Genomic analysis of inbreeding level, kinship and breed relationships in Creole cattle from South America.

The conservation of animal genetic resources refers to measures taken to prevent the loss of genetic diversity in livestock populations, including the protection of breeds from extinction. Creole cattle populations have suffered a drastic reduction in recent decades owing to absorbent crosses or replacement with commercial breeds of European or Indian origin. Genetic characterization can serve as a source of information for conservation strategies to maintain genetic variation. The objective of this work was to evaluate the levels of inbreeding and kinship through the use of genomic information. A total of 903 DNAs from 13 cattle populations from Argentina, Bolivia and Uruguay were genotyped using an SNP panel of 48 K. Also, a dataset of 76 K SNPs from Peruvian Creole was included. Two inbreeding indices (FROH and Fhat2) and kinship relationships were calculated. In addition, effective population size (Ne), linkage disequilibrium, population composition and phylogenetic relationships were estimated. In Creole cattle, FROH ranged from 0.14 to 0.03, and Fhat2 was close to zero. The inferred Ne trends exhibited a decline toward the present for all populations, whereas Creole cattle presented a lower magnitude of Ne than foreign breeds. Cluster analysis clearly differentiated the taurine and Zebu components (K2) and showed that Bolivian Creole cattle presented Zebu gene introgression. Despite the population reduction, Creole populations did not present extreme values of consanguinity and kinship and maintain high levels of genetic diversity. The information obtained in this work may be useful for planning conservation programmes for these valuable local animal genetic resources.

Analysis of Doberman Pinscher and Toy Poodle samples with targeted next-generation sequencing.

Next-generation sequencing (NGS) technologies have enabled the identification of many causal variants of genetic disorders, the development of parentage tests and the analysis of multiple traits in domestic animals. In this study, we evaluated the performance of a Canine Targeted Genotyping-by-Sequencing (GBS) custom panel (Thermo Fisher Scientific, Waltham, Ma, USA) in a cohort of 95 dog DNA samples, comprising 76 Doberman Pinschers and 19 Toy Poodles from Argentina. The used panel included 383 targets (228 parentage SNVs, 137 genetic disorder markers and 18 trait markers). While paternity analysis showed correct duo (97.4%; LOD > 2.98E+13) and trio (100%; LOD > 2.20E+15) parentage assignment, the panel resulted still insufficient for excluding close relatives in inbred populations. In this sense, close relatives were wrongly assigned as parents in 12.6% of duos and 0.3% of trios. We detected 17 polymorphic markers (genetic disorders, n = 4; hair type, n = 3; coat color, n = 10) and estimated their allele frequencies in the studied breeds. The accuracy of targeted GBS results were evaluated for three markers that were associated with Progressive rod-cone degeneration, von Willebrand disease type 1 and dilated cardiomyopathy by pyrosequencing and Sanger sequencing genotyping, showing 94-100% concordance among assays. The targeted GBS custom panel resulted cost-effective strategy to study the prevalence of genetic disorders and traits in a large number of samples and to analyze genetic interactions between previously reported variants. Once assays based on AgriSeq technology were standardized, their uses are a good strategy for large-scale routine genetic evaluation of animal populations.

Trace mineral mixture supplemented to in vitro maturation medium improves subsequent embryo development and embryo quality in cattle.

Trace minerals participate in reproductive processes and are crucial for oocyte maturation. The objective of the present study was to investigate the effect of combined supplementation with copper (Cu), manganese (Mn), selenium (Se) and zinc (Zn) during bovine in vitro maturation (IVM) on subsequent embryo development and quality. The IVM medium was supplemented as follows: a) Control (no mineral supplementation); b) MScz (6 ng/mL Mn + 100 ng/mL Se + 200 ng/mL Cu + 400 ng/mL Zn); c) MScZ (6 ng/mL Mn + 100 ng/mL Se + 200 ng/mL Cu + 1200 ng/mL Zn); d) MSCz (6 ng/mL Mn + 100 ng/mL Se + 600 ng/mL Cu + 400 ng/mL Zn). Supplementation with MScz and MSCz produced more blastocysts compared with the control. Total blastocyst cell number was higher when minerals were added at any combination. Day-8 blastocysts derived from oocytes treated with minerals had lower intracellular reactive oxygen species concentration and lipid content than the control. In conclusion, combined supplementation with Cu, Mn, Se and Zn during bovine oocyte IVM increased in vitro production performance, improving embryo developmental ability and quality.

