Pearson correlation test showed a positive association between chromosome length and the number of fragile sites (r = 0.54). On the contrary, 21 FS were identified on negative R bands while 9 FS were located on positive R bands

Analysis of mdr1-1Δ mutation of MDR1 gene in the “Cimarron Uruguayo” dog

ABSTRACT Objective. The aim of this paper is to analyze the frequency of the mdr1-1D mutation of the MDR1 gene in a dog sample of the Uruguayan Cimarron breed with the objective of increasing the knowledge of this breed’s genome. Materials and methods. Thirty-six animals of this breed were analyzed. The MDR1 gene region, which includes the location where the mutation would be present, was amplified by PCR. Results. The mutation was not detected in any of the analyzed Uruguayan Cimarron. Conclusions. The lack of described ivermectin intoxication cases in veterinary clinic in this breed is explained by the lack of the mutation object of this study. The sequence studied in Cimarron dogs is kept compared to other breeds, except Collies and related breeds (Border Collie, Bearded Collie, Old English sheepdog)

Descondensación de la heterocromatina en bovinos criollos portadores de la translocación robertsoniana (rob1; 29). acción del inductor 5 - azacitidina - C

La translocación Robertsoniana (rob1; 29) está ampliamente distribuida en razas comerciales y en reservas genéticas de bovinos Criollos americanos. Se ha descrito un enlentecimiento en el desarrollo de embriones portadores de esta aneuploidía, frente a embriones normales. La acción de la 5–aza–C, como agente desmetilante, permitiría descondensar la heterocromatina constitutiva o facultativa. En este trabajo se realiza inducción con el análogo de base 5–aza–C(10mM, 2 hrs) en cultivos linfocitarios de una hembra y un macho portadores de la rob1; 29, frente a bovinos Criollos normales. Se controla la acción desmetilante del inductor al identificar la despiralización del cromosoma X de replicación tardía en hembras y permitir el análisis de la despiralización de la cromatina en múltiples regiones de los autosomas (grandes, medianos, pequeños); de la rob1; 29 y del BTA1. Se discute la correlación existente entre regiones desmetiladas con la descondensación de la heterocromatina facultativa (condicional), relacionándola con la inestabilidad genómica, y la reprogramación epigenética.The Robertsonian translocation (rob1; 29) is widely spread in commercial breeds and specially in genetic reserve of American Creole cattle. It is also described a delay on embryo development in front of normal ones. The action of 5–aza–C, as an hypomethylated agent, could permitted to decondensate the constitutive or facultative heterochromatin. In this work we made induction with the 5–aza–C(10mM, 2 hrs) analogs, in lymphocyte cultures of female and male carriers and normal Creole cattle. The DNA hypometilation is found in the inactive X chromosome of late replication as it is incorporated during the last hours of cell culture. The decondensing effects of 5–aza–C analogs is observed in multiple regions of the autosomes chromatin, the rob1; 29 and the BTA1. A correlation between hypometilated regions and decondensed of facultative (conditional) heterochromatin is related with genomic instability, and epigenetic reprogramming

Marcadores de selección en cerdos Pampa Rocha: comparación con razas autóctonas de España y Portugal

Objective. The analysis of selection markers allows to obtain information about the evolutive story of a particular breed or line and allows also to evaluate the usefulness of those markers for breeding programs. We have analyzed SNPs in four genes of the creole pig breed Pampa Rocha and we have compared their allelic frequencies with the allelic frequencies of diverse autochthonous breeds of Spain and Portugal and also with Piétrain pigs and wild boars. Materials and methods. The SNPs were analyzed using diverse RT-PCR methods. Results. The results of the analysis show that Pampa Rocha pigs have similar allelic frequencies with the autochthonous breeds of Spain and Portugal especially in the case of IGF2 and also, but not so coincident, in the case of PEPCK-C. However, they differ considerably for MC4R, and also, but in a lower extent, for PRKAG3. We discuss in this work the usefulness of our results for breeding of Pampa Rocha pigs. Conclusions. Our results demonstrate the peculiarity of the Pampa Rocha breed regarding the markers studied.Objetivo. El análisis de marcadores de selección permite obtener datos de la vida evolutiva de una raza o línea y permite también evaluar la conveniencia o no de su uso en programas de mejora genética. Hemos evaluado SNPs en cuatro genes (IGF2, MC4R, PRKAG3 y PEPCK-C), que tienen importantes efectos fenotípicos, en cerdos de la raza Pampa Rocha, una raza criolla, y hemos comparado sus frecuencias alélicas con cerdos de diversas razas autóctonas y líneas de España y Portugal no sometidas a selección así como con jabalíes y cerdos de la raza Piétrain. Materiales y métodos. Los SNPs fueron analizados mediante diversa técnicas de RT-PCR. Resultados. Los resultados de los análisis muestran una similitud de frecuencias alélicas entre los cerdos de la raza Pampa Rocha y los cerdos autóctonos de la península ibérica sobre todo en el gen IGF2 y, en menor medida en el gen PEPCK-C. Sin embargo difieren considerablemente en el caso del marcador MC4R y, también en menor medida, en PRKAG3. En el trabajo se discute el uso potencial de los resultados obtenidos para orientar la selección genética de cerdos de la raza Pampa Rocha. Conclusiones. Nuestros resultados demuestran la peculiaridad de la raza Pampa Rocha con respecto a los marcadores estudiados