Jiajie Sun

Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome

The decrease in sequencing cost and increased sophistication of assembly algorithms for short-read platforms has resulted in a sharp increase in the number of species with genome assemblies. However, these assemblies are highly fragmented, with many gaps, ambiguities, and errors, impeding downstream applications. We demonstrate current state of the art for de novo assembly using the domestic goat (Capra hircus) based on long reads for contig formation, short reads for consensus validation, and scaffolding by optical and chromatin interaction mapping. These combined technologies produced what is, to our knowledge, the most continuous de novo mammalian assembly to date, with chromosome-length scaffolds and only 649 gaps. Our assembly represents a ∼400-fold improvement in continuity due to properly assembled gaps, compared to the previously published C. hircus assembly, and better resolves repetitive structures longer than 1 kb, representing the largest repeat family and immune gene complex yet produced for an individual of a ruminant species.

Porcine milk-derived exosomes promote proliferation of intestinal epithelial cells

Milk-derived exosomes were identified as a novel mechanism of mother-to-child transmission of regulatory molecules, but their functions in intestinal tissues of neonates are not well-studied. Here, we characterized potential roles of porcine milk-derived exosomes in the intestinal tract. In vitro, treatment with milk-derived exosomes (27 ± 3 ng and 55 ± 5 ng total RNA) significantly promoted IPEC-J2 cell proliferation by MTT, CCK8, EdU fluorescence and EdU flow cytometry assays. The qRT-PCR and Western blot analyses indicated milk-derived exosomes (0.27 ± 0.03 μg total RNA) significantly promoted expression of CDX2, IGF-1R and PCNA, and inhibited p53 gene expression involved in intestinal proliferation. Additionally, six detected miRNAs were significantly increased in IPEC-J2 cell, while FAS and SERPINE were significantly down-regulated relative to that in control. In vivo, treated groups (0.125 μg and 0.25 μg total RNA) significantly raised mice’ villus height, crypt depth and ratio of villus length to crypt depth of intestinal tissues, significantly increased CDX2, PCNA and IGF-1R’ expression and significantly inhibited p53′ expression. Our study demonstrated that milk-derived exosomes can facilitate intestinal cell proliferation and intestinal tract development, thus giving a new insight for milk nutrition and newborn development and health.

Single-molecule sequencing and conformational capture enable de novo mammalian reference genomes

The decrease in sequencing cost and increased sophistication of assembly algorithms for short-read platforms has resulted in a sharp increase in the number of species with genome assemblies. However, these assemblies are highly fragmented, with many gaps, ambiguities, and errors, impeding downstream applications. We demonstrate current state of the art for de novo assembly using the domestic goat (Capra hircus), based on long reads for contig formation, short reads for consensus validation, and scaffolding by optical and chromatin interaction mapping. These combined technologies produced the most contiguous de novo mammalian assembly to date, with chromosome-length scaffolds and only 663 gaps. Our assembly represents a >250-fold improvement in contiguity compared to the previously published C. hircus assembly, and better resolves repetitive structures longer than 1 kb, supporting the most complete repeat family and immune gene complex representation ever produced for a ruminant species.

Dietary protein-induced hepatic IGF-1 secretion mediated by PPARγ activation

Dietary protein or amino acid (AA) is a crucial nutritional factor to regulate hepatic insulin-like growth factor-1 (IGF-1) expression and secretion. However, the underlying intracellular mechanism by which dietary protein or AA induces IGF-1 expression remains unknown. We compared the IGF-1 gene expression and plasma IGF-1 level of pigs fed with normal crude protein (CP, 20%) and low-protein levels (LP, 14%). RNA sequencing (RNA-seq) was performed to detect transcript expression in the liver in response to dietary protein. The results showed that serum concentrations and mRNA levels of IGF-1 in the liver were higher in the CP group than in the LP group. RNA-seq analysis identified a total of 1319 differentially expressed transcripts (667 upregulated and 652 downregulated), among which the terms “oxidative phosphorylation”, “ribosome”, “gap junction”, “PPAR signaling pathway”, and “focal adhesion” were enriched. In addition, the porcine primary hepatocyte and HepG2 cell models also demonstrated that the mRNA and protein levels of IGF-1 and PPARγ increased with the increasing AA concentration in the culture. The PPARγ activator troglitazone increased IGF-1 gene expression and secretion in a dose dependent manner. Furthermore, inhibition of PPARγ effectively reversed the effects of the high AA concentration on the mRNA expression of IGF-1 and IGFBP-1 in HepG2 cells. Moreover, the protein levels of IGF-1 and PPARγ, as well as the phosphorylation of mTOR, significantly increased in HepG2 cells under high AA concentrations. mTOR phosphorylation can be decreased by the mTOR antagonist, rapamycin. The immunoprecipitation results also showed that high AA concentrations significantly increased the interaction of mTOR and PPARγ. In summary, PPARγ plays an important role in the regulation of IGF-1 secretion and gene expression in response to dietary protein.