Polledness in Argentinean Creole cattle, five centuries surviving.

Polledness has been shown to have autosomal Mendelian inheritance, with the polled locus being dominant to the horned locus. This trait was mapped to the BTA1 centromeric end in several breeds. One of the distinctive attributes of Creole cattle, such as the Argentinean Creole, is the presence of long, lyre-shaped horns. However, polled native animals were reported before the introduction of modern selected European breeds. Here, we studied the origin of the polled mutation, either independent or introgressed, in a Creole line from the Creole cattle founder group at the IIACS-INTA Leales Experimental Station (northwest Argentina). The study sample (65 animals: 26 horned and 39 polled) was genotyped using high-density SNP microarrays and three previously reported genetic markers (P202 ID , P80kb ID and PG ). A genome-wide association study, selection signatures, linkage disequilibrium analysis and copy number variations were used to detect the responsible region and the segregating haplotypes/alleles. The interval mapped in the Leales herd (1.23-2.13 Mb) overlapped with the region previously reported in several European cattle breeds, suggesting that the same locus could be segregating in this population. The previously reported variants PF and PG were not detected, thus dismissing the Holstein-Friesian and Nellore origins of the polled phenotype in this native breed. Conversely, the presence of the Celtic variant PC suggests an almost complete co-segregation. The cluster analysis rejected the hypothesis of recent introgression, which is compatible with the historical record of polled Creole cattle in northwest Argentina.

Computational prediction of nsSNPs effects on protein function and structure, a prioritization approach for further in vitro studies applied to bovine GSTP1.

The development of high-throughput technologies in the last decade produced an exponential increase in the amount of biological data available. The case of redox biology and apoptosis is not an exception, and nowadays there is a need to integrate information from multiple "omics" studies. Therefore, validation of proposed discoveries is essential. However, the study in biological systems of the effect of the massive amounts of sequence variation data generated with next-generation sequencing (NGS) technologies can be a very difficult and expensive process. In this context, the present study aimed to demonstrate the advantages of a computational methodology to systematically analyze the structural and functional effects of protein variants, in order to prioritize further studies. This approach stands out for its easy implementation, low costs and low time consumed. First, the possible impact of mutations on protein structure and function was tested by a combination of tools based on evolutionary and structural information. Next, homology modeling was performed to predict and compare the 3D protein structures of unresolved amino acid sequences obtained from genomic resequencing. This analysis applied to the bovine GSTP1 allowed to determine that some of amino acid substitutions may generate important changes in protein structure and function. Moreover, the haplotype analysis highlighted three structure variants worthwhile studying through in vitro or in vivo experiments.

Evidence of positive selection towards Zebuine haplotypes in the BoLA region of Brangus cattle.

