Retroviral Insertion Polymorphism (RIP) of Porcine Endogenous Retroviruses (PERVs) in Pig Genomes.

Endogenous retroviruses (ERVs) are one of the superfamilies of long terminal repeat retrotransposons (LTRs) in mice and humans. Approximately 8% of the pig genome is composed of sequences derived from LTRs. While the majority of ERVs in pigs have decayed, a small number of full-length copies can still mobilize within the genome. This study investigated the unexplored retroviral insertion polymorphisms (RIPs) generated by the mobilization of full-length ERVs (Fl-ERVs), and evaluated their impact on phenotypic variation to gain insights into the biological role of Fl-ERVs in pigs. Overall, 39 RIPs (insertions or deletions relative to the pig reference genome) generated by Fl-ERVs were predicted by comparative genomic analysis, and 18 of them were confirmed by PCR detection. Four RIP sites (D5, D14, D15, and D18) were further evaluated by population analysis, and all of them displayed polymorphisms in multiple breeds. The RIP site of ERV-D14, which is a Fl-ERV inserted in the STAB2-like gene, was further confirmed by sequencing. Population analysis of the polymorphic site of ERV-D14 reveals that it presents moderate polymorphism information in the Large White pig breed, and the association analysis reveals that the RIP of ERV-D14 is associated with age variations at 30 kg body weight (p < 0.05) and 100 kg body weight (p < 0.01) in the population of Large White pigs (N = 480). Furthermore, the ERV-D14 RIP is associated with changes in the expression of the target gene STAB2-like in the liver, backfat, and leaf fat in Sushan pigs. These data suggest that some Fl-ERVs are still mobilizing in the pig's genome, and contribute to genomic and phenotypic variations.

Integrated analysis of differently expressed microRNAs and mRNAs at different postnatal stages reveals intramuscular fat deposition regulation in goats (Capra hircus).

Intramuscular fat refers to the adipose tissue distributed in the muscle. It is an important indicator that affects the quality of goat meat, and can directly affect the tenderness and flavor of goat meat. Our previous study revealed the mRNA that may be crucial for intramuscular fat deposition during goat growth; however, how the microRNAs (miRNAs) are involved in the process is largely unclear. In the present study, a total of 401 known miRNAs and 120 goat novel miRNAs, including 110 differentially expressed (DE) miRNAs, were identified among longissimus dorsi from three growth stages (2, 9, and 24 months) by miRNA sequencing. Combining analysis of the DE mRNAs and DE miRNAs was then performed by miRDB and miRwalk, and miR-145-5p and FOXO1, miR-487b-3p, and PPARG coactivator 1 α (PPARGC1A), miR-345-3p, and solute carrier family 2 member 4 (SLC2A4), etc. were shown to closely associate with lipid metabolism, which was then validated by a correlation analysis. The final DE mRNAs were significantly enriched in fatty acid transmembrane transport, fatty acid homeostasis, apelin signaling pathway, glucagon signaling pathway, insulin signaling pathway, and AMPK signaling pathway by gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis. Besides, miR-145-5p showed a certain effect on goat intramuscular fat metabolism by acting on the possible target gene Forkhead Box O1 (FOXO1). These data provide some theoretical support for improving the quality of goat meat.

Insertion of 643bp Retrotransposon Upstream of PPARγ CDS Is Associated with Backfat of Large White Pigs.

PPARs are essential regulators of mammalian fatty acid and lipid metabolism. Although the effects of genetic variations, including single nucleotide polymorphisms (SNPs) in PPARs genes on the phenotype of domestic animals have been investigated, there is limited information on the impact of retrotransposon insertion polymorphisms (RIPs). In this study, a combined comparative genome and polymerase chain reaction (PCR) was used to excavate the RIPs in porcine PPARs. We also investigated the potential effects of retrotransposon insertion on phenotype and expression patterns. This study identified the two RIPs in PPARs genes, namely an ERV in intron 1 of PPARα and a combined retrotransposon in intron 2 of PPARγ, designated as PPARα-ERV-RIP and PPARγ-COM-RIP, respectively. These RIPs exhibited different distribution patterns among Chinese indigenous breeds and Western commercial breeds. Individuals with the PPARα-ERV-RIP+/+ genotype (+/+ indicated homozygous with insertion) among Large White pigs had significantly higher (p < 0.05) corrected backfat thickness compared to those with the other two genotypes. Similarly, those with the PPARγ-COM-RIP-/- genotype had significantly higher (p < 0.05) corrected backfat thickness than those with the other two genotypes in Large White pigs. Moreover, in 30-day-old Sujiang piglets, the PPARγ gene expression in the backfat of those with the PPARγ-COM-RIP-/- genotype (-/- indicated homozygous without insertion) was significantly greater (p < 0.01) than those with other genotypes. The dual luciferase reporter gene assay demonstrated that the combined retrotransposon insertion significantly reduced the activity of the MYC promoter in both C2C12 and 3T3-L1 cells (p < 0.01). Therefore, the combined retrotransposon insertion could function as a repressor to decrease the expression of PPARγ, making PPARγ-COM-RIP a valuable molecular marker for assisted selection of backfat thickness in pig breeding.

