Identification of novel cattle (Bos taurus) genes and biological insights of their function in pre-implantation embryo development.

BACKGROUND: Appropriate regulation of genes expressed in oocytes and embryos is essential for acquisition of developmental competence in mammals. Here, we hypothesized that several genes expressed in oocytes and pre-implantation embryos remain unknown. Our goal was to reconstruct the transcriptome of oocytes (germinal vesicle and metaphase II) and pre-implantation cattle embryos (blastocysts) using short-read and long-read sequences to identify putative new genes. RESULTS: We identified 274,342 transcript sequences and 3,033 of those loci do not match a gene present in official annotations and thus are potential new genes. Notably, 63.67% (1,931/3,033) of potential novel genes exhibited coding potential. Also noteworthy, 97.92% of the putative novel genes overlapped annotation with transposable elements. Comparative analysis of transcript abundance identified that 1,840 novel genes (recently added to the annotation) or potential new genes were differentially expressed between developmental stages (FDR < 0.01). We also determined that 522 novel or potential new genes (448 and 34, respectively) were upregulated at eight-cell embryos compared to oocytes (FDR < 0.01). In eight-cell embryos, 102 novel or putative new genes were co-expressed (|r|> 0.85, P < 1 × 10-8) with several genes annotated with gene ontology biological processes related to pluripotency maintenance and embryo development. CRISPR-Cas9 genome editing confirmed that the disruption of one of the novel genes highly expressed in eight-cell embryos reduced blastocyst development (ENSBTAG00000068261, P = 1.55 × 10-7). CONCLUSIONS: Our results revealed several putative new genes that need careful annotation. Many of the putative new genes have dynamic regulation during pre-implantation development and are important components of gene regulatory networks involved in pluripotency and blastocyst formation.

Protocol for the electroporation of CRISPR-Cas for DNA and RNA targeting in Bos taurus zygotes.

The use of CRISPR-Cas9 ribonucleoproteins has revolutionized manipulation of genomes. Here, we present a protocol for the electroporation of CRISPR-Cas for DNA and RNA targeting in Bos taurus zygotes. First, we describe steps for production and preparation of presumptive zygotes for electroporation. The first electroporation introduces ribonucleoproteins formed by Cas9D10A with two guide RNAs to target DNA, and the second introduces the same ribonucleoprotein complex to target DNA plus Cas13a with one guide RNA to target RNAs. For complete details on the use and execution of this protocol, please refer to Nix et al.1.

Ablation of OCT4 function in cattle embryos by double electroporation of CRISPR-Cas for DNA and RNA targeting (CRISPR-DART).

CRISPR-Cas ribonucleoproteins (RNPs) are important tools for gene editing in preimplantation embryos. However, the inefficient production of biallelic deletions in cattle zygotes has hindered mechanistic studies of gene function. In addition, the presence of maternal RNAs that support embryo development until embryonic genome activation may cause confounding phenotypes. Here, we aimed to improve the efficiency of biallelic deletions and deplete specific maternal RNAs in cattle zygotes using CRISPR-Cas editing technology. Two electroporation sessions with Cas9D10A RNPs targeting exon 1 and the promoter of OCT4 produced biallelic deletions in 91% of the embryos tested. In most cases, the deletions were longer than 1,000 nucleotides long. Electroporation of Cas13a RNPs prevents the production of the corresponding proteins. We electroporated Cas9D10A RNPs targeting exon 1, including the promoter region, of OCT4 in two sessions with inclusion of Cas13a RNPs targeting OCT4 mRNAs in the second session to ablate OCT4 function in cattle embryos. A lack of OCT4 resulted in embryos arresting development prior to blastocyst formation at a greater proportion (13%) than controls (31.6%, P < 0.001). The few embryos that developed past the morula stage did not form a normal inner cell mass. Transcriptome analysis of single blastocysts, confirmed to lack exon 1 and promoter region of OCT4, revealed a significant (False Discovery Rate, FDR < 0.1) reduction in transcript abundance of many genes functionally connected to stemness, including markers of pluripotency (CADHD1, DPPA4, GNL3, RRM2). The results confirm that OCT4 is a key regulator of genes that modulate pluripotency and is required to form a functional blastocyst in cattle.

Sexing of cattle embryos using RNA-sequencing data or polymerase chain reaction based on a complete sequence of cattle chromosome Y.

