Network analysis uncovers putative genes affecting resistance to tick infestation in Braford cattle skin.

BACKGROUND: Genetic resistance in cattle is considered a suitable way to control tick burden and its consequent losses for livestock production. Exploring tick-resistant (R) and tick-susceptible (S) hosts, we investigated the genetic mechanisms underlying the variation of Braford resistance to tick infestation. Skin biopsies from four-times-artificially infested R (n = 20) and S (n = 19) hosts, obtained before the first and 24 h after the fourth tick infestation were submitted to RNA-Sequencing. Differential gene expression, functional enrichment, and network analysis were performed to identify genetic pathways and transcription factors (TFs) affecting host resistance. RESULTS: Intergroup comparisons of hosts before (Rpre vs. Spre) and after (Rpost vs. Spost) tick infestation found 51 differentially expressed genes (DEGs), of which almost all presented high variation (TopDEGs), and 38 were redundant genes. Gene expression was consistently different between R and S hosts, suggesting the existence of specific anti-tick mechanisms. In the intragroup comparisons, Rpost vs. Rpre and Spost vs. Spre, we found more than two thousand DEGs in response to tick infestation in both resistance groups. Redundant and non-redundant TopDEGs with potential anti-tick functions suggested a role in the development of different levels of resistance within the same breed. Leukocyte chemotaxis was over-represented in both hosts, whereas skin degradation and remodeling were only found in TopDEGs from R hosts. Also, these genes indicated the participation of cytokines, such as IL6 and IL22, and the activation of Wingless (WNT)-signaling pathway. A central gene of this pathway, WNT7A, was consistently modulated when hosts were compared. Moreover, the findings based on a genome-wide association study (GWAS) corroborate the prediction of the WNT-signaling pathway as a candidate mechanism of resistance. The regulation of immune response was the most relevant pathway predicted for S hosts. Members of Ap1 and NF-kB families were the most relevant TFs predicted for R and S, respectively. CONCLUSION: This work provides indications of genetic mechanisms presented by Braford cattle with different levels of resistance in response to tick infestation, contributing to the search of candidate genes for tick resistance in bovine.

Identification of putative regulatory regions and transcription factors associated with intramuscular fat content traits.

BACKGROUND: Integration of high throughput DNA genotyping and RNA-sequencing data allows for the identification of genomic regions that control gene expression, known as expression quantitative trait loci (eQTL), on a whole genome scale. Intramuscular fat (IMF) content and carcass composition play important roles in metabolic and physiological processes in mammals because they influence insulin sensitivity and consequently prevalence of metabolic diseases such as obesity and type 2 diabetes. However, limited information is available on the genetic variants and mechanisms associated with IMF deposition in mammals. Thus, our hypothesis was that eQTL analyses could identify putative regulatory regions and transcription factors (TFs) associated with intramuscular fat (IMF) content traits. RESULTS: We performed an integrative eQTL study in skeletal muscle to identify putative regulatory regions and factors associated with intramuscular fat content traits. Data obtained from skeletal muscle samples of 192 animals was used for association analysis between 461,466 SNPs and the transcription level of 11,808 genes. This yielded 1268 cis- and 10,334 trans-eQTLs, among which we identified nine hotspot regions that each affected the expression of > 119 genes. These putative regulatory regions overlapped with previously identified QTLs for IMF content. Three of the hotspots respectively harbored the transcription factors USF1, EGR4 and RUNX1T1, which are known to play important roles in lipid metabolism. From co-expression network analysis, we further identified modules significantly correlated with IMF content and associated with relevant processes such as fatty acid metabolism, carbohydrate metabolism and lipid metabolism. CONCLUSION: This study provides novel insights into the link between genotype and IMF content as evident from the expression level. It thereby identifies genomic regions of particular importance and associated regulatory factors. These new findings provide new knowledge about the biological processes associated with genetic variants and mechanisms associated with IMF deposition in mammals.

Genomic structure and marker-derived gene networks for growth and meat quality traits of Brazilian Nelore beef cattle.