Two Retrotransposon Elements in Intron of Porcine BMPR1B Is Associated with Phenotypic Variation.

It has been established that through binding to bone morphogenetic proteins (BMPs), bone morphogenetic protein receptor I B (BMPR1B) can mediate transforming growth factor ß (TGF-ß) signal transduction, and is involved in the regulation of several biological processes, such as bone and muscle formation and homeostasis, as well as folliculogenesis. Also known as FecB, BMPR1B has been reported as the major gene for sheep prolificacy. A number of previous studies have analyzed the relationship between single nucleotide polymorphisms (SNPs) in this gene and its related performance. In recent years, with the illustration of the effect of retrotransposon insertion on the expression of the proximal genes or phenotypic variation, retrotransposon insertion polymorphisms (RIPs) have been used as a novel type of molecular marker in the evaluation of evolution, population structure and breeding of plant and domestic animals. In this study, the RIPs in porcine BMPR1B gene were excavated, and thereafter verified using a comparative genome and polymerase chain reaction (PCR). The potential effects of phenotype, gene expression and functions related to RIPs were also explored. The results showed that 13 distinct RIPs were identified in introns of porcine BMPR1B. Among these, only BMPR1B-SINE-RIP9 and BMPR1B-LINE-RIP13 displayed a close relationship with the growth traits of Large White pigs. Moreover, the total number of BMPR1B-SINE+/+-RIP9 individuals born was found to be significantly higher than that of SINE−/− (p < 0.05). These two RIPs showed an obvious distribution pattern among Chinese indigenous breeds and Western commercial breeds. The expression of BMPR1B in ovaries of adult BMPR1B-SINE+/+-RIP9 Sushan pigs was found to be significantly higher in comparison to those of BMPR1B-SINE−/−-RIP9 (p < 0.05). SINE insertion of BMPR1B-SINE-RIP9 and LINE insertion of BMPR1B-LINE-RIP13 were observed to significantly increase the activity of Octamer binding transcription factor 4 (OCT4) minipromoter in CHO and C2C12 cells (p < 0.01). Therefore, these two RIPs could serve as useful molecular markers for modulating the growth or reproductive traits in assisted selection of pig breeding, while the mechanisms of the insertion function should be studied further.

Retrotransposon Insertion Polymorphisms (RIPs) in Pig Reproductive Candidate Genes.

Retrotransposons account for more than one-third of the pig reference genome. On account of the genome variability in different breeds, structural variation (SV) caused by retrotranspos-on-generated deletion or insertion (indel) may have a function in the genome. Litter size is one of the most important reproductive traits and significantly impacts profitability in terms of pig production. We used the method of bioinformatics, genetics, and molecular biology to make an analysis among different pig genomes. Predicted 100 SVs were annotated as retrotransposon indel in 20 genes related to reproductive performance. The PCR detection based on these predicted SVs revealed 20 RIPs in 20 genes, that most RIPs (12) were generated by SINE indel, and eight RIPs were generated by the ERV indel. We selected 12 RIPs to make the second round PCR detection in 24 individuals among nine pig breeds. The PCR detection results revealed that the RIP-A1CF-4 insertion in the breed of Bama, Large White, and Meishan only had the homozygous genotype but low to moderately polymorphisms were present in other breeds. We found that RIP-CWH43-9, RIP-IDO2-9, RIP-PRLR-6, RIP-VMP1-12, and RIP-OPN-1 had a rich polymorphism in the breed of Large White pigs. The statistical analysis revealed that RIP-CWH43-9 had a SINE insertion profitable to the reproductive traits of TNB and NBA but was significantly affected (p < 0.01) and (p < 0.05) in the reproductive traits of litter birthweight (LW) in Large White. On the other hand, the SINE insertion in IDO2-9 may be a disadvantage to the reproductive traits of LW, which was significantly affected (p < 0.05) in Large White. These two RIPs are significant in pig genome research and could be useful molecular markers in the breeding system.

