Meiying Fang

Review on the development of genotyping methods for assessing farm animal diversity

Advances in molecular biotechnology have introduced new generations of molecular markers for use in the genetic improvement of farm animals. Consequently, more accurate genetic information can be obtained to better understand existing animal genetic resources. This review gives a brief summary on the development of genetic markers including both the classical genetic markers and more advanced DNA-based molecular markers. This review will help us better understand the characteristics of different genetic markers and the genetic diversity of animal genetic resources.

Transcriptome Profile at Different Physiological Stages Reveals Potential Mode for Curly Fleece in Chinese Tan Sheep

Tan sheep (Ovis aries), a Chinese indigenous breed, has special curly fleece after birth, especially at one month old. However, this unique phenotype disappears gradually with age and the underlying reasons of trait evolvement are still unknown. In this study, skin transcriptome data was used to study this issue. In total 51,215 transcripts including described transcripts and transfrags were identified. Pathway analysis of the top 100 most highly expressed transcripts, which included TCHH and keratin gene family members, such as KRT25, KRT5, KRT71, KRT14 and others, showed pathways known to be relevant to hair/fleece development and function. Six hundred differentially expressed (DE) transcripts were detected at two different physiological ages (one-month-old with curly fleece and 48-month-old without curly fleece) and were categorized into three major functional groups: cellular component, molecular function, and biological process. The top six functional categories included cell, cell part, cellular process, binding, intracellular, metabolic process. The detected differentially expressed genes were particularly involved in signal, signal peptide, disulfide bond, glycoprotein and secreted terms, respectively. Further splicing isoform analysis showed that the metallothionein 3 isoform was up-regulated in Tan lamb skin, indicating that it may be related to the conformation of curly fleece in Chinese Tan lamb. The hair-related important differentially expressed genes (SPINK4, FGF21, ESRα, EphA3, NTNG1 and GPR110) were confirmed by qPCR analysis. We deduced that the differences existed in expressed transcripts, splice isoforms and GO categories between the two different physiological stages, which might constitute the major reasons for explaining the trait evolvement of curly fleece in Chinese Tan sheep. This study provides some clues for elucidating the molecular mechanism of fleece change with age in Chinese Tan sheep, as well as supplying some potential values for understanding human hair disorder and texture changes.

Genome Wide Association Analysis Reveals New Production Trait Genes in a Male Duroc Population.

In this study, 796 male Duroc pigs were used to identify genomic regions controlling growth traits. Three production traits were studied: food conversion ratio, days to 100 KG, and average daily gain, using a panel of 39,436 single nucleotide polymorphisms. In total, we detected 11 genome-wide and 162 chromosome-wide single nucleotide polymorphism trait associations. The Gene ontology analysis identified 14 candidate genes close to significant single nucleotide polymorphisms, with growth-related functions: six for days to 100 KG (WT1, FBXO3, DOCK7, PPP3CA, AGPAT9, and NKX6-1), seven for food conversion ratio (MAP2, TBX15, IVL, ARL15, CPS1, VWC2L, and VAV3), and one for average daily gain (COL27A1). Gene ontology analysis indicated that most of the candidate genes are involved in muscle, fat, bone or nervous system development, nutrient absorption, and metabolism, which are all either directly or indirectly related to growth traits in pigs. Additionally, we found four haplotype blocks composed of suggestive single nucleotide polymorphisms located in the growth trait-related quantitative trait loci and further narrowed down the ranges, the largest of which decreased by ~60 Mb. Hence, our results could be used to improve pig production traits by increasing the frequency of favorable alleles via artificial selection.

A comprehensive microRNA expression profile of the backfat tissue from castrated and intact full-sib pair male pigs