The Brangus breed was developed to combine the superior characteristics of both of its founder breeds, Angus and Brahman. It combines the high adaptability to tropical and subtropical environments, disease resistance, and overall hardiness of Zebu cattle with the reproductive potential and carcass quality of Angus. It is known that the major histocompatibility complex (MHC, also known as bovine leucocyte antigen: BoLA), located on chromosome 23, encodes several genes involved in the adaptive immune response and may be responsible for adaptation to harsh environments. The objective of this work was to evaluate whether the local breed ancestry percentages in the BoLA locus of a Brangus population diverged from the estimated genome-wide proportions and to identify signatures of positive selection in this genomic region. For this, 167 animals (100 Brangus, 45 Angus and 22 Brahman) were genotyped using a high-density single nucleotide polymorphism array. The local ancestry analysis showed that more than half of the haplotypes (55.0%) shared a Brahman origin. This value was significantly different from the global genome-wide proportion estimated by cluster analysis (34.7% Brahman), and the proportion expected by pedigree (37.5% Brahman). The analysis of selection signatures by genetic differentiation (F st ) and extended haplotype homozygosity-based methods (iHS and Rsb) revealed 10 and seven candidate regions, respectively. The analysis of the genes located within these candidate regions showed mainly genes involved in immune response-related pathway, while other genes and pathways were also observed (cell surface signalling pathways, membrane proteins and ion-binding proteins). Our results suggest that the BoLA region of Brangus cattle may have been enriched with Brahman haplotypes as a consequence of selection processes to promote adaptation to subtropical environments.

Caracterización genética de cuatro poblaciones de ovinos criollos de Argentina / Genetic characterization of four populations of Argentinian creole sheep

Los ovinos criollos son los fundadores de la ganadería ovina en la Argentina y han contribuido de manera sostenida al desarrollo económico, social y cultural de algunas regiones del país. A pesar de ello, es un recurso zoogenético escasamente valorizado y por ende poco estudiado. En orden de caracterizar genéticamente a los ovinos criollos argentinos, se tomaron muestras de ADN de cuatro poblaciones representativas localizadas en las provincias de Buenos Aires, Corrientes, Santiago del Estero y Salta. Estas majadas se seleccionaron por ser grupos conservados, que presentan las características fenotípicas de la raza y no registran la introducción de animales de otras razas en el sistema de reproducción. Un total de 30 marcadores microsatélites y la región D-loop del ADN mitocondrial fueron analizados. El análisis de los microsatélites permitió evidenciar una alta diversidad genética intrapoblacional (Ho= 0,676; He= 0,685; PIC= 0,713). Dicha variabilidad es explicada por diferencias entre los patrones moleculares de los individuos estudiados que pueden clasificarse en 3 grupos de poblaciones significativamente diferentes: BA, SA, SE+CO. Dado que dichas poblaciones explican muy poco de la variabilidad total (7,6%), ellas deberían considerarse perteneciente a una misma raza. El análisis del D-loop mitocondrial demostró que los individuos analizados están relacionados con el haplogrupo asiático, el cual está ampliamente distribuido en las razas españolas que son las antecesoras de la raza criolla argentina. Los resultados obtenidos en este trabajo proveerán información para establecer criterios de manejo de este recurso genético de Argentina con el fin de implementar planes de conservación, recuperación y/o mejora de los programas.

Creole sheep are the founders of sheep farming in Argentina and have contributed in a sustained way to the economic, social and cultural development of some regions of this country. However, it is a scarcely valorised and poorly studied genetic resource. In order to genetically characterize the Argentinian Creole sheep, DNA samples were taken from four representative populations located in the provinces of Buenos Aires, Corrientes, Santiago del Estero and Salta. These flocks were selected because they are considered to be conserved groups, they have the phenotypic characteristics of the creole breed and there are no records about the introduction of animals of other breeds into those systems. A total of 30 microsatellites and the D-loop region of mitochondrial DNA were analysed. Microsatellite analysis showed high level of genetic diversity within populations (Ho= 0.676; He= 0.685; PIC= 0.713). This variability is explained by differences between molecular patterns of the studied individuals, which can be classified into three significantly different population groups: BA, SA, SE+CO. Since these populations explain very little of the total variability (7.6%), it can be considered that they belong to a same race. The analysis of the mitochondrial D-loop showed that Argentinian Creole sheep have haplotypes belonging to the Asian haplogroup, which is widely distributed in the Spanish breeds, which are considered to be their ancestors. The results obtained in the present study will provide information to develop management criteria for this genetic resource in Argentina, in order to implement their conservation, recovery and/or to develop breeding programs.

