Identification of novel genes in cattle (Bos taurus) and biological insights into their function in embryo development

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. We identified 274,342 transcript sequences, and 3,033 of those transcripts do not match a gene present in an annotation, 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<1x10-8) with several genes annotated with gene ontology 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=1.55x10-7). In conclusion, 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.