BACKGROUND: Nelore is the major beef cattle breed in Brazil with more than 130 million heads. Genome-wide association studies (GWAS) are often used to associate markers and genomic regions to growth and meat quality traits that can be used to assist selection programs. An alternative methodology to traditional GWAS that involves the construction of gene network interactions, derived from results of several GWAS is the AWM (Association Weight Matrices)/PCIT (Partial Correlation and Information Theory). With the aim of evaluating the genetic architecture of Brazilian Nelore cattle, we used high-density SNP genotyping data (~770,000 SNP) from 780 Nelore animals comprising 34 half-sibling families derived from highly disseminated and unrelated sires from across Brazil. The AWM/PCIT methodology was employed to evaluate the genes that participate in a series of eight phenotypes related to growth and meat quality obtained from this Nelore sample. RESULTS: Our results indicate a lack of structuring between the individuals studied since principal component analyses were not able to differentiate families by its sires or by its ancestral lineages. The application of the AWM/PCIT methodology revealed a trio of transcription factors (comprising VDR, LHX9 and ZEB1) which in combination connected 66 genes through 359 edges and whose biological functions were inspected, some revealing to participate in biological growth processes in literature searches. CONCLUSIONS: The diversity of the Nelore sample studied is not high enough to differentiate among families neither by sires nor by using the available ancestral lineage information. The gene networks constructed from the AWM/PCIT methodology were a useful alternative in characterizing genes and gene networks that were allegedly influential in growth and meat quality traits in Nelore cattle.

Global liver gene expression differences in Nelore steers with divergent residual feed intake phenotypes.

BACKGROUND: Efficiency of feed utilization is important for animal production because it can reduce greenhouse gas emissions and improve industry profitability. However, the genetic basis of feed utilization in livestock remains poorly understood. Recent developments in molecular genetics, such as platforms for genome-wide genotyping and sequencing, provide an opportunity to identify genes and pathways that influence production traits. It is known that transcriptional networks influence feed efficiency-related traits such as growth and energy balance. This study sought to identify differentially expressed genes in animals genetically divergent for Residual Feed Intake (RFI), using RNA sequencing methodology (RNA-seq) to obtain information from genome-wide expression profiles in the liver tissues of Nelore cattle. RESULTS: Differential gene expression analysis between high Residual Feed Intake (HRFI, inefficient) and low Residual Feed Intake (LRFI, efficient) groups was performed to provide insights into the molecular mechanisms that underlie feed efficiency-related traits in beef cattle. A total of 112 annotated genes were identified as being differentially expressed between animals with divergent RFI phenotypes. These genes are involved in ion transport and metal ion binding; act as membrane or transmembrane proteins; and belong to gene clusters that are likely related to the transport and catalysis of molecules through the cell membrane and essential mechanisms of nutrient absorption. Genes with functions in cellular signaling, growth and proliferation, cell death and survival were also differentially expressed. Among the over-represented pathways were drug or xenobiotic metabolism, complement and coagulation cascades, NRF2-mediated oxidative stress, melatonin degradation and glutathione metabolism. CONCLUSIONS: Our data provide new insights and perspectives on the genetic basis of feed efficiency in cattle. Some previously identified mechanisms were supported and new pathways controlling feed efficiency in Nelore cattle were discovered. We potentially identified genes and pathways that play key roles in hepatic metabolic adaptations to oxidative stress such as those involved in antioxidant mechanisms. These results improve our understanding of the metabolic mechanisms underlying feed efficiency in beef cattle and will help develop strategies for selection towards the desired phenotype.

Detection of quantitative trait loci for mineral content of Nelore longissimus dorsi muscle.

