FZD7, Regulated by Non-CpG Methylation, Plays an Important Role in Immature Porcine Sertoli Cell Proliferation.

The regulatory role of non-CpG methylation in mammals has been important in whole-genome bisulfite sequencing. It has also been suggested that non-CpG methylation regulates gene expression to affect the development and health of mammals. However, the dynamic regulatory mechanisms of genome-wide, non-CpG methylation during testicular development still require intensive study. In this study, we analyzed the dataset from the whole-genome bisulfite sequencing (WGBS) and the RNA-seq of precocious porcine testicular tissues across two developmental stages (1 and 75 days old) in order to explore the regulatory roles of non-CpG methylation. Our results showed that genes regulated by non-CpG methylation affect the development of testes in multiple pathways. Furthermore, several hub genes that are regulated by non-CpG methylation during testicular development-such as VEGFA, PECAM1, and FZD7-were also identified. We also found that the relative expression of FZD7 was downregulated by the zebularine-induced demethylation of the first exon of FZD7. This regulatory relationship was consistent with the results of the WGBS and RNA-seq analysis. The immature porcine Sertoli cells were transfected with RNAi to mimic the expression patterns of FZD7 during testicular development. The results of the simulation test showed that cell proliferation was significantly impeded and that cell cycle arrest at the G2 phase was caused by the siRNA-induced FZD7 inhibition. We also found that the percentage of early apoptotic Sertoli cells was decreased by transfecting them with the RNAi for FZD7. This indicates that FZD7 is an important factor in linking the proliferation and apoptosis of Sertoli cells. We further demonstrated that Sertoli cells that were treated with the medium collected from apoptotic cells could stimulate proliferation. These findings will contribute to the exploration of the regulatory mechanisms of non-CpG methylation in testicular development and of the relationship between the proliferation and apoptosis of normal somatic cells.

FSH promotes immature porcine Sertoli cell proliferation by activating the CCR7/Ras-ERK signaling axis.

In brief: The appropriate growth and functions of Sertoli cells are crucial to testis development and spermatogenesis in mammals. This study reveals a novel mechanism of follicle-stimulating hormone in immature porcine Sertoli cell proliferation. Abstract: Follicle-stimulating hormone (FSH) is a major Sertoli cell mitogen that binds to the FSH receptor. Sertoli cells are indispensable for testis development and spermatogenesis. However, the regulatory mechanisms of FSH in immature Sertoli cell proliferation have not been determined, particularly in domestic animals. In the present study, we identified the regulatory mechanisms of FSH during immature porcine Sertoli cell proliferation. Transcriptome analysis revealed 114 differentially expressed genes that were induced by FSH treatment, which contains 68 upregulated and 46 downregulated genes. These differentially expressed genes were enriched in multiple pathways, including the Ras signaling pathway. Knockdown of the CC-chemokine receptor 7 (CCR7) gene, which was upregulated by FSH, inhibited cell cycle progression by arresting cells in the G1 phase and reduced the cell proliferation and ERK1/2 phosphorylation. In addition, Kobe0065 inhibited Ras signaling in a similar manner as CCR7 knockdown. Furthermore, FSH abolished the effects of Ras signaling pathway inhibition and CCR7 knockdown. Collectively, FSH promotes immature porcine Sertoli cell proliferation by activating the CCR7/Ras-ERK signaling axis. Our results provide novel insights into the regulatory mechanism of FSH in porcine testis development and spermatogenesis by deciding the fate of immature porcine Sertoli cells.

Traditional Chinese herbal medicine complex supplementation improves reproductive performance, serum biochemical parameters, and anti-oxidative capacity in periparturient dairy cows.