When necessary, RNA-sequencing data or polymerase chain reaction (PCR) assays can be used to determine the presence of the chromosome Y (ChrY) in samples. This information allows for biological variation due to sexual dimorphism to be studied. A prime example is when researchers conduct RNA-sequencing of single embryos, or conceptuses, prior to the development of gonads. A recent publication of a complete sequence of the ChrY has removed limitations for the development of these procedures in cattle, otherwise imposed by the absence of a ChrY in the reference genome. Using the sequence of the cattle ChrY and transcriptome data, we conducted a systematic search for genes in the ChrY that are exclusively expressed in male tissues. The genes ENSBIXG00000029763, ENSBIXG00000029774, ENSBIXG00000029788, and ENSBIXG00000029892 were consistently expressed across male tissues and lowly expressed or absent in female samples. We observed that the cumulative values of counts per million were 2688-fold greater in males than the equivalent values in female samples. Thus, we deemed these genes suitable for the sexing of samples using RNA-sequencing data. We successfully used this set of genes to infer the sex of 22 cattle blastocysts (8 females and 14 males). Additionally, the completed sequence of the cattle ChrY has segments in the male-specific region that are not repeated. We designed a pair of oligonucleotides that targets one of these non-repeated regions in the male-specific sequence of the ChrY. Using this pair of oligonucleotides, in a multiplexed PCR assay with oligonucleotides that anneal to an autosome chromosome, we accurately identified the sex of cattle blastocysts. We developed efficient procedures for the sexing of samples in cattle using either transcriptome data or their DNA. The procedures using RNA-sequencing will greatly benefit researchers who work with samples limited in cell numbers which are only sufficient to produce transcriptome data. The oligonucleotides used for the accurate sexing of samples using PCR are transferable to other cattle tissue samples.

Identification of Runs of Homozygosity Islands and Genomic Estimated Inbreeding Values in Caqueteño Creole Cattle (Colombia).

The Caqueteño Creole (CAQ) is a native breed of cattle from the Caquetá department (Colombia), adapted to tropical conditions, which is extremely important to production systems in those regions. However, CAQ is poorly studied. In this sense, population structure studies associated with runs of homozygosity (ROH) analysis would allow for a better understanding of CAQ. Through ROH analysis, it is possible to reveal genetic relationships between individuals, measure genome inbreeding levels, and identify regions associated with traits of economic interest. Samples from a CAQ population (n = 127) were genotyped with the Bovine HD BeadChip (777,000 SNPs) and analyzed with the PLINK 1.9 program to estimate FROH and ROH islands. We highlighted a decrease in inbreeding frequency for FROH 4−8 Mb, 8−16 Mb, and >16 Mb classes, indicating inbreeding control in recent matings. We also found genomic hotspot regions on chromosomes 3, 5, 6, 8, 16, 20, and 22, where chromosome 20 harbored four hotspots. Genes in those regions were associated with fertility and immunity traits, muscle development, and environmental resistance, which may be present in the CAQ breed due to natural selection. This indicates potential for production systems in tropical regions. However, further studies are necessary to elucidate the CAQ production objective.

Transcriptome Profile Reveals Genetic and Metabolic Mechanisms Related to Essential Fatty Acid Content of Intramuscular Longissimus thoracis in Nellore Cattle.

Beef is a source of essential fatty acids (EFA), linoleic (LA) and alpha-linolenic (ALA) acids, which protect against inflammatory and cardiovascular diseases in humans. However, the intramuscular EFA profile in cattle is a complex and polygenic trait. Thus, this study aimed to identify potential regulatory genes of the essential fatty acid profile in Longissimus thoracis of Nellore cattle finished in feedlot. Forty-four young bulls clustered in four groups of fifteen animals with extreme values for each FA were evaluated through differentially expressed genes (DEG) analysis and two co-expression methodologies (WGCNA and PCIT). We highlight the ECHS1, IVD, ASB5, and ERLIN1 genes and the TF NFIA, indicated in both FA. Moreover, we associate the NFYA, NFYB, PPARG, FASN, and FADS2 genes with LA, and the RORA and ELOVL5 genes with ALA. Furthermore, the functional enrichment analysis points out several terms related to FA metabolism. These findings contribute to our understanding of the genetic mechanisms underlying the beef EFA profile in Nellore cattle finished in feedlot.

Transcriptomic profile of longissimus thoracis associated with fatty acid content in Nellore beef cattle.