BACKGROUND: Beef cattle require dietary minerals for optimal health, production and reproduction. Concentrations of minerals in tissues are at least partly genetically determined. Mapping genomic regions that affect the mineral content of bovine longissimus dorsi muscle can contribute to the identification of genes that control mineral balance, transportation, absorption and excretion and that could be associated to metabolic disorders. METHODS: We applied a genome-wide association strategy and genotyped 373 Nelore steers from 34 half-sib families with the Illumina BovineHD BeadChip. Genome-wide association analysis was performed for mineral content of longissimus dorsi muscle using a Bayesian approach implemented in the GenSel software. RESULTS: Muscle mineral content in Bos indicus cattle was moderately heritable, with estimates ranging from 0.29 to 0.36. Our results suggest that variation in mineral content is influenced by numerous small-effect QTL (quantitative trait loci) but a large-effect QTL that explained 6.5% of the additive genetic variance in iron content was detected at 72 Mb on bovine chromosome 12. Most of the candidate genes present in the QTL regions for mineral content were involved in signal transduction, signaling pathways via integral (also called intrinsic) membrane proteins, transcription regulation or metal ion binding. CONCLUSIONS: This study identified QTL and candidate genes that affect the mineral content of skeletal muscle. Our findings provide the first step towards understanding the molecular basis of mineral balance in bovine muscle and can also serve as a basis for the study of mineral balance in other organisms.

Identification of genomic regions associated with feed efficiency in Nelore cattle.

BACKGROUND: Feed efficiency is jointly determined by productivity and feed requirements, both of which are economically relevant traits in beef cattle production systems. The objective of this study was to identify genes/QTLs associated with components of feed efficiency in Nelore cattle using Illumina BovineHD BeadChip (770 k SNP) genotypes from 593 Nelore steers. The traits analyzed included: average daily gain (ADG), dry matter intake (DMI), feed-conversion ratio (FCR), feed efficiency (FE), residual feed intake (RFI), maintenance efficiency (ME), efficiency of gain (EG), partial efficiency of growth (PEG) and relative growth rate (RGR). The Bayes B analysis was completed with Gensel software parameterized to fit fewer markers than animals. Genomic windows containing all the SNP loci in each 1 Mb that accounted for more than 1.0% of genetic variance were considered as QTL region. Candidate genes within windows that explained more than 1% of genetic variance were selected by putative function based on DAVID and Gene Ontology. RESULTS: Thirty-six QTL (1-Mb SNP window) were identified on chromosomes 1, 2, 3, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 19, 20, 21, 22, 24, 25 and 26 (UMD 3.1). The amount of genetic variance explained by individual QTL windows for feed efficiency traits ranged from 0.5% to 9.07%. Some of these QTL minimally overlapped with previously reported feed efficiency QTL for Bos taurus. The QTL regions described in this study harbor genes with biological functions related to metabolic processes, lipid and protein metabolism, generation of energy and growth. Among the positional candidate genes selected for feed efficiency are: HRH4, ALDH7A1, APOA2, LIN7C, CXADR, ADAM12 and MAP7. CONCLUSIONS: Some genomic regions and some positional candidate genes reported in this study have not been previously reported for feed efficiency traits in Bos indicus. Comparison with published results indicates that different QTLs and genes may be involved in the control of feed efficiency traits in this Nelore cattle population, as compared to Bos taurus cattle.

Identification of KCNJ11 as a functional candidate gene for bovine meat tenderness.

The potassium inwardly rectifying channel, subfamily J, member 11 (KCNJ11) gene was investigated as a candidate for meat tenderness based on the effects reported on muscle for KCNJ11 gene knockout in rat models and its position in a quantitative trait locus (QTL) for meat tenderness in the bovine genome. Sequence variations in the KCNJ11 gene were described by sequencing six amplified fragments, covering almost the entire gene. We identified single nucleotide polymorphisms (SNP) and validated them by different approaches, taking advantage of simultaneous projects that are being developed with the same Nelore population. By sequencing the KCNJ11 in Nelore steers representing extreme phenotypes for Warner-Bratzler shear force (WBSF), it was possible to identify 22 SNPs. We validated two of the identified markers by genotyping the whole population (n = 460). Analysis of association between genotypes and WBSF values revealed a significant additive effect of a SNP at different meat aging times (P ≤ 0.05). In addition, an association between the expression levels of KCNJ11 and WBSF was found, with lower expression levels of KCNJ11 associated with more tender meat (P ≤ 0.05). The results showed that the KCNJ11 gene is a candidate mapped to a QTL for meat tenderness previously identified on BTA15 and may be useful to identify animals with genetic potential to produce tender meat. The effect of KCNJ11 observed on muscle is potentially due to changes in activity of KATP channels, which in turn influence the flow of potassium in the intracellular space, allowing establishment of the membrane potential necessary for muscle contraction.

