Identification of candidate lethal haplotypes and genomic association with post-natal mortality and reproductive traits in Nellore cattle.

The wide use of genomic information has enabled the identification of lethal recessive alleles that are the major genetic causes of reduced conception rates, longer calving intervals, or lower survival for live-born animals. This study was carried out to screen the Nellore cattle genome for lethal recessive haplotypes based on deviation from the expected population homozygosity, and to test SNP markers surrounding the lethal haplotypes region for association with heifer rebreeding (HR), post-natal mortality (PNM) and stayability (STAY). This approach requires genotypes only from apparently normal individuals and not from affected embryos. A total of 62,022 animals were genotyped and imputed to a high-density panel (777,962 SNP markers). Expected numbers of homozygous individuals were calculated, and the probabilities of observing 0 homozygotes was obtained. Deregressed genomic breeding values [(G)EBVs] were used in a GWAS to identify candidate genes and biological mechanisms affecting HR, STAY and PNM. In the functional analyses, genes within 100 kb down and upstream of each significant SNP marker, were researched. Thirty haplotypes had high expected frequency, while no homozygotes were observed. Most of the alleles present in these haplotypes had a negative mean effect for PNM, HR and STAY. The GWAS revealed significant SNP markers involved in different physiological mechanisms, leading to harmful effect on the three traits. The functional analysis revealed 26 genes enriched for 19 GO terms. Most of the GO terms found for biological processes, molecular functions and pathways were related to tissue development and the immune system. More phenotypes underlying these putative regions in this population could be the subject of future investigation. Tests to find putative lethal haplotype carriers could help breeders to eliminate them from the population or manage matings in order to avoid homozygous.

Transcriptome Profiling of the Liver in Nellore Cattle Phenotypically Divergent for RFI in Two Genetic Groups.

The identification and selection of genetically superior animals for residual feed intake (RFI) could enhance productivity and minimize environmental impacts. The aim of this study was to use RNA-seq data to identify the differentially expressed genes (DEGs), known non-coding RNAs (ncRNAs), specific biomarkers and enriched biological processes associated with RFI of the liver in Nellore cattle in two genetic groups. In genetic group 1 (G1), 24 extreme RFI animals (12 low RFI (LRFI) versus 12 high RFI (HRFI)) were selected from a population of 60 Nellore bulls. The RNA-seq of the samples from their liver tissues was performed using an Illumina HiSeq 2000. In genetic group 2 (G2), 20 samples of liver tissue of Nellore bulls divergent for RFI (LRFI, n = 10 versus HRFI, n = 10) were selected from 83 animals. The raw data of the G2 were chosen from the ENA repository. A total of 1811 DEGs were found for the G1 and 2054 for the G2 (p-value ≤ 0.05). We detected 88 common genes in both genetic groups, of which 33 were involved in the immune response and in blocking oxidative stress. In addition, seven (B2M, ADSS, SNX2, TUBA4A, ARHGAP18, MECR, and ABCF3) possible gene biomarkers were identified through a receiver operating characteristic analysis (ROC) considering an AUC > 0.70. The B2M gene was overexpressed in the LRFI group. This gene regulates the lipid metabolism protein turnover and inhibits cell death. We also found non-coding RNAs in both groups. MIR25 was up-regulated and SNORD16 was down-regulated in the LRFI for G1. For G2, up-regulated RNase_MRP and SCARNA10 were found. We highlight MIR25 as being able to act by blocking cytotoxicity and oxidative stress and RMRP as a blocker of mitochondrial damage. The biological pathways associated with RFI of the liver in Nellore cattle in the two genetic groups were for energy metabolism, protein turnover, redox homeostasis and the immune response. The common transcripts, biomarkers and metabolic pathways found in the two genetic groups make this unprecedented work even more relevant, since the results are valid for different herds raised in different ways. The results reinforce the biological importance of these known processes but also reveal new insights into the complexity of the liver tissue transcriptome of Nellore cattle.

Small genetic variation affecting mRNA isoforms associated with marbling and meat color in beef cattle.