The beef fatty acid (FA) profile has the potential to impact human health, and displays polygenic and complex features. This study aimed to identify the transcriptomic FA profile in the longissimus thoracis muscle in Nellore beef cattle finished in feedlot. Forty-four young bulls were sampled to assess the beef FA profile by considering 14 phenotypes and including differentially expressed genes (DEG), co-expressed (COE), and differentially co-expressed genes (DCO) analyses. All samples (n = 44) were used for COE analysis, whereas 30 samples with extreme phenotypes for the beef FA profile were used for DEG and DCO. A total of 912 DEG were identified, and the polyunsaturated (n = 563) and unsaturated ω-3 (n = 346) FA sums groups were the most frequently observed. The COE analyses identified three modules, of which the blue module (n = 1776) was correlated with eight of 14 FA phenotypes. Also, 759 DCO genes were listed, and the oleic acid (n = 358) and monounsaturated fatty acids sum (n = 120) were the most frequent. Furthermore, 243 and 13, 319 and seven, and 173 and 12 gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways were enriched respectively for the DEG, COE, and DCO analyses. Combining the results, we highlight the unexplored GIPC2, ASB5, and PPP5C genes in cattle. Besides LIPE and INSIG2 genes in COE modules, the ACSL3, ECI1, DECR2, FITM1, and SDHB genes were signaled in at least two analyses. These findings contribute to understand the genetic mechanisms underlying the beef FA profile in Nellore beef cattle finished in feedlot.

Genome-wide scans for signatures of selection in Mangalarga Marchador horses using high-throughput SNP genotyping.

BACKGROUND: The detection of signatures of selection in genomic regions provides insights into the evolutionary process, enabling discoveries regarding complex phenotypic traits. In this research, we focused on identifying genomic regions affected by different selection pressures, mainly highlighting the recent positive selection, as well as understanding the candidate genes and functional pathways associated with the signatures of selection in the Mangalarga Marchador genome. Besides, we seek to direct the discussion about genes and traits of importance in this breed, especially traits related to the type and quality of gait, temperament, conformation, and locomotor system. RESULTS: Three different methods were used to search for signals of selection: Tajima's D (TD), the integrated haplotype score (iHS), and runs of homozygosity (ROH). The samples were composed of males (n = 62) and females (n = 130) that were initially chosen considering well-defined phenotypes for gait: picada (n = 86) and batida (n = 106). All horses were genotyped using a 670 k Axiom® Equine Genotyping Array​ (Axiom MNEC670). In total, 27, 104 (chosen), and 38 candidate genes were observed within the signatures of selection identified in TD, iHS, and ROH analyses, respectively. The genes are acting in essential biological processes. The enrichment analysis highlighted the following functions: anterior/posterior pattern for the set of genes (GLI3, HOXC9, HOXC6, HOXC5, HOXC4, HOXC13, HOXC11, and HOXC10); limb morphogenesis, skeletal system, proximal/distal pattern formation, JUN kinase activity (CCL19 and MAP3K6); and muscle stretch response (MAPK14). Other candidate genes were associated with energy metabolism, bronchodilator response, NADH regeneration, reproduction, keratinization, and the immunological system. CONCLUSIONS: Our findings revealed evidence of signatures of selection in the MM breed that encompass genes acting on athletic performance, limb development, and energy to muscle activity, with the particular involvement of the HOX family genes. The genome of MM is marked by recent positive selection. However, Tajima's D and iHS results point also to the presence of balancing selection in specific regions of the genome.

Fine-scale estimation of inbreeding rates, runs of homozygosity and genome-wide heterozygosity levels in the Mangalarga Marchador horse breed.

With the availability of high-density SNP panels and the establishment of approaches for characterizing homozygosity and heterozygosity sites, it is possible to access fine-scale information regarding genomes, providing more than just comparisons of different inbreeding coefficients. This is the first study that seeks to access such information for the Mangalarga Marchador (MM) horse breed on a genomic scale. To this end, we aimed to assess inbreeding levels using different coefficients, as well as to characterize homozygous and heterozygous runs in the population. Using Axiom ® Equine Genotyping Array-670k SNP (Thermo Fisher), 192 horses were genotyped. Our results showed different estimates: inbreeding from genomic coefficients (FROH ) = 0.16; pedigree-based (FPED ) = 0.008; and a method based on excess homozygosity (FHOM ) = 0.010. The correlations between the inbreeding coefficients were low to moderate, and some comparisons showed negative correlations, being practically null. In total, 85,295 runs of homozygosity (ROH) and 10,016 runs of heterozygosity (ROHet) were characterized for the 31 horse autosomal chromosomes. The class with the highest percentage of ROH was 0-2 Mbps, with 92.78% of the observations. In the ROHet results, only the 0-2 class presented observations, with chromosome 11 highlighted in a region with high genetic variability. Three regions from the ROHet analyses showed genes with known functions: tripartite motif-containing 37 (TRIM37), protein phosphatase, Mg2+ /Mn2+ dependent 1E (PPM1E) and carbonic anhydrase 10 (CA10). Therefore, our findings suggest moderate inbreeding, possibly attributed to breed formation, annulling possible recent inbreeding. Furthermore, regions with high variability in the MM genome were identified (ROHet), associated with the recent selection and important events in the development and performance of MM horses over generations.