Transcriptome analysis of the spider Phoneutria pertyi venom glands reveals novel venom components for the genus Phoneutria.

Phoneutria nigriventer spider venom has been studied for more than 40 years and several components with pharmacological potential have been described in it. However, studies on venoms from other species of the Phoneutria genus are scarce. In this work, a conventional cDNA library from the species Phoneutria pertyi venom glands was constructed, aiming to identify novel putative cysteine-rich peptide toxins for the genus Phoneutria. 296 unique sequences were identified and 51 sequences corresponded to putative cysteine-rich peptide toxins. Besides cysteine-rich peptide toxins, other putative venom components such as protease inhibitors, defensins and serine proteinases were identified. Furthermore, by manual curation of the sequences with no match at UniProt, we were able to identify glycine-rich proteins (GRP), a class of venom component never described in Phoneutria genus. This work describes the first complete sequences of toxins from the venom of P. pertyi and reveals that, despite most of the retrieved toxins show a high identity to toxins identified in Phoneutria genus, novel putative toxins remains to be described.

An overview of Phoneutria nigriventer spider venom using combined transcriptomic and proteomic approaches.

Phoneutria nigriventer is one of the largest existing true spiders and one of the few considered medically relevant. Its venom contains several neurotoxic peptides that act on different ion channels and chemical receptors of vertebrates and invertebrates. Some of these venom toxins have been shown as promising models for pharmaceutical or biotechnological use. However, the large diversity and the predominance of low molecular weight toxins in this venom have hampered the identification and deep investigation of the less abundant toxins and the proteins with high molecular weight. Here, we combined conventional and next-generation cDNA sequencing with Multidimensional Protein Identification Technology (MudPIT), to obtain an in-depth panorama of the composition of P. nigriventer spider venom. The results from these three approaches showed that cysteine-rich peptide toxins are the most abundant components in this venom and most of them contain the Inhibitor Cysteine Knot (ICK) structural motif. Ninety-eight sequences corresponding to cysteine-rich peptide toxins were identified by the three methodologies and many of them were considered as putative novel toxins, due to the low similarity to previously described toxins. Furthermore, using next-generation sequencing we identified families of several other classes of toxins, including CAPs (Cysteine Rich Secretory Protein-CRiSP, antigen 5 and Pathogenesis-Related 1-PR-1), serine proteinases, TCTPs (translationally controlled tumor proteins), proteinase inhibitors, metalloproteinases and hyaluronidases, which have been poorly described for this venom. This study provides an overview of the molecular diversity of P. nigriventer venom, revealing several novel components and providing a better basis to understand its toxicity and pharmacological activities.

The Bos taurus-Bos indicus balance in fertility and milk related genes.

Numerical approaches to high-density single nucleotide polymorphism (SNP) data are often employed independently to address individual questions. We linked independent approaches in a bioinformatics pipeline for further insight. The pipeline driven by heterozygosity and Hardy-Weinberg equilibrium (HWE) analyses was applied to characterize Bos taurus and Bos indicus ancestry. We infer a gene co-heterozygosity network that regulates bovine fertility, from data on 18,363 cattle with genotypes for 729,068 SNP. Hierarchical clustering separated populations according to Bos taurus and Bos indicus ancestry. The weights of the first principal component were subjected to Normal mixture modelling allowing the estimation of a gene's contribution to the Bos taurus-Bos indicus axis. We used deviation from HWE, contribution to Bos indicus content and association to fertility traits to select 1,284 genes. With this set, we developed a co-heterozygosity network where the group of genes annotated as fertility-related had significantly higher Bos indicus content compared to other functional classes of genes, while the group of genes associated with milk production had significantly higher Bos taurus content. The network analysis resulted in capturing novel gene associations of relevance to bovine domestication events. We report transcription factors that are likely to regulate genes associated with cattle domestication and tropical adaptation. Our pipeline can be generalized to any scenarios where population structure requires scrutiny at the molecular level, particularly in the presence of a priori set of genes known to impact a phenotype of evolutionary interest such as fertility.