The aim of this study was to identify mRNA isoforms and small genetic variants that may be affecting marbling and beef color in Nellore cattle. Longissimus thoracis muscle samples from 20 bulls with different phenotypes (out of 80 bulls set) for marbling (moderate (n = 10) and low (n = 10) groups) and beef color (desirable (n = 10) and undesirable (n = 9) group) traits were used to perform transcriptomic analysis using RNA sequencing. Fourteen and 15 mRNA isoforms were detected as differentially expressed (DE) (P-value ≤ 0.001) between divergent groups for marbling and meat color traits, respectively. Some of those DE mRNA isoforms have shown sites of splicing modified by small structural variants as single nucleotide variant (SNV), insertion, and/or deletion. Enrichment analysis identified metabolic pathways, such as O2/CO2 exchange in erythrocytes, tyrosine biosynthesis, and phenylalanine degradation. The results obtained suggest potential key regulatory genes associated with these economically important traits for the beef industry and for the consumer.

Identification of novel mRNA isoforms associated with meat tenderness using RNA sequencing data in beef cattle.

The Warner-Bratzler shear force (WBSF) and myofibrillar fragmentation index (MFI) are complementary methodologies used to measure beef tenderness. Longissimus thoracis samples from the 20 most extreme bulls (out of 80 bulls set) for WBSF (tender (n = 10) and tough (n = 10)) and MFI (high (n = 10) and low (n = 10)) traits were collected to perform transcriptomic analysis using RNA-Sequencing. All analysis were performed through CLC Genomics Workbench. A total of 39 and 27 transcripts for WBSF and MFI phenotypes were DE, respectively. The possible DE novel mRNA isoforms, for WBSF and MFI traits, are myosin encoders (e.g. MYL1 and MYL6). In addition, we identified potential mRNA isoforms related to genes affecting the speed fibers degradation during the meat aging process. The DE novel transcripts are transcripted by genes with biological functions related to oxidative process, energy production and striated muscle contraction. The results suggest that the identified mRNA isoforms could be used as potential candidate to select animals in order to improve meat tenderness.

Use of gene expression profile to identify potentially relevant transcripts to myofibrillar fragmentation index trait.

The myofibrillar fragmentation index (MFI) is an indicative trait for meat tenderness. Longissimus thoracis muscle samples from the 20 most extreme bulls (out of 80 bulls set) for MFI (high (n = 10) and low (n = 10) groups) trait were used to perform transcriptomic analysis, using RNA Sequencing (RNA-Seq). An average of 24.616 genes was expressed in the Nellore muscle transcriptome analysis. A total of 96 genes were differentially expressed (p value ≤ 0.001) between the two groups of divergent bulls for MFI. The HEBP2 and BDH1 genes were overexpressed in animals with high MFI. The MYBPH and MYL6, myosin encoders, were identified. The differentially expressed genes were related to increase mitochondria efficiency, especially in cells under oxidative stress conditions, and these also were related to zinc and calcium binding, membrane transport, and muscle constituent proteins, such as actin and myosin. Most of those genes were involved in metabolic pathways of oxidation-reduction, transport of lactate in the plasma membrane, and muscle contraction. This is the first study applying MFI phenotypes in transcriptomic studies to identify and understand differentially expressed genes for beef tenderness. These results suggest that differences detected in gene expression between high and low MFI animals are related to reactive mechanisms and structural components of oxidative fibers under the condition of cellular stress. Some genes may be selected as positional candidate genes to beef tenderness, MYL6, MYBPH, TRIM63, TRIM55, TRIOBP, and CHRNG genes. The use of MFI phenotypes could enhance results of meat tenderness studies.

Gene expression profiling and identification of hub genes in Nellore cattle with different marbling score levels.