This study was conducted to investigate the effects of a traditional Chinese herbal medicine complex (TCHMC) on the productive performance of periparturient dairy cows. Eighteen non-lactating pregnant Holstein dairy cows with similar body conditions with 1 to 2 parity were randomly divided into three groups (n = 6), receiving a basal diet with 0 (CON group), 200 (T-200 group), and 300 (T-300 group) g TCHMC per day from 14 to 9 days prepartum. The results demonstrated that TCHMC treatments decreased the days of gestation, calving to first service, and calving to first visible estrus. Compared with CON at specific time points, TCHMC treatments increased the concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2), whereas progesterone (P4) and E2 concentrations decreased. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (CK) concentrations were downregulated, whereas that of globulin (GLB) and immunoglobulin G (IgG) were upregulated by TCHMC treatments around the time of calving. Compared with CON and T-200 treatments, the T-300 treatment increased the serum concentrations of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) and decreased the malondialdehyde (MDA) concentration from 7 d prepartum to 21 d postpartum when. In addition, although TCHMC treatment had no effect on average birth weight, heart rate, respiratory rate, and body temperature of calves, the T-300 treatment increased serum albumin (ALB) and IgG concentrations in calves from 3 to 14 days postpartum. The addition of TCHMC used in the present study could serve as a potential effective strategy to improve the health and productive performance of periparturient dairy cows, and the optimal dose should be set at 300 g per day.

circBTBD7 Promotes Immature Porcine Sertoli Cell Growth through Modulating miR-24-3p/MAPK7 Axis to Inactivate p38 MAPK Signaling Pathway.

Sertoli cells are the crucial coordinators to guarantee normal spermatogenesis and male fertility. Although circular RNAs (circRNAs) exhibit developmental-stage-specific expression in porcine testicular tissues and have been thought of as potential regulatory molecules in spermatogenesis, their functions and mechanisms of action remain largely unknown, especially in domestic animals. A novel circBTBD7 was identified from immature porcine Sertoli cells using reverse transcription PCR, Sanger sequencing, and fluorescence in situ hybridization assays. Functional assays illustrated that circBTBD7 overexpression promoted cell cycle progression and cell proliferation, as well as inhibited cell apoptosis in immature porcine Sertoli cells. Mechanistically, circBTBD7 acted as a sponge for the miR-24-3p and further facilitated its target mitogen-activated protein kinase 7 (MAPK7) gene. Overexpression of miR-24-3p impeded cell proliferation and induced cell apoptosis, which further attenuated the effects of circBTBD7 overexpression. siRNA-induced MAPK7 deficiency resulted in a similar effect to miR-24-3p overexpression, and further offset the effects of miR-24-3p inhibition. Both miR-24-3p overexpression and MAPK7 knockdown upregulated the p38 phosphorylation activity. The SB202190 induced the inhibition of p38 MAPK pathway and caused an opposite effect to that of miR-24-3p overexpression and MAPK7 knockdown. Collectively, circBTBD7 promotes immature porcine Sertoli cell growth through modulating the miR-24-3p/MAPK7 axis to inactivate the p38 MAPK signaling pathway. This study expanded our knowledge of noncoding RNAs in porcine normal spermatogenesis through deciding the fate of Sertoli cells.

miR-126 Controls the Apoptosis and Proliferation of Immature Porcine Sertoli Cells by Targeting the PIK3R2 Gene through the PI3K/AKT Signaling Pathway.

The quantity of Sertoli cells in the adult testis decides the daily gamete formation, and accumulating evidence indicates that epigenetic factors regulate the proliferation of Sertoli cells. Research on the function and regulatory mechanism of microRNAs (miRNAs) in Sertoli cells has not been comprehensive yet, especially on domestic animals. In this article, we report that miR-126 controls the proliferation and apoptosis of immature porcine Sertoli cells based on previous studies. Our results confirmed that miR-126 elevation promotes cell cycle progression, cell proliferation and represses cell apoptosis; on the contrary, the inhibitory effects of miR-126 result in the opposite. The phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2) gene, a member of the PI3K family, was verified as a direct target of miR-126 using the dual-luciferase reporter analysis. miR-126 negatively regulated the mRNA and protein expression level of PIK3R2 in immature porcine Sertoli cells. siRNA-induced PIK3R2 inhibition caused similar effects as miR-126 overexpression and eliminated the influences of miR-126 knockdown in immature porcine Sertoli cells. In addition, both miR-126 overexpression and PIK3R2 inhibition elevated the phosphorylation of PI3K and AKT, whereas the miR-126 knockdown demonstrated the contrary result. In short, miR-126 controls the proliferation and apoptosis of immature porcine Sertoli cells by targeting the PIK3R2 gene through the PI3K/AKT signaling pathway. The research supplies a theoretical and practical foundation for exploring the functional parts of miR-126 in swine sperm by defining the destiny of immature Sertoli cells.