SINE Insertion May Act as a Repressor to Affect the Expression of Pig LEPROT and Growth Traits.

Retrotransposon is an important component of the mammalian genome. Previous studies have shown that the expression of protein-coding genes was affected by the insertion of retrotransposon into the proximal genes, and the phenotype variations would be related to the retrotransposon insertion polymorphisms (RIPs). In this study, leptin (LEP), leptin receptor (LEPR), and leptin receptor overlapping transcript (LEPROT), which play important roles in the regulation of fat synthesis and body weight, were screened to search for the RIPs and their effect on phenotype and gene expression, as well as to further study the function of the insertion. The results showed that three RIPs located in intron 1 of LEPROT and intron 2 and 21 of LEPR were identified, and they were all SINEA1, which was one type of retrotransposon. The SINE insertion at the LEPROT was the dominant allele in native pig breeds. The age of 100 kg body weight of SINE+/+ Large White individuals was significantly higher than those of SINE+/− and SINE−/− individuals (p < 0.05). The LEPROT gene expression in the liver and suet of 30-day-old SINE−/− Sujiang piglets were significantly higher than those of SINE+/+ and SINE+/− piglets (p < 0.01). The dual-luciferase reporter gene assay showed that SINE insertion in PK15 and 3T3-L1 cells significantly reduced the promoter activity of the LEPROT gene (p < 0.01). Therefore, SINE insertion can be a repressor to reduce the expression of LEPROT and could be a useful molecular marker for assisted selection of growth traits in pig breeding.

Retrotransposon Insertion Polymorphisms (RIPs) in Pig Coat Color Candidate Genes.

The diversity of livestock coat color results from human positive selection and represents an indispensable part of breed identity. As an important biodiversity resource, pigs have many special characteristics, including the most visualized feature, coat color, and excellent adaptation, and the coat color represents an important phenotypic characteristic of the pig breed. Exploring the genetic mechanisms of phenotypic characteristics and the melanocortin system is of considerable interest in domestic animals because their energy metabolism and pigmentation have been under strong selection. In this study, 20 genes related to coat color in mammals were selected, and the structural variations (SVs) in these genic regions were identified by sequence alignment across 17 assembled pig genomes, from representing different types of pigs (miniature, lean, and fat type). A total of 167 large structural variations (>50 bp) of coat-color genes, which overlap with retrotransposon insertions (>50 bp), were obtained and designated as putative RIPs. Finally, 42 RIPs were confirmed by PCR detection. Additionally, eleven RIP sites were further evaluated for their genotypic distributions by PCR in more individuals of eleven domesticated breeds representing different coat color groups. Differential distributions of these RIPs were observed across populations, and some RIPs may be associated with breed differences.

Gene Cloning, Tissue Expression Profiles and Antiviral Activities of Interferon-ß from Two Chinese Miniature Pig Breeds.

The porcine interferon (PoIFN) complex represents an ideal model for studying IFN evolution which has resulted from viral pressure during domestication. Bama and Banna miniature pigs are the two Chinese miniature pig breeds that have been developed as laboratory animal models for studying virus infection, pathogenesis, and vaccine evaluation. However, the PoIFN complex of such miniature pig breeds remains to be studied. In the present study, we cloned PoIFN-ß genes from Bama and Banna miniature pigs, detected their PoIFN-ß tissue expression profiles, prepared recombinant PoIFN-ß (rPoIFN-ß) using the E. coli expression system, and measured their antiviral activities against three different pig viruses. At the amino acid sequence level, PoIFN-ßs of the two miniature pig breeds were identical, which shared 100% identity with that of Congjiang Xiang pigs, 99.4-100% identity with that of domestic pigs, and 99.5% identity with that of three species of African wild boars. The tissue expression profiles of PoIFN-ß mRNA differed not only between the two miniature pig breeds but between miniature pigs and domestic pigs as well. The four promoter domains of PoIFN-ß of the two miniature pig breeds were identical with that of humans, domestic pigs, and three species of African wild boars. The recombinant PoIFN-ß prepared from the two miniature pig breeds showed dose-dependent pre-infection and post-infection antiviral activities against vesicular stomatitis virus, porcine respiratory and reproductive syndrome virus, and pig pseudorabies virus. This study provided evidence for the high sequence conservation of PoIFN-ß genes within the Suidae family with different tissue expression profiles and antiviral activities.

Gene cloning, prokaryotic expression and antiviral activities of interferon-αω from Chinese Bama miniature pigs.