BackgroundIt is widely known that castration has a significant effect on the accumulation of adipose tissue. microRNAs (miRNAs) are known to be involved in fat deposition and to be regulated by the androgen-induced androgen receptor (AR). However, there is little understanding of the relationship between miRNAs and fat deposition after castration. In this study, the high-throughput SOLiD sequencing approach was used to identify and characterize miRNA expression in backfat from intact and castrated full-sib male 23-week-old pigs. The patterns of adipogenesis and fat deposition were compared between castrated and intact male pigs.ResultsA total of 366 unique miRNA genes were identified, comprising 174 known pre-miRNAs and 192 novel pre-miRNAs. One hundred and sixty-seven pre-miRNAs were common to both castrated (F3) and intact (F4) male pig small RNA libraries. The novel pre-miRNAs encoded 153 miRNAs/miRNA*s and 141 miRNAs/miRNA*s in the F3 and F4 libraries, respectively. One hundred and seventy-seven miRNAs, including 45 up- and 132 down-regulated, had more than 2-fold differential expression between the castrated and intact male pigs (p-value < 0.001). Thirty-five miRNAs were further selected, based on the expression abundance and differentiation between the two libraries, to predict their targets in KEGG pathways. KEGG pathway analyses suggested that miRNAs differentially expressed between the castrated and intact male pigs are involved in proliferation, apoptosis, differentiation, migration, adipose tissue development and other important biological processes. The expression patterns of eight arbitrarily selected miRNAs were validated by stem-loop reverse-transcription quantitative polymerase chain reaction. These data confirmed the expression tendency observed with SOLiD sequencing. miRNA isomiRs and mirtrons were also investigated in this study. Mirtrons are a recently described category of miRNA relying on splicing rather than processing by the microprocessor complex to generate the RNAi pathway. The functions of miRNAs important for regulating fat deposition were also investigated in this study.ConclusionsThis study expands the number of fat-deposition-related miRNAs in pig. The results also indicate that castration can significantly affect the expression patterns of fat-related miRNAs. The differentially expressed miRNAs may play important roles in fat deposition after castration.

Investigation of Testosterone, Androstenone, and Estradiol Metabolism in HepG2 Cells and Primary Culture Pig Hepatocytes and Their Effects on 17βHSD7 Gene Expression

Steroid metabolism is important in various species. The accumulation of androgen metabolite, androstenone, in pig adipose tissue is negatively associated with pork flavor, odour and makes the meat unfit for human consumption. The 17β-hydroxysteroid dehydrogenase type 7 (17βHSD7) expressed abundantly in porcine liver, and it was previously suggested to be associated with androstenone levels. Understanding the enzymes and metabolic pathways responsible for androstenone as well as other steroids metabolism is important for improving the meat quality. At the same time, metabolism of steroids is known to be species- and tissue-specific. Therefore it is important to investigate between-species variations in the hepatic steroid metabolism and to elucidate the role of 17βHSD7 in this process. Here we used an effective methodological approach, liquid chromatography coupled with mass spectrometry, to investigate species-specific metabolism of androstenone, testosterone and beta-estradiol in HepG2 cell line, and pig cultured hepatocytes. Species- and concentration-depended effect of steroids on 17βHSD7 gene expression was also investigated. It was demonstrated that the investigated steroids can regulate the 17βHSD7 gene expression in HepG2 and primary cultured porcine hepatocytes in a concentration-dependent and species-dependent pattern. Investigation of steroid metabolites demonstrated that androstenone formed a 3′-hydroxy compound 3β-hydroxy-5α-androst-16-ene. Testosterone was metabolized to 4-androstene-3,17-dione. Estrone was found as the metabolite for β-estradiol. Inhibition study with 17βHSD inhibitor apigenin showed that apigenin didn’t affect androstenone metabolism. Apigenin at high concentration (50 µM) tends to inhibit testosterone metabolism but this inhibition effect was negligible. Beta-estradiol metabolism was notably inhibited with apigenin at high concentration. The study also established that the level of testosterone and β-estradiol metabolites was markedly increased after co-incubation with high concentration of apigenin. This study established that 17βHSD7 is not the key enzyme responsible for androstenone and testosterone metabolism in porcine liver cells.

Splice variant identification and expression analysis of the fat mass and obesity-associated (FTO) gene in intact and castrated male pigs.

The fat mass and obesity-associated gene (FTO) has previously been associated with human body mass index and porcine intramuscular fat content. In this study, the potential alternative splicing pattern of the FTO gene was investigated, and three novel splice variants were identified in Large White and indigenous Chinese Tibetan pigs. To explore the molecular effect of the FTO gene on the fat deposition caused by castration, 40 paired full-sibling male pigs were divided into intact and castrated groups, and the trait data showed that abdominal fat weight, percentage of abdominal fat weight, and backfat thickness in the intact group were significantly lower than in the castrated group (p < 0.05). FTO mRNA expression in the hypothalamus was significantly increased in the castrated group compared with the intact group (p < 0.05), but was not different in the abdominal fat or backfat tissues. Moreover, the relative expression of the FTO gene was shown to have a higher level in the hypothalamus when compared with expression in the fat tissues (p < 0.05). Our results suggest that there are different patterns of alternative splicing and FTO expression among pig breeds. This study will provide clues for obtaining a better understanding of the porcine FTO gene function.