Bovine thyroglobulin gene polymorphisms and their association with sexual precocity in Guzerat bulls.

Puberty is a stage of sexual development determined by the interaction of environmental factors and genetic mechanisms. Among them, thyroid function plays a key role in sexual development and spermatogenic function and is under the control of several genes, including the well-described thyroglobulin gene (TG). Previous reports have shown genetic association between thyroid function and selected single nucleotide polymorphisms (SNPs) in taurine cattle. Therefore, the identification of genetic mechanisms involved in the regulation of this trait can assist with the selection for early pubertal bulls, thus improving genetic progress in livestock breeding. The aim of this study was to validate the association between TG SNPs and age at puberty in zebuine bulls. Three SNPs (rs110406764, rs109662686, rs109057985) were genotyped in 159 Guzerat animals using SEQUENOM technology. Results showed a significant association (p < .05) between the studied SNPs and puberty age, in agreement with our previous reports in a taurine breed. Interestingly, allele frequencies were different from those already reported, being GAT the most favourable allele for age at puberty in Guzerat (94.4 days lower). Overall, our findings corroborate previous reports and reinforce the importance of genetic influence in the regulation of sexual development and puberty through a thyroid pathway in zebuine cattle.

DNA profile of dog feces as evidence to solve a homicide.

Dog fecal samples were collected at the crime scene and from the shoes of the suspect to see whether they could be linked. DNA was genotyped using a 145bp fragment containing a 60bp hotspot region of the mitochondrial DNA (mtDNA) control region. Once the species origin was identified, sequences were aligned with the 23 canine haplotypes defined, showing that evidence and reference had 100% identity with haplotype 5. The frequency of haplotype 5 and the exclusion power of the reference population were 0.056 and 0.89, respectively. The forensic index showed that it was 20 times more likely that the evidence belonged to the reference dog than to some other unknown animal. The results support that the mtDNA hypervariable region 1 (HV1) is a good alternative for typing in trace or degraded casework samples when the STR panel fails, and demonstrate the utility of domestic animal samples to give additional information to solve human legal cases.

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.

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.

Characterization of bovine MHC class II DRB3 diversity in South American Holstein cattle populations.

Holstein cattle dominate the global milk production industry because of their outstanding milk production, however, this breed is susceptible to tropical endemic pathogens and suffers from heat stress and thus fewer Holstein populations are raised in tropical areas. The bovine major histocompatibility complex (BoLA)-DRB3 class II gene is used as a marker for disease and immunological traits, and its polymorphism has been studied extensively in Holstein cattle from temperate and cold regions. We studied the genetic diversity of the BoLA-DRB3 gene in South American Holstein populations to determine whether tropical populations have diverged from those bred in temperate and cold regions by selection and/or crossbreeding with local native breeds. We specifically studied Exon 2 of this gene from 855 South American Holstein individuals by a polymerase chain reaction (PCR) sequence-based typing method. We found a high degree of gene diversity at the allelic (Na > 20 and He > 0.87) and molecular (π > 0.080) levels, but a low degree of population structure (FST = 0.009215). A principal components analysis and tree showed that the Bolivian subtropical population had the largest genetic divergence compared with Holsteins bred in temperate or cold regions, and that this population was closely related to Bolivian Creole cattle. Our results suggest that Holstein genetic divergence can be explained by selection and/or gene introgression from local germplasms. This is the first examination of BoLA-DRB3 in Holsteins adapted to tropical environments, and contributes to an ongoing effort to catalog bovine MHC allele frequencies by breed and location.

Study of the influence of genes related to muscle oxidative processes on beef color.