MiR-191 promotes the porcine immature Sertoli cell proliferation by targeting the BDNF gene through activating the PI3K/AKT signaling pathway.

The number of Sertoli cells in the testis is a major regulator on the sperm production capacity. MicroRNAs (miRNAs) participate in regulating the proliferation and apoptosis of porcine immature Sertoli cells. However, the functions and mechanisms of action of most identified miRNAs in porcine Sertoli cells remain largely unknown. In the present study, based on our previous results from an EdU-based high-content screening assay, we further studied the mechanism of action of miR-191 on the proliferation and apoptosis of porcine immature Sertoli cells through flow cytometry, Western blotting, and dual-luciferase activity analyses. The results demonstrated that overexpression of miR-191 promoted cell cycle progression from G1 phase to the S and G2 phases, enhanced cell proliferation, and inhibited apoptosis in the porcine immature Sertoli cells, whereasmiR-191 inhibition resulted in the opposite effects. The results from a luciferase reporter assay showed that miR-191 directly targeted the 3'-UTR of theBDNF gene. BDNF knockdown also promoted cell cycle progression to the S phase, cell proliferation and inhibited cell apoptosis, which were consistent with the effects of the miR-191overexpression. A co-transfection experiment showed that BDNF knockdown abolished the effects of miR-191 inhibition. Furthermore, both miR-191 overexpression and BDNFinhibition elevated the phosphorylation of PI3K and AKT, the key components of the PI3K/AKT signaling pathway, whereas BDNFinhibition offset the effects of the miR-191 knockdown. Overall, these data indicated that miR-191 promotes cell proliferation and inhibits apoptosis in porcine immature Sertoli cells by targeting theBDNF gene through activating the PI3K/AKT signaling pathway. This study provides a novel scientific basis for further investigation on the biological functions of miR-191 on porcine spermatogenesis.

miR-222 Suppresses Immature Porcine Sertoli Cell Growth by Targeting the GRB10 Gene Through Inactivating the PI3K/AKT Signaling Pathway.

Sertoli cells are central and essential coordinators of spermatogenesis. Accumulating evidence has demonstrated that miRNAs participate in the regulation of Sertoli cell growth. However, the functions and the regulatory mechanisms of miRNAs in Sertoli cells of domestic animals remain largely unknown. Here we report that miR-222 overexpression repressed cell cycle progression and proliferation and promoted the apoptosis of immature porcine Sertoli cells, whereas miR-222 inhibition resulted in the opposite result. miR-222 directly targeted the 3'-UTR of the GRB10 gene and inhibited its mRNA abundance. An siRNA-induced GRB10 knockdown showed similar effects as did miR-222 overexpression on cell proliferation and apoptosis and further attenuated the role of miR-222 inhibition. Furthermore, both miR-222 overexpression and GRB10 inhibition repressed the phosphorylation of PI3K and AKT, the key elements of the PI3K/AKT signaling pathway, whereas GRB10 inhibition offsets the effects of the miR-222 knockdown. Overall, we concluded that miR-222 suppresses immature porcine Sertoli cell growth by targeting the GRB10 gene through inactivation of the PI3K/AKT signaling pathway. This study provides novel insights into the epigenetic regulation of porcine spermatogenesis by determining the fate of Sertoli cells.

miR-130a promotes immature porcine Sertoli cell growth by activating SMAD5 through the TGF-ß-PI3K/AKT signaling pathway.

