Juan Pedro Lirón

Comparison of the effectiveness of microsatellites and SNP panels for genetic identification, traceability and assessment of parentage in an inbred Angus herd

During the last decade, microsatellites (short tandem repeats or STRs) have been successfully used for animal genetic identification, traceability and paternity, although in recent year single nucleotide polymorphisms (SNPs) have been increasingly used for this purpose. An efficient SNP identification system requires a marker set with enough power to identify individuals and their parents. Genetic diagnostics generally include the analysis of related animals. In this work, the degree of information provided by SNPs for a consanguineous herd of cattle was compared with that provided by STRs. Thirty-six closely related Angus cattle were genotyped for 18 STRs and 116 SNPs. Cumulative SNPs exclusion power values (Q) for paternity and sample matching probability (MP) yielded values greater than 0.9998 and 4.32E−42, respectively. Generally 2–3 SNPs per STR were needed to obtain an equivalent Q value. The MP showed that 24 SNPs were equivalent to the ISAG (International Society for Animal Genetics) minimal recommended set of 12 STRs (MP ∼ 10−11). These results provide valuable genetic data that support the consensus SNP panel for bovine genetic identification developed by the Parentage Recording Working Group of ICAR (International Committee for Animal Recording).

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.

Association between GNRHR, LHR and IGF1 polymorphisms and timing of puberty in male Angus cattle

BackgroundIn bovines, there are significant differences within and among beef breeds in the time when bulls reach puberty. Although the timing of puberty is likely to be a multigenic trait, previous studies indicate that there may also be single genes that exert major effects on the timing of puberty within the general population. Despite its economic importance, there are not many SNPs or genetic markers associated with the age of puberty in male cattle. In the present work, we selected three candidate genes, GNRHR, LHR and IGF1, and associated their polymorphisms with the age of puberty in Angus male cattle.ResultsAfter weaning, 276 Angus males were measured every month for weight (W), scrotal circumference (SC), sperm concentration (C) and percentage of motility (M). A total of 4 SNPs, two within GNRHR, one in LHR and one in IGF1 were genotyped using the pyrosequencing technique. IGF1-SnaBI SNP was significant associated (P < 0.01) with age at SC 28 cm, but it were not associated with age at M 10% and C 50 million. Genotype CC exhibited an average age at SC 28 cm of 7 and 11 days higher than CT (p = 0.037) and TT (p = 0.012), respectively. This SNP explained 1.5% of the genetic variance of age of puberty at SC28. LHR-I499L, GNRHR-SNP5 and GNRHR-SNP6 were not associated with any of the measurements. However, GNRHR haplotypes showed a suggestive association with age at SC 28 cm.ConclusionsThe findings presented here could support the hypothesis that IGF1 is a regulator of the arrival to puberty in male calves and is involved in the events that precede and initiate puberty in bull calves. Given that most studies in cattle, as well as in other mammals, were done in female, the present results are the first evidence of markers associated with age at puberty in male cattle.

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.

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.

Assignment of paternity in a judicial dispute between two neighbor Holstein dairy farmers.

DNA profiling was used as evidence to assign paternity in a dispute between two neighbors in a judicial case of undue appropriation of cattle offspring from five alleged Holstein sires. Five offspring were genotyped using ten genetic markers (nine microsatellites and the BOLA-DRB3 locus). The computer program CERVUS was used to estimate the LOD score values and the confidence of paternity assignments. The results presented here show that three out of five paternity cases were assigned at 95% of confidence to a single sire with a LOD score ranging from 2.53 to 3.55. A fourth male was assigned using its delta value. Finally, all alleged sires were excluded from the paternity of the fifth offspring, probably due to the existence of an non-sampled male in the studied population. We concluded that the likelihood-based approach, included into CERVUS program, was a powerful tool in cattle kinship analysis when dealing with judicial dispute particularly when the dams genotype was absent, allowing the assignments of paternity at 95% level of confidence in situations usually used by dairy and beef cattle producers in Argentine (e.g., multi-sire pasture mating).

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.

Assessing the association of single nucleotide polymorphisms in thyroglobulin gene with age of puberty in bulls

Puberty is a stage of sexual development determined by the interaction of many loci and environmental factors. Identification of genes contributing to genetic variation in this character can assist with selection for early pubertal bulls, improving genetic progress in livestock breeding. Thyroid hormones play an important role in sexual development and spermatogenic function. The objective of this study was to evaluate the association between single nucleotide polymorphisms (SNPs) located in thyroglobulin(TG) gene with age of puberty in Angus bulls. Four SNPs were genotyped in 273 animals using SEQUENOM technology and the association between markers and puberty age was analyzed. Results showed a significant association (P < 0.05) between these markers and puberty age estimated at a sperm concentration of 50 million and a progressive motility of 10%. This is the first report of an association of TG polymorphisms with age of puberty in bulls, and results suggest the importance of thyroidal regulation in bovine sexual development and arrival to puberty.

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.

Implication of Population Structure in the Resolution of Cattle Stealing Cases

Abstract:  Estimation of population subdivision using genetic markers shows that genetic differentiation in livestock and pet breeds is significantly higher than in human populations. Nevertheless, the influence of population substructure and sample size on match probability has not been extensively analyzed in domestic species. To evaluate the magnitude of the subpopulation effect on estimation of match probabilities in bovine robbery cases, we calculated and compared the match probabilities obtained from cattle breed databases using both real, adjudicated cases from the Buenos Aires Province (Argentina), as well as simulated data. While the Balding and Nichols’ correction, when applied to the population database used in the case, produce a more conservative value favorable to the defendant, the match probabilities calculated using the simple product estimator produce a value favorable to the prosecution. We suggest an alternative procedure that can be used. The method consists of choosing the highest value from all match probabilities calculated from the database of each breed. This approach represents an intermediate and more accurate estimation of match probability, although it still produces a slight conservative value favorable to the defense.