The biochemical bases of meat color are determined by the concentration and redox state of myoglobin, hemoglobin, cytochromes, and other pigments. Post-mortem depletion of cellular oxygen results in oxidative stresses that consume NADH and affects reducing activity, while enzymatic detoxification influences the cellular oxidative processes, both affecting meat color. The aim of this work was to study the influence of several genes related to cellular oxidative processes that could affect CIELAB meat color parameters. The study was performed in steers that received a grass-based diet combined with grain, hays and silages. Results suggest a possible link between colorimetric parameters (a*, b* and chroma) and SNPs in the GSTP1 gene (P<0.05). Although the influence of the enzymes, encoded by GSTP1 gene, on meat color has been proposed previously at biochemical level and protein expression level, further association studies in different populations and functional studies of proteins are needed to confirm the genetic determination of that gene on meat color.

Assessment of biodiversity in Chilean cattle using the distribution of major histocompatibility complex class II BoLA-DRB3 allele.

Bovine leukocyte antigens (BoLAs) are used extensively as markers for bovine disease and immunological traits. In this study, we estimated BoLA-DRB3 allele frequencies using 888 cattle from 10 groups, including seven cattle breeds and three crossbreeds: 99 Red Angus, 100 Black Angus, 81 Chilean Wagyu, 49 Hereford, 95 Hereford × Angus, 71 Hereford × Jersey, 20 Hereford × Overo Colorado, 113 Holstein, 136 Overo Colorado, and 124 Overo Negro cattle. Forty-six BoLA-DRB3 alleles were identified, and each group had between 12 and 29 different BoLA-DRB3 alleles. Overo Negro had the highest number of alleles (29); this breed is considered in Chile to be an 'Old type' European Holstein Friesian descendant. By contrast, we detected 21 alleles in Holstein cattle, which are considered to be a 'Present type' Holstein Friesian cattle. Chilean cattle groups and four Japanese breeds were compared by neighbor-joining trees and a principal component analysis (PCA). The phylogenetic tree showed that Red Angus and Black Angus cattle were in the same clade, crossbreeds were closely related to their parent breeds, and Holstein cattle from Chile were closely related to Holstein cattle in Japan. Overall, the tree provided a thorough description of breed history. It also showed that the Overo Negro breed was closely related to the Holstein breed, consistent with historical data indicating that Overo Negro is an 'Old type' Holstein Friesian cattle. This allelic information will be important for investigating the relationship between major histocompatibility complex (MHC) and disease.

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.

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.

ELA-DRA polymorphisms are not associated with Equine Arteritis Virus infection in horses from Argentina.

Polymorphisms at Major Histocompatibility Complex (MHC) genes have been associated with resistance/susceptibility to infectious diseases in domestic animals. The aim of this investigation was to evaluate whether polymorphisms of the DRA gene the Equine Lymphocyte Antigen is associated with susceptibility to Equine Arteritis Virus (EAV) infection in horses in Argentina. The equine DRA gene was screened for polymorphisms using Pyrosequencing® Technology which allowed the detection of three ELA-DRA exon 2 alleles. Neither allele frequencies nor genotypic differentiation exhibited any statistically significant (P-values=0.788 and 0.745) differences between the EAV-infected and no-infected horses. Fisher's exact test and OR calculations did not show any significant association. As a consequence, no association could be established between the serological condition and ELA-DRA.

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.

Eca20 microsatellite polymorphisms in equine viral arteritis-infected horses from Argentina.

We investigated the association of equine arteritis virus (EAV) infection and three short tandem repeat (STR) polymorphisms located within or in close proximity to equine lymphocyte antigen (ELA) region. We used a case-control design as a first approach before proceeding to select candidate genes. One hundred and sixty-five Silla Argentino horses were taken in 2002 from positive serological detections of EAV in Argentina, to determine whether STR genotypes were correlated to genetic susceptibility to EVA. Allele frequency distribution did not show significant differences between both groups (P = 0.0781). However, in particular alleles, Fisher exact test and odds ratio calculations showed significant values >1 for TKY08 and LEX52, and <1 for UM011, TKY08, LEX52 and VHL20. Interestingly, TKY08 STR is located in ELA class I region.