Sertoli cells play vital roles in normal spermatogenesis, and microRNAs (miRNAs) participate in regulating Sertoli cell development. However, the functions and mechanisms of action of most identified miRNAs in porcine Sertoli cells remain largely unknown. Herein, we primarily explored the regulatory roles of miR-130a in immature porcine Sertoli cells using EdU-based high-content screening assay. The results demonstrated that 27 miRNAs have potential roles in the promotion of immature porcine Sertoli cell proliferation, and miR-130a was identified as a promising candidate. miR-130a promoted cell cycle progression and cell proliferation, whereas it impeded cell apoptosis in immature porcine Sertoli cells. It also contributed to Sertoli cell proliferation and testis development in vivo. A TMT-based proteomics approach revealed that miR-130a regulated the expression of 91 proteins and multiple pathways, including the TGF-ß and PI3K/AKT signaling. miR-130a did not directly target the 3'-UTR of SMAD5; however, it increased SMAD5 phosphorylation. Moreover, miR-130a enhanced TGF-ß signaling by activating SMAD5 protein, and TGF-ß signaling further activated the PI3K/AKT signaling pathway to promote cell proliferation and inhibit cell apoptosis in porcine immature Sertoli cells. Collectively, miR-130a promoted immature porcine Sertoli cell growth by activating SMAD5 through the TGF-ß-PI3K/AKT signaling pathway. This study, therefore, provides novel insights into the effects of miR-130a on porcine spermatogenesis through the regulation of immature Sertoli cell proliferation and apoptosis.

The application of genomic selection in pig cross breeding.

Genomic selection is a form of marker-assisted selection in which genetic markers covering the entire genome are used so that all quantitative trait loci are in linkage disequilibrium with at least one marker. Genomic selection improves the efficiency and accuracy of breeding and it is widely used in purebred breeding across many animal species. However, some studies indicate that the accuracy of genome selection in cross breeding needs to be improved,especially in cross population. As one of the most extensive breeding methods employed in the swine industry, cross breeding has significant, potential research and economic value to further improve its performance by combining with genomic selection. In this review, we summarize the application of genomic selection in pigs, and elucidate the genomic selection deficiencies in breeding hybrid pigs. This review will also provide valuable insights for the future application and improvement of genomic selection in pig cross breeding.

miR-362 knock-down promotes proliferation and inhibits apoptosis in porcine immature Sertoli cells by targeting the RMI1 gene.

Immature Sertoli cell proliferation determines the total number of mature Sertoli cells and further regulates normal spermatogenesis. Accumulating evidence demonstrates that microRNAs (miRNAs) play regulatory roles in immature Sertoli cell proliferation, while the functions and mechanisms of the Sertoli cells of domestic animals are poorly understood. In the present study, we aimed to investigate the roles of miR-362 in cell proliferation and apoptosis of porcine immature Sertoli cells. The results showed that miR-362 inhibition promoted the entrance of cells into the S phase and increased the expressions of cell cycle-related genes c-MYC, CNNE1, CCND1 and CDK4. Knock-down of miR-362 also promoted cell proliferation and inhibited apoptosis, which was demonstrated by the results from cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) and Annexin V-FITC/PI staining assays. The recQ-mediated genome instability protein 1 (RMI1) gene was identified as a potential target gene of miR-362 via luciferase reporter assay, and miR-362 repressed the protein expression of RMI1 in porcine immature Sertoli cells. siRNA-induced RMI1 knock-down further abolished the effects of miR-362 inhibition on porcine immature Sertoli cells. Collectively, we concluded that miR-362 knock-down promotes proliferation and inhibits apoptosis in porcine immature Sertoli cells by targeting the RMI1 gene, which indicates that miR-362 determines the fate of immature Sertoli cells.

miR-499 promotes immature porcine Sertoli cell growth through the PI3K/AKT pathway by targeting the PTEN gene.

Sertoli cells are indispensable for normal spermatogenesis, and increasing evidence has shown that miRNAs participate in the regulation of Sertoli cell growth. However, the functions and regulatory mechanisms of miRNAs in Sertoli cells of domestic animals have not been fully investigated. In the present study, we mainly investigated the regulatory roles of miR-499 in immature porcine Sertoli cells. The results showed that miR-499 was mainly located in the basement section of seminiferous tubules of prepubertal porcine testicular tissue. Overexpression of miR-499 promoted cell proliferation and inhibited apoptosis, whereas miR-499 inhibition resulted in the opposite effect. The PTEN gene was directly targeted by miR-499, and the expression of mRNA and protein was also negatively regulated by miR-499 in immature porcine Sertoli cells. siRNA-induced PTEN knockdown resulted in a similar effect as an overexpression of miR-499 and abolished the effects of miR-499 inhibition on immature porcine Sertoli cells. Moreover, both miR-499 overexpression and the PTEN knockdown activated the PI3K/AKT signaling pathway, whereas inhibition of the PI3K/AKT signaling pathway caused immature porcine Sertoli cell apoptosis and inhibited cell proliferation. Overall, miR-499 promotes proliferation and inhibits apoptosis in immature porcine Sertoli cells through the PI3K/AKT pathway by targeting the PTEN gene. This study provides novel insights into the effects of miR-499 in spermatogenesis through the regulation of immature Sertoli cell proliferation and apoptosis.

miR-26a inhibits proliferation and promotes apoptosis in porcine immature Sertoli cells by targeting the PAK2 gene.