The marbling rate evaluation is difficult and expensive, requiring slaughter of the animal or ultrasound measurement. Thus, this trait is generally not included in animal breeding programs. The use of molecular techniques to elucidate intramuscular fat deposition may help improve this trait. In this respect, transcriptome studies and differential gene expression analysis by RNA-Seq can contribute to advances in this area. The objective of this study was to use RNA-Seq to identify differentially expressed genes (DEGs) in muscle tissue (longissimus thoracis) of Nellore cattle divergently ranked on marbling, in order to increase our understanding of genes involved in the expression of this trait. The results revealed 49 DEGs and three hub genes (CISH, UFM1, TSHZ1), all of them involved in insulin and diabetes mellitus metabolism. These results indicating key genes and pathways, which may help to develop strategies designed to select animals with greater marbling.

Prediction of hub genes associated with intramuscular fat content in Nelore cattle.

BACKGROUND: The aim of this study was to use transcriptome RNA-Seq data from longissimus thoracis muscle of uncastrated Nelore males to identify hub genes based on co-expression network obtained from differentially expressed genes (DEGs) associated with intramuscular fat content. RESULTS: A total of 30 transcriptomics datasets (RNA-Seq) obtained from longissimus thoracis muscle were selected based on the phenotypic value of divergent intramuscular fat content: 15 with the highest intramuscular fat content (HIF) and 15 with the lowest intramuscular fat content (LIF). The transcriptomics datasets were aligned with a reference genome and 65 differentially expressed genes (DEGs) were identified, including 21 upregulated and 44 downregulated genes in HIF animals. The normalized count data from DEGs was then used for co-expression network construction. From the co-expression network, four modules were identified. The topological properties of the network were analyzed; those genes engaging in the most interactions (maximal clique centrality method) with other DEGs were predicted to be hub genes (PDE4D, KLHL30 and IL1RAP), which consequently may play a role in cellular and/or systemic lipid biology in Nelore cattle. Top modules screened from the gene co-expression network were identify. The two candidate modules had clear associated biological pathways related to fat development, cell adhesion, and muscle differentiation, immune system, among others. The hub genes belonged in top modules and were downregulated in HIF animals. PDE4D and IL1RAP have known effects on lipid metabolism and the immune system through the regulation of cAMP signaling. Given that cAMP is known to play a role in lipid systems, PDE4D and IL1RAP downregulation may contribute to increased levels of intracellular cAMP and thus may have effects on IF content differences in Nelore cattle. KLHL30 may have effects on muscle metabolism. Klhl protein families play a role in protein degradation. However, the downregulation of this gene and its role in lipid metabolism has not yet been clarified. CONCLUSIONS: The results reported in this study indicate candidate genes and molecular mechanisms involved in IF content difference in Nelore cattle.

Differences in global gene expression in muscle tissue of Nellore cattle with divergent meat tenderness.

BACKGROUND: Meat tenderness is the consumer's most preferred sensory attribute. This trait is affected by a number of factors, including genotype, age, animal sex, and pre- and post-slaughter management. In view of the high percentage of Zebu genes in the Brazilian cattle population, mainly Nellore cattle, the improvement of meat tenderness is important since the increasing proportion of Zebu genes in the population reduces meat tenderness. However, the measurement of this trait is difficult once it can only be made after animal slaughtering. New technologies such as RNA-Seq have been used to increase our understanding of the genetic processes regulating quantitative traits phenotypes. The objective of this study was to identify differentially expressed genes related to meat tenderness, in Nellore cattle in order to elucidate the genetic factors associated with meat quality. Samples were collected 24 h postmortem and the meat was not aged. RESULTS: We found 40 differentially expressed genes related to meat tenderness, 17 with known functions. Fourteen genes were up-regulated and 3 were down-regulated in the tender meat group. Genes related to ubiquitin metabolism, transport of molecules such as calcium and oxygen, acid-base balance, collagen production, actin, myosin, and fat were identified. The PCP4L1 (Purkinje cell protein 4 like 1) and BoLA-DQB (major histocompatibility complex, class II, DQ beta) genes were validated by qRT-PCR. The results showed relative expression values similar to those obtained by RNA-Seq, with the same direction of expression (i.e., the two techniques revealed higher expression of PCP4L1 in tender meat samples and of BoLA-DQB in tough meat samples). CONCLUSIONS: This study revealed the differential expression of genes and functions in Nellore cattle muscle tissue, which may contain potential biomarkers involved in meat tenderness.