Genome-wide association study to identify genomic regions and positional candidate genes associated with male fertility in beef cattle

[EN] Fertility plays a key role in the success of calf production, but there is evidence that reproductive efficiency in beef cattle has decreased during the past half-century worldwide. Therefore, identifying animals with superior fertility could significantly impact cow-calf production efficiency. The objective of this research was to identify candidate regions affecting bull fertility in beef cattle and positional candidate genes annotated within these regions. A GWAS using a weighted single-step genomic BLUP approach was performed on 265 crossbred beef bulls to identify markers associated with scrotal circumference (SC) and sperm motility (SM). Eight windows containing 32 positional candidate genes and five windows containing 28 positional candidate genes explained more than 1% of the genetic variance for SC and SM, respectively. These windows were selected to perform gene annotation, QTL enrichment, and functional analyses. Functional candidate gene prioritization analysis revealed 14 prioritized candidate genes for SC of which MAP3K1 and VIP were previously found to play roles in male fertility. A different set of 14 prioritized genes were identified for SM and five were previously identified as regulators of male fertility (SOD2, TCP1, PACRG, SPEF2, PRLR). Significant enrichment results were identified for fertility and body conformation QTLs within the candidate windows. Gene ontology enrichment analysis including biological processes, molecular functions, and cellular components revealed significant GO terms associated with male fertility. The identification of these regions contributes to a better understanding of fertility associated traits and facilitates the discovery of positional candidate genes for future investigation of causal mutations and their implications.SIThe authors acknowledge financial support from the (FDE.13.17) Sustainable Beef and Forage Science Cluster funded by the Canadian Beef Cattle Check-Off, Beef Cattle Research Council (BCRC), Alberta Beef Producers, Alberta Cattle Feeders’ Association, Beef Farmers of Ontario, La Fédération des Productuers de bovins du Québec, and Agriculture and Agri-Food Canada’s Canadian Agricultural Partnership. This study was also supported by the Ontario Ministry of Agriculture, Food, and Rural Affairs (OMAFRA), Ontario Ministry of Research and Innovation, Agriculture and Agri-Food Canada (AAFC), and Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant. Hannah Sweett was supported by the OMAFRA Highly Qualified Personnel Scholarship Program

A new tetra-primer ARMS-PCR for genotyping bovine kappa-casein polymorphisms

[EN] Kappa-casein (κ-casein) is one of the most abundant milk proteins. Its main function is to avoid the aggregation of casein micelles, keeping them, and therefore calcium phosphate, in pockets in solution. In bovines, a κ-casein functional polymorphism has been associated with fat, calcium, and protein milk contents and faster curd contraction in cheese production. Quicker curd contraction reduces the loss of milk solids, enhancing cheese yield. This polymorphism induces a double amino acid substitution (Thr136Ile and Ala148Asp). The polymorphism is normally detected by PCR-RFLP, which is a laborious method. An interesting methodological alternative is the tetra-primer amplification refractory mutation system PCR (tetra-primer ARMS-PCR). A tetra-primer ARMS-PCR for the detection of this κ-casein polymorphism has been described. However, specificity was not achieved, probably due to problems with primer design. We developed a new tetra-primer ARMS-PCR for the detection of the κ-casein polymorphism. This new method was validated in a double-blind test, by comparison with the results obtained for 50 Guzerá bulls formerly genotyped by PCR-RFLP. This new method achieved 100% sensitivity and specificity. We conclude that this method is a useful, cost-efficient alternative for the detection of functional κ-casein polymorphismsSIResearch supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Pesquisa (CNPq), Pró-Reitoria de Pesquisa de Universidade Federal de Minas gerais (PRPq/UFMG), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). MRSC, PASF, MAM, and RSV have CNPq fellowships. IRC has a CAPES fellowship. We are grateful to Professor Vânia Maldini Pena and Ms. Ariane Figueiredo Menicucci from Colégio Brasileiro de Criadores do Guzerá, for providing biological sample

Transcriptome analysis of perirenal fat from Spanish Assaf suckling lamb carcasses showing different levels of kidney knob and channel fat

