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.

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.

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.