Genome-Enabled Prediction of Breeding Values for Feedlot Average Daily Weight Gain in Nelore Cattle.

Nelore is the most economically important cattle breed in Brazil, and the use of genetically improved animals has contributed to increased beef production efficiency. The Brazilian beef feedlot industry has grown considerably in the last decade, so the selection of animals with higher growth rates on feedlot has become quite important. Genomic selection (GS) could be used to reduce generation intervals and improve the rate of genetic gains. The aim of this study was to evaluate the prediction of genomic-estimated breeding values (GEBV) for average daily weight gain (ADG) in 718 feedlot-finished Nelore steers. Analyses of three Bayesian model specifications [Bayesian GBLUP (BGBLUP), BayesA, and BayesCπ] were performed with four genotype panels [Illumina BovineHD BeadChip, TagSNPs, and GeneSeek High- and Low-density indicus (HDi and LDi, respectively)]. Estimates of Pearson correlations, regression coefficients, and mean squared errors were used to assess accuracy and bias of predictions. Overall, the BayesCπ model resulted in less biased predictions. Accuracies ranged from 0.18 to 0.27, which are reasonable values given the heritability estimates (from 0.40 to 0.44) and sample size (568 animals in the training population). Furthermore, results from Bos taurus indicus panels were as informative as those from Illumina BovineHD, indicating that they could be used to implement GS at lower costs.

Bovine NR1I3 gene polymorphisms and its association with feed efficiency traits in Nellore cattle.

The Nuclear receptor 1 family I member 3 (NR1I3), also known as the Constitutive Androstane Receptor (CAR), was initially characterized as a key regulator of xenobiotic metabolism. However, recent biochemical and structural data suggest that NR1I3 is activated in response to metabolic and nutritional stress in a ligand-independent manner. Thus, we prospected the Bovine NR1I3 gene for polymorphisms and studied their association with feed efficiency traits in Nellore cattle. First, 155 purebred Nellore bulls were individually measured for Residual Feed Intake (RFI) and the 25 best (High Feed Efficiency group, HFE) and the 25 worst animals (Low Feed Efficiency group, LFE) were selected for DNA extraction. The entire Bovine NR1I3 gene was amplified and polymorphisms were identified by sequencing. Then, one SNP different between HFE and LFE groups was genotyped in all the 155 animals and in another 288 animals totalizing 443 Nellore bulls genotyped for association of NR1I3 SNPs with feed efficiency traits. We found 24 SNPs in the NR1I3 gene and choose a statistically different SNP between HFE and LFE groups for further analysis. Genotyping of the 155 animals showed a significant association within SNP and RFI (p = 0.04), Residual Intake and BW Gain (p = 0.04) and Dry Matter Intake (p = 0.01). This SNP is located in the 5'flanking promoter region of NR1I3 gene and different alleles alter the binding site for predicted transcriptional factors as HNF4alpha, CREM and c-MYB, leading us to conclude that NR1I3 expression and regulation might be important to feed efficiency.

An overview of Phoneutria nigriventer spider venom using combined transcriptomic and proteomic approaches