[EN] Introduction: Suckling lamb meat is highly appreciated in European Mediterranean countries because of its mild flavor and soft texture. In suckling lamb carcasses, perirenal and pelvic fat depots account for a large fraction of carcass fat accumulation, and their proportions are used as an indicator of carcass quality. Material and Methods: This study aimed to characterize the genetic mechanisms that regulate fat deposition in suckling lambs by evaluating the transcriptomic differences between Spanish Assaf lambs with significantly different proportions of kidney knob and channel fat (KKCF) depots in their carcasses (4 High-KKCF lambs vs. 4 Low-KKCF lambs). Results: The analyzed fat tissue showed overall dominant expression of white adipose tissue gene markers, although due to the young age of the animals (17–36 days), the expression of some brown adipose tissue gene markers (e.g., UCP1, CIDEA) was still identified. The transcriptomic comparison between the High-KKCF and Low-KKCF groups revealed a total of 80 differentially expressed genes (DEGs). The enrichment analysis of the 49 DEGs with increased expression levels in the Low-KKCF lambs identified significant terms linked to the biosynthesis of lipids and thermogenesis, which may be related to the higher expression of the UCP1 gene in this group. In contrast, the enrichment analysis of the 31 DEGs with increased expression in the High-KKCF lambs highlighted angiogenesis as a key biological process supported by the higher expression of some genes, such as VEGF-A and THBS1, which encode a major angiogenic factor and a large adhesive extracellular matrix glycoprotein, respectively. Discussion: The increased expression of sestrins, which are negative regulators of the mTOR complex, suggests that the preadipocyte differentiation stage is being inhibited in the High-KKCF group in favor of adipose tissue expansion, in which vasculogenesis is an essential process. All of these results suggest that the fat depots of the High-KKCF animals are in a later stage of development than those of the Low-KKCF lambs. Further genomic studies based on larger sample sizes and complementary analyses, such as the identification of polymorphisms in the DEGs, should be designed to confirm these results and achieve a deeper understanding of the genetic mechanisms underlying fat deposition in suckling lambsSIThe research described here has been funded by the project EpiMilksheep (RTI2018-093535-B-100) funded by the Spanish Ministry of Science and Innovation. MA-G is funded by a predoctoral fellowship from the Junta de Castilla and León Government and the European Social Fun

Evaluación preliminar de los entornos interactivos de aprendizaje basados en problemas en la enseñanza universitaria

[ES] Uno de los grandes retos de los cursos de enseñanza universitaria que abordan temas relacionados con la genética y la genómica consiste en mostrar a los estudiantes, de forma simplificada, cómo la genómica puede dar respuesta a determinados problemas mediante la integración de información procedente de distintas fuentes y metodologías. Esto también es necesario para garantizar que los futuros profesionales de la medicina o la veterinaria sean capaces de integrar la genómica en aplicaciones clínicas o de diagnóstico cotidianas. Así, el objetivo principal de este estudio fue evaluar la aceptación por parte del alumnado de enseñanza superior de entornos interactivos de aprendizaje relacionados con la docencia de la genética y la genómica. Así, como grupo de innovación docente de la Universidad de León (ULE) VetGeneULE, hemos desarrollado dos entornos interactivos de aprendizaje basado en problemas, dirigidos a estudiantes de una asignatura de Grado y otra de Máster de titulaciones de la ULE, utilizando la plataforma Genially, uno para facilitar la compresión de las bases de la genética mendeliana y otro poniendo al estudiante al frente de la resolución de un caso de estudio genómico explotando una estrategia de gamificación. En ambos casos, la presentación interactiva ofrece a los estudiantes un entorno teórico-práctico guiado donde el aprendizaje tiene lugar gracias a metodologías pedagógicas alternativas como son la gamificación y el aprendizaje basado en problemas. Tras la realización de la presentación, los estudiantes de forma voluntaria contestaron una encuesta para conocer el grado de interés que les había suscitado este tipo de presentaciones interactivas. Como resultado más importante obtuvimos una muy diferente participación por parte de ambos grupos de alumnos. Sin embargo, todos ellos estaban de acuerdo en que este tipo de presentaciones interactivas eran un complemento muy útil a las clases teóricas para un aprendizaje más profundo y activo

Integrated analyses of the methylome and transcriptome to unravel sex differences in the perirenal fat from suckling lambs

