Network Analyses Predict Small RNAs That Might Modulate Gene Expression in the Testis and Epididymis of Bos indicus Bulls.

Spermatogenesis relies on complex molecular mechanisms, essential for the genesis and differentiation of the male gamete. Germ cell differentiation starts at the testicular parenchyma and finishes in the epididymis, which has three main regions: head, body, and tail. RNA-sequencing data of the testicular parenchyma (TP), head epididymis (HE), and tail epididymis (TE) from four bulls (three biopsies per bull: 12 samples) were subjected to differential expression analyses, functional enrichment analyses, and co-expression analyses. The aim was to investigate the co-expression and infer possible regulatory roles for transcripts involved in the spermatogenesis of Bos indicus bulls. Across the three pairwise comparisons, 3,826 differentially expressed (DE) transcripts were identified, of which 384 are small RNAs. Functional enrichment analysis pointed to gene ontology (GO) terms related to ion channel activity, detoxification of copper, neuroactive receptors, and spermatogenesis. Using the regulatory impact factor (RIF) algorithm, we detected 70 DE small RNAs likely to regulate the DE transcripts considering all pairwise comparisons among tissues. The pattern of small RNA co-expression suggested that these elements are involved in spermatogenesis regulation. The 3,826 DE transcripts (mRNAs and small RNAs) were further subjected to co-expression analyses using the partial correlation and information theory (PCIT) algorithm for network prediction. Significant correlations underpinned the co-expression network, which had 2,216 transcripts connected by 158,807 predicted interactions. The larger network cluster was enriched for male gamete generation and had 15 miRNAs with significant RIF. The miRNA bta-mir-2886 showed the highest number of connections (601) and was predicted to down-regulate ELOVL3, FEZF2, and HOXA13 (negative co-expression correlations and confirmed with TargetScan). In short, we suggest that bta-mir-2886 and other small RNAs might modulate gene expression in the testis and epididymis, in Bos indicus cattle.

Exploring the Regulatory Potential of Long Non-Coding RNA in Feed Efficiency of Indicine Cattle.

Long non-coding RNA (lncRNA) can regulate several aspects of gene expression, being associated with complex phenotypes in humans and livestock species. In taurine beef cattle, recent evidence points to the involvement of lncRNA in feed efficiency (FE), a proxy for increased productivity and sustainability. Here, we hypothesized specific regulatory roles of lncRNA in FE of indicine cattle. Using RNA-Seq data from the liver, muscle, hypothalamus, pituitary gland and adrenal gland from Nellore bulls with divergent FE, we submitted new transcripts to a series of filters to confidently predict lncRNA. Then, we identified lncRNA that were differentially expressed (DE) and/or key regulators of FE. Finally, we explored lncRNA genomic location and interactions with miRNA and mRNA to infer potential function. We were able to identify 126 relevant lncRNA for FE in Bos indicus, some with high homology to previously identified lncRNA in Bos taurus and some possible specific regulators of FE in indicine cattle. Moreover, lncRNA identified here were linked to previously described mechanisms related to FE in hypothalamus-pituitary-adrenal axis and are expected to help elucidate this complex phenotype. This study contributes to expanding the catalogue of lncRNA, particularly in indicine cattle, and identifies candidates for further studies in animal selection and management.

Systems Biology Reveals NR2F6 and TGFB1 as Key Regulators of Feed Efficiency in Beef Cattle.

Systems biology approaches are used as strategy to uncover tissue-specific perturbations and regulatory genes related to complex phenotypes. We applied this approach to study feed efficiency (FE) in beef cattle, an important trait both economically and environmentally. Poly-A selected RNA of five tissues (adrenal gland, hypothalamus, liver, skeletal muscle and pituitary) of eighteen young bulls, selected for high and low FE, were sequenced (Illumina HiSeq 2500, 100 bp, pared-end). From the 17,354 expressed genes considering all tissues, 1,335 were prioritized by five selection categories (differentially expressed, harboring SNPs associated with FE, tissue-specific, secreted in plasma and key regulators) and used for network construction. NR2F6 and TGFB1 were identified and validated by motif discovery as key regulators of hepatic inflammatory response and muscle tissue development, respectively, two biological processes demonstrated to be associated with FE. Moreover, we indicated potential biomarkers of FE, which are related to hormonal control of metabolism and sexual maturity. By using robust methodologies and validation strategies, we confirmed the main biological processes related to FE in Bos indicus and indicated candidate genes as regulators or biomarkers of superior animals.

Candidate mutations used to aid the prediction of genetic merit for female reproductive traits in tropical beef cattle

In this study, we aimed to provide a wet laboratory validation for a set of single nucleotide polymorphisms (SNP), which had been identified as candidate functional variants in silico. Genotyping for candidate SNP was performed in Brahman and Tropical Composite cattle. After quality control, 29 SNP were first investigated individually for their association with female reproductive traits and then used as a panel for genomic predictions. The reproductive traits studied were age at first corpus luteum (AGECL; days), post-partum anoestrus interval (PPAI; days), and a binary trait that described if the cow had ovulated before weaning the first calf or not (PW, 0-1). Single nucleotide polymorphisms in six genes (FOXA2, TRAF4, IRF2, IRF1, BPTF, and CPEB1) were found to be significantly associated with reproduction traits . The genomic prediction method used was BayesR, to accommodate the 29 new SNP and compare their performance with predictions based on 50K genotypes (Illumina SNP chip). When new SNP and PLAG1 mutation rs109231213 were included in the genomic predictions for female reproductive traits their accuracies improved. The best predictions were obtained by combining the new SNP and the 50K SNP using BayesR analysis, with a 4% improvement in accuracy. The proportion of the genetic variance explained by the new SNP together was 0.07 for AGECL, 0.03 for PPAI, and 0.02 for PW. It would be favourable to include these new SNP in future versions of bovine SNP chips to target selection for female reproductive traits. These new SNP are likely to improve genomic predictions for female reproductive traits in tropical beef cattle breeds, with varying degrees of Bos indicus content.(AU)

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.