Integrated analysis of non-coding RNA and mRNA expression profiles of 2 pig breeds differing in muscle traits

Production of high-quality meat is important to satisfy the consumer and allow the pork industry to be competitive. It is evident that different muscle fiber types in different breeds greatly influence the pork quality, but the underlying molecular mechanism remains unclear. We used Ribo-Zero RNA-Seq and miRNA-Seq to examine global expressions of protein-coding transcripts and non-coding RNAs including miRNA, lncRNA, and circRNA in the longissimus dorsi of Landrace and Lantang pigs. Of the 22,469 identified coding transcripts, only 547 candidates were differentially expressed, including 461 upregulated and 86 downregulated transcripts in the Lantang pigs compared with Landrace. Gene ontology analysis of these differentially-expressed transcripts further revealed 17 genes involved in myogenesis. In addition, 5,566 lncRNA and 4,360 circRNA candidates were found to be differentially expressed. Of these, 3,976 lncRNAs and 1,401 circRNAs were upregulated in the Lantang library, while 1,590 lncRNAs and 2,959 circRNAs were downregulated. Of the differentially expressed circRNAs, 236 candidates were edited from 93 functional hosting-genes related to myogenesis. We found 96 showed upregulation and 140 showed downregulation. By analyzing Ribo-Zero RNA-Seq data in combination with matched miRNA profiles, we identified 68 sponge modulators participating in 26 miRNA-mediated ceRNA interactions, including 19 lncRNAs, 40 circRNAs, and 9 mRNAs. Our study uncovered a novel post-transcriptional regulation layer which could help in the understanding of the mechanisms that underlie porcine myofiber development in different breeds.

In low protein diets, microRNA-19b regulates urea synthesis by targeting SIRT5

Ammonia detoxification, which takes place via the hepatic urea cycle, is essential for nitrogen homeostasis and physiological well-being. It has been reported that a reduction in dietary protein reduces urea nitrogen. MicroRNAs (miRNAs) are major regulatory non-coding RNAs that have significant effects on several metabolic pathways; however, little is known on whether miRNAs regulate hepatic urea synthesis. The objective of this study was to assess the miRNA expression profile in a low protein diet and identify miRNAs involved in the regulation of the hepatic urea cycle using a porcine model. Weaned 28-days old piglets were fed a corn-soybean normal protein diet (NP) or a corn-soybean low protein diet (LP) for 30 d. Hepatic and blood samples were collected, and the miRNA expression profile was assessed by sequencing and qRT-PCR. Furthermore, we evaluated the possible role of miR-19b in urea synthesis regulation. There were 25 differentially expressed miRNAs between the NP and LP groups. Six of these miRNAs were predicted to be involved in urea cycle metabolism. MiR-19b negatively regulated urea synthesis by targeting SIRT5, which is a positive regulator of CPS1, the rate limiting enzyme in the urea cycle. Our study presented a novel explanation of ureagenesis regulation by miRNAs.

The effect of dietary ginseng polysaccharide supplementation on the immune responses involved in porcine milk-derived esRNAs

Ginseng and its polysaccharides (GPS) have been well known as an immune modulator. This study was conducted to investigate the effects of dietary supplemental GPS on the immune responses involved in sow’s milk-derived exosomal shuttle RNAs (esRNAs) using RNA-Seq and miRNA-Seq. Of the 213 identified miRNA types, a total of 26 conserved miRNAs were differently expressed in response to GPS supplementation, including 10 up-regulated and 16 down-regulated miRNAs in GPS feeding group. In addition, exosomal transcriptome analysis identified 14,696 protein-coding genes in sow’s milk exosomes, and 283 genes with 204 and 79 candidates showing up and down-regulation were significantly responded to GPS supplementation. Integrated analysis of each differently expressed miRNA with significantly expressed genes further revealed the presence of 51 highly conserved miRNA-gene interactions that were annotated to be related to immunoregulatory functions. This work provided an important advance in the functional identification of dietary GPS supplementation and more fundamental information about how GPS promoted the immune response and healthy growth of the infant from mothers at molecular levels.