[EN] In sheep, differences were observed regarding fat accumulation and fatty acid (FA) composition between males and females, which may impact the quality and organoleptic characteristics of the meat. The integration of different omics technologies is a relevant approach for investigating biological and genetic mechanisms associated with complex traits. Here, the perirenal tissue of six male and six female Assaf suckling lambs was evaluated using RNA sequencing and whole-genome bisulfite sequencing (WGBS). A multiomic discriminant analysis using multiblock (s)PLS-DA allowed the identification of 314 genes and 627 differentially methylated regions (within these genes), which perfectly discriminate between males and females. These candidate genes overlapped with previously reported QTLs for carcass fat volume and percentage of different FAs in milk and meat from sheep. Additionally, differentially coexpressed (DcoExp) modules of genes between males (nine) and females (three) were identified that harbour 22 of these selected genes. Interestingly, these DcoExp were significantly correlated with fat percentage in different deposits (renal, pelvic, subcutaneous and intramuscular) and were associated with relevant biological processes for adipogenesis, adipocyte differentiation, fat volume and FA composition. Consequently, these genes may potentially impact adiposity and meat quality traits in a sex-specific manner, such as juiciness, tenderness and flavourSIThis research work was financially supported by the RTI2018-093535-B-I00 project funded by the “Agencia Estatal de Investigación” of the Spanish Ministry of Science and Innovation (Madrid, Spain). MA-G is funded by the Junta de Castilla y Leon fellowship. This research has used the high- performance computing resources of the Castilla y León Supercomputing Center (SCAYLE, www.scayle.es; León, Spain). PF is the beneficiary of a Maria Zambrano Grant of the University of Leon funding by the Ministry of Universities (Madrid, Spain) and financed by the European Union-Next Generation E

Genetic mechanisms underlying spermatic and testicular traits within and among cattle breeds: systematic review and prioritization of GWAS results1

[EN] Reduced bull fertility imposes economic losses in bovine herds. Specifically, testicular and spermatic traits are important indicators of reproductive efficiency. Several genome-wide association studies (GWAS) have identified genomic regions associated with these fertility traits. The aims of this study were as follows: 1) to perform a systematic review of GWAS results for spermatic and testicular traits in cattle and 2) to identify key functional candidate genes for these traits. The identification of functional candidate genes was performed using a systems biology approach, where genes shared between traits and studies were evaluated by a guilt by association gene prioritization (GUILDify and ToppGene software) in order to identify the best functional candidates. These candidate genes were integrated and analyzed breeds. Results showed that GWAS for testicular-related traits have been developed for beef breeds only, whereas the majority of GWAS for spermatic-related traits were conducted using dairy breeds. comparing traits measured within the same study, the highest number of genes shared between different traits was observed, indicating a high impact of the population genetic structure and environmental effects. Several chromosomal regions were enriched for functional candidate genes associated with fertility traits. Moreover, multiple functional candidate genes were enriched for markers in a species-specific basis, taurine (Bos taurus) or indicine (Bos indicus). For the different candidate regions identified in the GWAS in the literature, functional candidate genes were detected as follows: B. Taurus chromosome X (BTX) (TEX11, IRAK, CDK16, ATP7A, ATRX, HDAC6, FMR1, L1CAM, MECP2, etc.), BTA17 (TRPV4 and DYNLL1), and BTA14 (MOS, FABP5, ZFPM2). These genes are responsible for regulating metabolic pathways or biological processes associated with fertility, such as progression of spermatogenesis, control of ciliary activity, development of Sertoli cells, DNA integrity in spermatozoa, and homeostasis of testicular cells. This study represents the first systematic review on male fertility traits in cattle using a system biology approach to identify key candidate genes for these traits.S

Identification of Candidate Genes for Reactivity in Guzerat (Bos indicus) Cattle: A Genome-Wide Association Study

[EN] Temperament is fundamental to animal production due to its direct influence on the animalherdsman relationship. When compared to calm animals, the aggressive, anxious or fearful ones exhibit less weight gain, lower reproductive efficiency, decreased milk production and higher herd maintenance costs, all of which contribute to reduced profits. However, temperament is a trait that is complex and difficult to assess. Recently, a new quantitative system, REATEST®, for assessing reactivity, a phenotype of temperament, was developed. Herein, we describe the results of a Genome-wide association study for reactivity, assessed using REATEST® with a sample of 754 females from five dual-purpose (milk and meat production) Guzerat (Bos indicus) herds. Genotyping was performed using a 50k SNP chip and a twostep mixed model approach (Grammar-Gamma) with a one-by-one marker regression was used to identify QTLs. QTLs for reactivity were identified on chromosomes BTA1, BTA5, BTA14, and BTA25. Five intronic and two intergenic markers were significantly associated with reactivity. POU1F1, DRD3, VWA3A, ZBTB20, EPHA6, SNRPF and NTN4 were identified as candidate genes. Previous QTL reports for temperament traits, covering areas surrounding the SNPs/genes identified here, further corroborate these associations. The seven genes identified in the present study explain 20.5% of reactivity variance and give a better understanding of temperament biology. IntroductionSIWe thank the farmers, who allowed the development of this project in their facilities. We thank to Mr. Peter Laspina for performing language review and for the valuable comments. This study was supported by Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Empresa Brasileira de Pesquisa Agropecuária (Embrapa). Maria Gabriela Campolina Diniz Peixoto was supported by the Fapemig—CVZ APQ 01353 e CVZ APQ 3182–5.04/07. MRSC has a fellowship from the CNPq– 307975/2010-0 and was supported by CNPq– 312068/2015-8 and 481018/2008-5 projects. MGCDP, RVV, MAM have fellowships from FAPEMIG. PASF has CNPq fellowship, FCS and ICR have CAPES fellowships

