Assembly of a pangenome for global cattle reveals missing sequences and novel structural variations, providing new insights into their diversity and evolutionary history.

A cattle pangenome representation was created based on the genome sequences of 898 cattle representing 57 breeds. The pangenome identified 83 Mb of sequence not found in the cattle reference genome, representing 3.1% novel sequence compared with the 2.71-Gb reference. A catalog of structural variants developed from this cattle population identified 3.3 million deletions, 0.12 million inversions, and 0.18 million duplications. Estimates of breed ancestry and hybridization between cattle breeds using insertion/deletions as markers were similar to those produced by single nucleotide polymorphism-based analysis. Hundreds of deletions were observed to have stratification based on subspecies and breed. For example, an insertion of a Bov-tA1 repeat element was identified in the first intron of the APPL2 gene and correlated with cattle breed geographic distribution. This insertion falls within a segment overlapping predicted enhancer and promoter regions of the gene, and could affect important traits such as immune response, olfactory functions, cell proliferation, and glucose metabolism in muscle. The results indicate that pangenomes are a valuable resource for studying diversity and evolutionary history, and help to delineate how domestication, trait-based breeding, and adaptive introgression have shaped the cattle genome.

Characterization of extrachromosomal circular DNA in cattle using 676 whole genome sequencing datasets.

Extrachromosomal circular DNA (eccDNA) is an important fraction of the genome. Recent studies proved that eccDNA plays important roles in genetic variation, aging and environmental adaptation, which have drawn wide attention. However, the characteristics of eccDNA in cattle remain unclear. Here, we studied eccDNAs from 676 cattle of 58 breeds using whole genome sequencing datasets. In total, 47 355 high-confidence eccDNAs were identified and covered 4.6% of the cattle autosomes in length. Similarly to other species, the cattle eccDNA preferentially located in the genic and repeat sequences. Cattle eccDNAs contained complete sequences of 661 genes, which were significantly (p < 0.05) enriched in immunity-related functions. The eccDNA was further proved to have inverted repeats on the boundaries, which contained a high proportion of A/T and ranged from 4 to 17 bp. Interestingly, we successfully separated animals according to their geographical distributions and their tissues where DNA was isolated. This implied possible roles for eccDNA in cattle selection and tissue development. Our study supplies basic knowledges on eccDNAs in cattle, which will promote understanding of extrachromosomal DNA.

New insights into transcriptome variation during cattle adipocyte adipogenesis by direct RNA sequencing.

We performed direct RNA sequencing (DRS) together with PCR-amplified cDNA long and short read sequencing for cattle adipocyte at different stages. We proved that the DRS was with advantages to avoid artificial transcripts and questionable exitrons. Totally, we obtained 68,124 transcripts with information of alternative splicing, poly (A) length and mRNA modification. The number of transcripts for adipogenesis was expanded by alternative splicing, which lead regulation mechanisms far more complex than ever known. We detected 891 differentially expressed genes (DEGs). However, 62.78% transcripts of DEGs were not significantly differentially expressed, and 248 transcripts showed opposite changing directions with their genes. The poly (A) tail became globally shorter in differentiated adipocyte than in primary adipocyte, and had a weak negative correlation with gene/transcript expression. Moreover, the study of different mRNA modifications implied their potential roles in gene expression and alternative splicing. Overall, our study promoted better understanding of adipogenesis mechanisms in cattle adipocytes.

Over-Expression of Two Different Isoforms of Cattle TUSC5 Showed Opposite Effects on Adipogenesis.

(1) Background: Adipogenesis is an important issue in human health and livestock meat quality that has received widespread attention and extensive study. However, alternative splicing events may generate multiple isoforms with different functions. This will lead to known knowledge being far more complex than before. (2) Methods: We studied the effects of two different TUSC5 isoforms (TUSC5A and TUSC5B) in cattle on adipogenesis by constructing over-expression cell models and RNA-sequencing methods. (3) Results: We discovered that over-expression of TUSC5A promotes the process of adipogenesis while over-expression of TUSC5B suppresses it. Eight important genes (PPARG, ACC1, FASN, SCD1, LPL, FABP4, GPDH, and GLUT4) during adipogenesis were significantly promoted (student's t-test, p < 0.05) by TUSC5A and suppressed by TUSC5B both before and after cell differentiation. By performing a comprehensive analysis using a RNA-seq strategy, we found that both up-regulated differentially expressed genes (DEGs, |log2FoldChange| ≥ 1, p ≤ 0.05) of TUSC5A and down-regulated DEGs of TUSC5B were significantly enriched in the adipogenesis related GO terms, and the PPAR signaling pathway may play important role in those differences. (4) Conclusions: Our study proved that over-expression of two TUSC5 isoforms would regulate adipogenesis in the opposite direction. It is important to understand the function of the TUSC5 gene correctly.