Characterization of faecal microbial communities of dairy cows fed diets containing ensiled Moringa oleifera fodder.

Moringa oleifera (M. oleifera) is a remarkable species with high nutritional value and good biomass production, which can be used as livestock fodder. In this study, we examined changes in the faecal microbiota of thirty dairy cows in response to alternative M. oleifera diets and their effects on nutrient digestion, milk traits and the faecal concentrations of short-chain fatty acids. No differences in milk yield and constituents were found between the control and the M. oleifera alternative groups. Cows fed M. oleifera silage had lower dry matter digestibility, as well as the propionate and isovalerate concentrations in M. oleifera treated group. Using 16S rDNA gene sequencing, 1,299,556 paired-end reads were obtained. Clustering analysis revealed 13 phyla and 93 genera across all samples. Firmicutes and Bacteroidetes were the co-dominant phyla. Ten taxa displayed a significant difference in response to the high M. oleifera diet. In addition, strong correlations between Akkermansia and Prevotella with milk yield and protein indicated that some bacterial groups could be used to improve milk traits. Our results provided an insight into the microbiome-associated responses to M. oleifera in livestock diets, and could aid the development of novel applications of M. oleifera.

Revelation of mRNAs and proteins in porcine milk exosomes by transcriptomic and proteomic analysis

BackgroundMilk is a complex liquid that provides nutrition to newborns. Recent reports demonstrated that milk is enriched in maternal-derived exosomes that are involved in fetal physiological and pathological conditions by transmission of exosomal mRNAs, miRNAs and proteins. Until now, there is no such research relevant to exosomal mRNAs and proteins in porcine milk, therefore, we have attempted to investigate porcine milk exosomal mRNAs and proteins using RNA-sequencing and proteomic analysis.ResultsA total of 16,304 (13,895 known and 2,409 novel mRNAs) mRNAs and 639 (571 known, 66 candidate and 2 putative proteins) proteins were identified. GO and KEGG annotation indicated that most proteins were located in the cytoplasm and participated in many immunity and disease-related pathways, and some mRNAs were closely related to metabolisms, degradation and signaling pathways. Interestingly, 19 categories of proteins were tissue-specific and detected in placenta, liver, milk, plasma and mammary. COG analysis divided the identified mRNAs and proteins into 6 and 23 categories, respectively, 18 mRNAs and 10 proteins appeared to be involved in cell cycle control, cell division and chromosome partitioning. Additionally, 14 selected mRNAs were identified by qPCR, meanwhile, 10 proteins related to immunity and cell proliferation were detected by Western blot.ConclusionsThese results provide the first insight into porcine milk exosomal mRNA and proteins, and will facilitate further research into the physiological significance of milk exosomes for infants.

Systematic profiling of short tandem repeats in the cattle genome

Short tandem repeats (STRs), or microsatellites, are genetic variants with repetitive 2–6 base pair motifs in many mammalian genomes. Using high-throughput sequencing and experimental validations, we systematically profiled STRs in five Holsteins. We identified a total of 60,106 microsatellites and generated the first high-resolution STR map, representing a substantial pool of polymorphism in dairy cattle. We observed significant STRs overlap with functional genes and quantitative trait loci (QTL). We performed evolutionary and population genetic analyses using over 20,000 common dinucleotide STRs. Besides corroborating the well-established positive correlation between allele size and variance in allele size, these analyses also identified dozens of outlier STRs based on two anomalous relationships that counter expected characteristics of neutral evolution. And one STR locus overlaps with a significant region of a summary statistic designed to detect STR-related selection. Additionally, our results showed that only 57.1% of STRs located within SNP-based linkage disequilibrium (LD) blocks whereas the other 42.9% were out of blocks. Therefore, a substantial number of STRs are not tagged by SNPs in the cattle genome, likely due to STRs distinct mutation mechanism and elevated polymorphism. This study provides the foundation for future STR-based studies of cattle genome evolution and selection.