Feet and legs malformation in Nellore cattle: genetic analysis and prioritization of GWAS results

Beef cattle affected by feet and legs malformations (FLM) cannot perform their productive and reproductive functions satisfactorily, resulting in significant economic losses. Accelerated weight gain in young animals due to increased fat deposition can lead to ligaments, tendon and joint strain and promote gene expression patterns that lead to changes in the normal architecture of the feet and legs. The possible correlated response in the FLM due to yearling weight (YW) selection suggest that this second trait could be used as an indirect selection criterion. Therefore, FLM breeding values and the genetic correlation between FLM and yearling weight (YW) were estimated for 295,031 Nellore animals by fitting a linear-threshold model in a Bayesian approach. A genome-wide association study was performed to identify genomic windows and positional candidate genes associated with FLM. The effects of single nucleotide polymorphisms (SNPs) on FLM phenotypes (affected or unaffected) were estimated using the weighted single-step genomic BLUP method, based on genotypes of 12,537 animals for 461,057 SNPs. Twelve non-overlapping windows of 20 adjacent SNPs explaining more than 1% of the additive genetic variance were selected for candidate gene annotation. Functional and gene prioritization analysis of candidate genes identified six genes (ATG7, EXT1, ITGA1, PPARD, SCUBE3, and SHOX) that may play a role in FLM expression due to their known role in skeletal muscle development, aberrant bone growth, lipid metabolism, intramuscular fat deposition and skeletogenesis. Identifying genes linked to foot and leg malformations enables selective breeding for healthier herds by reducing the occurrence of these conditions. Genetic markers can be used to develop tests that identify carriers of these mutations, assisting breeders in making informed breeding decisions to minimize the incidence of malformations in future generations, resulting in greater productivity and animal welfare

Biotinidase deficiency: Genotype-biochemical phenotype association in Brazilian patients

[EN] The association between the BTD genotype and biochemical phenotype [profound biotinidase deficiency (BD), partial BD or heterozygous activity] is not always consistent. This study aimed to investigate the genotype-biochemical phenotype association in patients with low biotinidase activity. Methods All exons, the 5'UTR and the promoter of the BTD gene were sequenced in 72 Brazilian individuals who exhibited low biotinidase activity. For each patient, the expected biochemical phenotype based on the known genotype was compared with the observed biochemical phenotype. Additional non-genetic factors that could affect the biotinidase activity were also analysed. Most individuals were identified by neonatal screening (n = 66/72). When consecutive results for the same patient were compared, age, prematurity and neonatal jaundice appeared to affect the level of biotinidase activity. The biochemical phenotype at the time of the second blood collection changed in 11/22 patients compared to results from the first sample. Three novel variants were found: c.1337T>C (p.L446P), c.1466A>G (p.N489S) and c.962G>A (p.W321*). Some patients with the same genotype presented different biochemical phenotypes. The expected and observed biochemical phenotypes agreed in 68.5% of cases (concordant patients). The non-coding variants c.-183G>A, c.-315A>G and c.-514C>T were present in heterozygosis in 5/17 discordant patients. In addition, c.- 183G>A and c.-514C>T were also present in 10/37 concordant patients. The variants found in the promoter region do not appear to have a strong impact on biotinidase activity. Since there is a disparity between the BTD genotype and biochemical phenotype, and biotinidase activity may be affected by both genetic and non-genetic factors, we suggest that the diagnosis of BD should be based on more than one measurement of plasma biotinidase activity. DNA analysis can be of additional relevance to differentiate between partial BD and heterozygosity.SIThis study received financial support from Fundo de Incentivo à Pesquisa e Eventos/Hospital de Clínicas de Porto Alegre (FIPE-HCPA) for research materials and publication fee. Post Graduate Program in Genetics and Molecular Biology (Universidade Federal do Rio Grande do Sul) funded the translation. ECN has a commercial affiliation (CTN Diagnósticos) which did not have any role or financial contribution to this research. TB have fellowship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes). FS had fellowship from the Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS). IVDS, MRSC and PASF have fellowships from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). HB receives a research grant of Orphan Europe. The funders did no provide support in the form of salaries for any author, and did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section