Phoneutria nigriventer is one of the largest existing true spiders and one of the few considered medically relevant. Its venom contains several neurotoxic peptides that act on different ion channels and chemical receptors of vertebrates and invertebrates. Some of these venom toxins have been shown as promising models for pharmaceutical or biotechnological use. However, the large diversity and the predominance of low molecular weight toxins in this venom have hampered the identification and deep investigation of the less abundant toxins and the proteins with high molecular weight. Here, we combined conventional and next-generation cDNA sequencing with Multidimensional Protein Identification Technology (MudPIT), to obtain an in-depth panorama of the composition of P. nigriventerspider venom. The results from these three approaches showed that cysteine-rich peptide toxins are the most abundant components in this venom and most of them contain the Inhibitor Cysteine Knot (ICK) structural motif. Ninety-eight sequences corresponding to cysteine-rich peptide toxins were identified by the three methodologies and many of them were considered as putative novel toxins, due to the low similarity to previously described toxins. Furthermore, using next-generation sequencing we identified families of several other classes of toxins, including CAPs (Cysteine Rich Secretory Protein-CRiSP, antigen 5 and Pathogenesis-Related 1-PR-1), serine proteinases, TCTPs (translationally controlled tumor proteins), proteinase inhibitors, metalloproteinases and hyaluronidases, which have been poorly described for this venom. This study provides an overview of the molecular diversity of P. nigriventervenom, revealing several novel components and providing a better basis to understand its toxicity and pharmacological activities.

Genome-wide association for growth traits in Canchim beef cattle.

Studies are being conducted on the applicability of genomic data to improve the accuracy of the selection process in livestock, and genome-wide association studies (GWAS) provide valuable information to enhance the understanding on the genetics of complex traits. The aim of this study was to identify genomic regions and genes that play roles in birth weight (BW), weaning weight adjusted for 210 days of age (WW), and long-yearling weight adjusted for 420 days of age (LYW) in Canchim cattle. GWAS were performed by means of the Generalized Quasi-Likelihood Score (GQLS) method using genotypes from the BovineHD BeadChip and estimated breeding values for BW, WW, and LYW. Data consisted of 285 animals from the Canchim breed and 114 from the MA genetic group (derived from crossings between Charolais sires and ½ Canchim + ½ Zebu dams). After applying a false discovery rate correction at a 10% significance level, a total of 4, 12, and 10 SNPs were significantly associated with BW, WW, and LYW, respectively. These SNPs were surveyed to their corresponding genes or to surrounding genes within a distance of 250 kb. The genes DPP6 (dipeptidyl-peptidase 6) and CLEC3B (C-type lectin domain family 3 member B) were highlighted, considering its functions on the development of the brain and skeletal system, respectively. The GQLS method identified regions on chromosome associated with birth weight, weaning weight, and long-yearling weight in Canchim and MA animals. New candidate regions for body weight traits were detected and some of them have interesting biological functions, of which most have not been previously reported. The observation of QTL reports for body weight traits, covering areas surrounding the genes (SNPs) herein identified provides more evidence for these associations. Future studies targeting these areas could provide further knowledge to uncover the genetic architecture underlying growth traits in Canchim cattle.

An overview of Phoneutria nigriventer spider venom using combined transcriptomic and proteomic approaches

Phoneutria nigriventer is one of the largest existing true spiders and one of the few considered medically relevant. Its venom contains several neurotoxic peptides that act on different ion channels and chemical receptors of vertebrates and invertebrates. Some of these venom toxins have been shown as promising models for pharmaceutical or biotechnological use. However, the large diversity and the predominance of low molecular weight toxins in this venom have hampered the identification and deep investigation of the less abundant toxins and the proteins with high molecular weight. Here, we combined conventional and next-generation cDNA sequencing with Multidimensional Protein Identification Technology (MudPIT), to obtain an in-depth panorama of the composition of P. nigriventerspider venom. The results from these three approaches showed that cysteine-rich peptide toxins are the most abundant components in this venom and most of them contain the Inhibitor Cysteine Knot (ICK) structural motif. Ninety-eight sequences corresponding to cysteine-rich peptide toxins were identified by the three methodologies and many of them were considered as putative novel toxins, due to the low similarity to previously described toxins. Furthermore, using next-generation sequencing we identified families of several other classes of toxins, including CAPs (Cysteine Rich Secretory Protein-CRiSP, antigen 5 and Pathogenesis-Related 1-PR-1), serine proteinases, TCTPs (translationally controlled tumor proteins), proteinase inhibitors, metalloproteinases and hyaluronidases, which have been poorly described for this venom. This study provides an overview of the molecular diversity of P. nigriventervenom, revealing several novel components and providing a better basis to understand its toxicity and pharmacological activities.