Porcine interferon (PoIFN) complex represents an ideal model for studying IFN evolution that resulted from viral pressure during domestication. IFN-αω is an emergent subtype of type I IFNs which has been primarily characterized in domestic pigs. In this study, the PoIFN-αω cDNA was cloned from Chinese Bama miniature pigs by RT-PCR, and its tissue expression profile was analyzed by real-time RT-PCR. The cDNA was expressed in Escherichia coli as a His-tagged protein and purified by nickel affinity chromatography. The antiviral activities of recombinant PoIFN-αω (rPoIFN-αω) against four different pig viruses were measured using cytopathic effect (CPE) inhibition assay. Although the PoIFN-αω sequence of Bama miniature pigs was identical to that of domestic pigs, the tissue expression profiles differed significantly between the two pig species. The rPoIFN-αω showed dose-dependent pre-infection antiviral activities against porcine pseudorabies virus, vesicular stomatitis virus and porcine reproductive and respiratory syndrome virus, but not against porcine circovirus type 2. When used as treatment post infection with the three viruses, rPoIFN-αω showed the efficacy in decreasing CPE in the infected cells in a time-dependent manner. Therefore, the expressed rPoIFN-αω could be used as an antiviral agent against pig virus infections.

Effect of vitrification on meiotic maturation, mitochondrial distribution and glutathione synthesis in immature silver fox cumulus oocyte complexes.

The present study was designed to investigate the effects of vitrifying oocytes obtained from silver foxes on nuclear maturation, mitochondrial distribution and glutathione (GSH) synthesis after in vitro culture for 72 h. Immature oocytes were randomly divided into three groups: (1) fresh GV (germinal vesicle) oocytes (Control group), (2) exposure to the equilibration and vitrification solution but without being plunged into liquid nitrogen (exposed group), and (3) vitrification by the cryoloop method (vitrified-warmed group). The number of survival oocytes was not decreased by either being exposed to the cryoprotectant or being vitrified-warmed compared with the control group (P > 0.05). After IVM, the percentage of resumption of meiosis for vitrified-warmed oocytes (41.9%) was significantly lower than in the control (81.2%) and exposed (79.1%) groups (P < 0.05). However, the proportion of oocytes reaching the metaphase II (MII) stage was similar among the different groups (11.4%, 9.3% and 5.2%, respectively, P > 0.05). The translocation of active mitochondria during fox oocyte maturation was revealed using MitoTracker Red staining and confocal laser microscopy. For fresh oocytes at the GV stage, active mitochondria were distributed around the entire cortex with small granulations and various-sized cavities (no MitoTracker signals). After IVM, the mitochondria formed large granulations and clumps throughout the cytoplasm. Vitrification significantly decreased the proportion of MII oocytes with normal mitochondrial distribution compared with the control and exposed groups (35.4%, 71.9% and 59.2%, respectively, P < 0.05). Similarly, the GSH content was significantly lower in vitrified-warmed oocytes compared with the control and exposed oocytes after IVM (3.4, 5.7 and 4.7 pM/oocyte, respectively, P < 0.05). However, no significant difference was observed between the cryoprotectant exposed and control groups with regard to the normal mitochondrial distribution or GSH content (P > 0.05). These results indicate that vitrification of fox immature oocytes using a cryoloop allows them to resume meiosis and develop to the MII stage. The damage to mitochondria and the GSH synthesis deficiency may be associated with the reduced developmental competence of cryopreserved oocytes.

Comparative Transcriptome Analysis of Raccoon Dog Skin to Determine Melanin Content in Hair and Melanin Distribution in Skin.

The raccoon dog (Nyctereutes procyonoides) is an important canid fur-bearing animal species worldwide. Chinese raccoon dogs that present a white mutation, especially those with a white coat. Exploring melanin biosynthesis in the hair and skin of raccoon dogs is important for understanding the survival and evolutionary mechanisms of them. In this study, we measured the content of melanin in the hair of two types of raccoon dog and generated stained slices of skin tissue. The results indicated that melanin biosynthesis occurs in the wild-type (W) and white-type (B) raccoon dog skin, although less melanin is produced in B skin. We then sequenced the skin transcriptomes of W and B, compared the similarities and differences in expressed genes. A comparison of the gene expression showed 60 up-regulated genes and 127 down-regulated genes in B skin. We analyzed the unigenes and pathways related to the melanogenesis pathway and found that TYR, TYRP1, MC1R, SLC24a5, SLC45a2 and OCA2 were significantly down-regulated in B skin and these results were verified via qRT-PCR. We surmised that the phenotypic characteristics of the white mutation might be caused by the reduced expression of these genes and this finding provides new insights for future experiments in raccoon dogs.