Accumulating reports have demonstrated that microRNAs (miRNAs) participate in regulating the complex processes of animal testis development and spermatogenesis; yet, the mechanisms by which miRNAs regulate spermatogenesis are poorly understood. miR-26a was identified as a miRNA that is differentially expressed among different pig testicular tissue developmental stages in our previous study. In this study, p21 activated kinase 2 (PAK2) gene was determined as one target gene of miR-26a by luciferase reporter assay, and miR-26a repressed the PAK2 mRNA abundance in porcine Sertoli cells. The Cell Counting Kit-8 (CCK8) assay, 5-Ethynyl-2'-deoxyuridine (EdU) assay and annexin V-FITC/PI staining assay results showed that miR-26a overexpression inhibited proliferation and promoted apoptosis in porcine Sertoli cells. These phenomena were similar to the siRNA-mediated knockdown of the PAK2 gene. Taken together, our results demonstrate that miR-26a inhibits proliferation and promotes apoptosis in porcine Sertoli cells by targeting the PAK2 gene, which may be a regulator of porcine spermatogenesis.

Genome-wide analysis of long non-coding RNAs and their role in postnatal porcine testis development.

A comprehensive and systematic understanding of the roles of lncRNAs in the postnatal development of the pig testis has still not been achieved. In the present study, we obtained more than one billion clean reads and identified 15,528 lncRNA transcripts; these transcripts included 5032 known and 10,496 novel porcine lncRNA transcripts and corresponded to 10,041 lncRNA genes. Pairwise comparisons identified 449 known and 324 novel lncRNAs that showed differential expression patterns. GO and KEGG pathway enrichment analyses revealed that the targeted genes were involved in metabolic pathways regulating testis development and spermatogenesis, such as the TGF-beta pathway, the PI3K-Akt pathway, the Wnt/ß-catenin pathway, and the AMPK pathway. Using this information, we predicted some lncRNAs and coding gene pairs were predicted that may function in testis development and spermatogenesis; these are listed in detail. This study has provided the most comprehensive catalog to date of lncRNAs in the postnatal pig testis and will aid our understanding of their functional roles in testis development and spermatogenesis.

The role of ubiquitin-proteasome pathway in spermatogenesis.

Ubiquitin-proteasome pathway (UPP) is the main pathway of protein degradation in eukaryotic cells. The UPP plays very important roles in cell cycle progression, apoptosis, stress response and growth and development through regulating protein interaction, protein activity, protein localization and signal transduction. Previous studies have shown that the UPP is essential for regulating acrosome and tail biogenesis during spermatogenesis in human and animals. The dysregulation of UPP during spermatogenesis results in sperm deformity and reduced sperm motility and leads to reproductive system diseases such as oligospermatism, infertility and testicular tumors. In this review, we summarized the signal transduction and regulation mechanism of UPP in spermatogenesis, which may provide references for future studies.

Systematic Identification of Long Noncoding RNAs in Immature and Mature Porcine Testes.

Thousands of long noncoding RNAs (lncRNAs) have been identified in mouse, rat, and human testes, some of which play important roles in testis development and spermatogenesis. However, systematic analysis of lncRNAs expressed in postnatal pig testes has not been reported. Thus, in this study, we present the expression and characterization of lncRNAs in immature (30-day-old [D30]) and mature (180-day-old [D180]) pig testes. A total of 90 440 168 (85.75%) and 97 001 700 (95.35%) 150-base-pair paired-end clean reads were generated in D30 and D180 cDNA libraries, respectively, using the Illumina HiSeq 4000 platform; 36 727 transcripts were assembled in those two libraries, 777 lncRNA transcripts from 752 lncRNA gene loci were identified using the highly stringent pipeline, and 101 of those lncRNA transcripts were significantly differentially expressed. Those lncRNAs shared some characteristics with other mammals, including fewer exons, shorter length and exon length, and lower expression level compared with those of protein-coding genes; 402 protein-coding genes (<10 kb) were found as nearest neighbors of 294 out of 752 lncRNA genes, and gene ontology enrichment showed that they were enriched in transcription- and development-related processes. Fifteen differentially expressed and 10 novel lncRNAs were randomly selected and validated by quantitative polymerase chain reaction (PCR) and reverse transcriptase PCR. In addition, one of the 10 novel lncRNAs was further confirmed using RACE clone technology. This study provides a catalog of porcine testes lncRNAs for further understanding their regulation roles in pig testis development and spermatogenesis.