Non-synonymous mutations mapped to chromosome X associated with andrological and growth traits in beef cattle.

BACKGROUND: Previous genome-wide association analyses identified QTL regions in the X chromosome for percentage of normal sperm and scrotal circumference in Brahman and Tropical Composite cattle. These traits are important to be studied because they are indicators of male fertility and are correlated with female sexual precocity and reproductive longevity. The aim was to investigate candidate genes in these regions and to identify putative causative mutations that influence these traits. In addition, we tested the identified mutations for female fertility and growth traits. RESULTS: Using a combination of bioinformatics and molecular assay technology, twelve non-synonymous SNPs in eleven genes were genotyped in a cattle population. Three and nine SNPs explained more than 1% of the additive genetic variance for percentage of normal sperm and scrotal circumference, respectively. The SNPs that had a major influence in percentage of normal sperm were mapped to LOC100138021 and TAF7L genes; and in TEX11 and AR genes for scrotal circumference. One SNP in TEX11 was explained ~13% of the additive genetic variance for scrotal circumference at 12 months. The tested SNP were also associated with weight measurements, but not with female fertility traits. CONCLUSIONS: The strong association of SNPs located in X chromosome genes with male fertility traits validates the QTL. The implicated genes became good candidates to be used for genetic evaluation, without detrimentally influencing female fertility traits.

Low frequency of Y anomaly detected in Australian Brahman cow-herds.

Indicine cattle have lower reproductive performance in comparison to taurine. A chromosomal anomaly characterized by the presence Y markers in females was reported and associated with infertility in cattle. The aim of this study was to investigate the occurrence of the anomaly in Brahman cows. Brahman cows (n = 929) were genotyped for a Y chromosome specific region using real time-PCR. Only six out of 929 cows had the anomaly (0.6%). The anomaly frequency was much lower in Brahman cows than in the crossbred population, in which it was first detected. It also seems that the anomaly doesn't affect pregnancy in the population. Due to the low frequency, association analyses couldn't be executed. Further, SNP signal of the pseudoautosomal boundary region of the Y chromosome was investigated using HD SNP chip. Pooled DNA of "non-pregnant" and "pregnant" cows were compared and no difference in SNP allele frequency was observed. Results suggest that the anomaly had a very low frequency in this Australian Brahman population and had no effect on reproduction. Further studies comparing pregnant cows and cows that failed to conceive should be executed after better assembly and annotation of the Y chromosome in cattle.

Genome-wide mapping of loci explaining variance in scrotal circumference in Nellore cattle.

The reproductive performance of bulls has a high impact on the beef cattle industry. Scrotal circumference (SC) is the most recorded reproductive trait in beef herds, and is used as a major selection criterion to improve precocity and fertility. The characterization of genomic regions affecting SC can contribute to the identification of diagnostic markers for reproductive performance and uncover molecular mechanisms underlying complex aspects of bovine reproductive biology. In this paper, we report a genome-wide scan for chromosome segments explaining differences in SC, using data of 861 Nellore bulls (Bos indicus) genotyped for over 777,000 single nucleotide polymorphisms. Loci that excel from the genome background were identified on chromosomes 4, 6, 7, 10, 14, 18 and 21. The majority of these regions were previously found to be associated with reproductive and body size traits in cattle. The signal on chromosome 14 replicates the pleiotropic quantitative trait locus encompassing PLAG1 that affects male fertility in cattle and stature in several species. Based on intensive literature mining, SP4, MAGEL2, SH3RF2, PDE5A and SNAI2 are proposed as novel candidate genes for SC, as they affect growth and testicular size in other animal models. These findings contribute to linking reproductive phenotypes to gene functions, and may offer new insights on the molecular biology of male fertility.

Genome-wide association study for birth weight in Nellore cattle points to previously described orthologous genes affecting human and bovine height.

BACKGROUND: Birth weight (BW) is an economically important trait in beef cattle, and is associated with growth- and stature-related traits and calving difficulty. One region of the cattle genome, located on Bos primigenius taurus chromosome 14 (BTA14), has been previously shown to be associated with stature by multiple independent studies, and contains orthologous genes affecting human height. A genome-wide association study (GWAS) for BW in Brazilian Nellore cattle (Bos primigenius indicus) was performed using estimated breeding values (EBVs) of 654 progeny-tested bulls genotyped for over 777,000 single nucleotide polymorphisms (SNPs). RESULTS: The most significant SNP (rs133012258, PGC = 1.34 × 10-9), located at BTA14:25376827, explained 4.62% of the variance in BW EBVs. The surrounding 1 Mb region presented high identity with human, pig and mouse autosomes 8, 4 and 4, respectively, and contains the orthologous height genes PLAG1, CHCHD7, MOS, RPS20, LYN, RDHE2 (SDR16C5) and PENK. The region also overlapped 28 quantitative trait loci (QTLs) previously reported in literature by linkage mapping studies in cattle, including QTLs for birth weight, mature height, carcass weight, stature, pre-weaning average daily gain, calving ease, and gestation length. CONCLUSIONS: This study presents the first GWAS applying a high-density SNP panel to identify putative chromosome regions affecting birth weight in Nellore cattle. These results suggest that the QTLs on BTA14 associated with body size in taurine cattle (Bos primigenius taurus) also affect birth weight and size in zebu cattle (Bos primigenius indicus).