The complete mitochondrial genome of Jintang black goat (Capra hircus).

Jintang black goat (Capra hircus) is an indigenous breed of Sichuan province of China. It is the first time that the complete mitochondrial genome sequence of Jintang black goat is reported in this work, which is determined through the PCR-based method. The total length of the mitogenome is 16,813 bp, which contains two ribosomal RNA genes, 22 tRNA genes, 13 PCGs, and a control region (D-loop region). The total base composition of Jintang black goat mitochondrial genome is 33.52% A, 13.13% G, 27.35% T, and 26.00% C, and in the order A > T > C > G. The complete mitochondrial genome of Jintang black goat provides an important data in genetic mechanism and the evolution genomes.

The complete mitochondrial genome of Congjiang miniature pig (Sus scrofa).

Congjiang miniature pig is one of the most important local pig breeds in China. It is the first time that the complete mitochondrial genome sequence of Lantang pig is reported in this work, which is determined through the PCR-based method. The total length of the mitogenome is 16,772 bp, which contains 1 control region (D-loop region), 2 ribosomal RNA genes, 13 PCGs and 22 tRNA genes. The total base composition of Congjiang miniature pig mitochondrial genome is 34.78% for A, 26.21% for C, 25.78% for T and 13.22% for G and in the order A>C>T>G. The complete mitochondrial genome of Congjiang miniature pig provides an important data in genetic mechanism and the evolution genomes.

The complete sequence of the mitochondrial genome of Luchuan pig (Sus scrofa).

Luchuan pig is one of the famous native breeds in China. In this study, we report the complete mitochondrial genome sequence of Luchuan pig for the first time, which is determined through the PCR-based method. The total length of the mitogenome is 16,710 bp with the base composition of 34.67% A, 13.33% G, 25.82% T and 26.18% C, and an A + T (60.48%)-rich feature is detected, which contains 1 control region (D-loop region), 2 ribosomal RNA genes, 13 PCGs and 22 tRNA genes. The complete mitochondrial genome of Luchuan pig provides an important data in genetic mechanism and the evolution genomes.

The complete sequence of the mitochondrial genome of Guanling pig (Sus scrofa).

Guanling pig is one of the native breeds in Guizhou Province in China. The compete mitochondrial genome of Guanling pig was determined by polymerase chain reaction (PCR). The result shows that the compete mitochondrial genome of Guanling pig is 16,731 bp, and it contains a major non-coding control region (D-Loop region), 2 ribosomal RNA genes, 13 protein-coding genes (PCGs) and 22 transfer RNA genes. The mitochondrial DNA control region of the Guanling pig contains repeat motif TAC ACG TGC G, 5' nucleotide of the first repeat is at the position 814 bp, and the repeat number is 13. The mitochondrial genome of Guanling pig subsequently provides important information in genetic mechanism and the evolution genomes.

The complete sequence of the mitochondrial genome of Lantang pig (Sus scrofa).

Lantang pig is a native breed of Guangzhou Province in China. It is the first time that the complete mitochondrial genome sequence of Lantang pig is reported in this work, which is determined through the PCR-based method. The total length of the mitognome is 16,709 bp, which contains 2 ribosomal RNA genes, 22 tRNA genes, 13 PCGs and 1 conntrol region (D-loop region, Table 1). The total base composition of Lantang pig mitochondrial genome is 34.69% for A, 26.18% for C, 25.82% for T and 13.31% for G, in the order A>C>T>G. The complete mitochondrial genome of Lantang pig provides an important data in genetic mechanism and the evolution genomes.