Differential expression of CYB5A in Chinese and European pig breeds due to genetic variations in the promoter region

Cytochrome b5 (CYB5A) is an important electron transfer protein with homologues in a number of different organisms. In pigs, CYB5A is related to boar taint because of its role in androstenone biosynthesis. To determine the variety of CYB5A expression in pig breeds, genetic variations in the porcine CYB5A promoter region in both Chinese and European pig breeds were examined. Three single nucleotide polymorphisms (NC_010443.4:g.165901487delG, g.165901767T>C and g.165902078C>T) were identified in the porcine CYB5A promoter region. These SNPs occurred in different frequencies in Chinese and European pigs. Chinese pigs were primarily haplotype B (denoted as delG-C-T: the position of nt 165901487 of the CYB5 gene is a G deletion, nt 165901767 is C and nt 165902078 is T), except for Licha black pigs, which were primarily haplotype A (denoted as G-T-C: nt 165901487 is G, nt 165901767 is T and nt 165902078 is C), similar to European pigs. Quantitative PCR data from liver tissues demonstrated that haplotype B individuals had higher CYB5A expression than did those with haplotype A. This was confirmed by in vitro cell transfection assays, in which haplotype B individuals had higher reporter activity than did those with haplotype A. In silico analysis predicted that Myc-associated zinc-finger protein (MAZ) is a potential transcription factor at position 165901767. Electrophoretic mobility shift assays showed this polymorphism affects the stable binding of transcription factors to the CYB5A promoter, which in turn affects the expression levels of this gene. Therefore, this variation of the porcine CYB5A promoter region may explain the differences in androstenone accumulation between Chinese and European pig breeds and may also prove useful as a genetic marker to distinguish the origin of different pig breeds.

Metabolism of Androstenone, 17β-Estradiol and Dihydrotestosterone in Primary Cultured Pig Hepatocytes and the Role of 3β-Hydroxysteroid Dehydrogenase in This Process

Steroids metabolism plays an important role in mammals and contributes to quality of pig meat. The main objective of this study was to identify metabolites of androstenone, 17β-estradiol and dihydrotestosterone using primary cultured pig hepatocytes as a model system. The role of 3β-hydroxysteroid dehydrogenase (3βHSD) in regulation of steroid metabolism was also validated using trilostane, a specific 3βHSD inhibitor. Steroid glucuronide conjugated metabolites were detected by liquid chromatography time of flight mass spectrometry (LC-TOF-MS). 3βHSD enzyme was essential for metabolism of androstenone to 5α-androst-16-en-3β-ol, which then formed androstenone glucuronide conjugate. Metabolism of 17β-estradiol was accompanied by formation of glucuronide-conjugated estrone and glucuronide-conjugated estradiol. The ratio of the two metabolites was around 5∶1. 3βHSD enzyme was not involved in 17β-estradiol metabolism. 5α-Dihydrotestosterone-17β-glucuronide was identified as a dihydrotestosterone metabolite, and this metabolism was related to 3βHSD enzyme activity as demonstrated by inhibition study.

Genetic variations and sequences analysis of MTATP6 and MTATP8 genes among different Chinese pig breeds

To investigate the genetic variations of mtATP6 and mtATP8 genes among different Chinese pig breeds, two fragments of 425 and 743 bp containing the whole coding region of mtATP8 and mtATP6 genes were amplified with 805 individuals from 23 Chinese local pig breeds, three types of Chinese wild boars and three European pig breeds. Sequence comparison identified a total number of 17 substitutions including six variable sites in mtATP8 and eleven substitutions in mtATP6 gene. The restriction enzyme Fok I revealed four polymorphic sites (nt8086, 8176, 8514 and 7784), and four RFLP haplotype patterns (A, B, C and D) were identified in mtATP6 and mtATP8 genes among all tested samples. Our data showed AC combined haplotype originated from Asia and BD was regarded as European origin. The average frequency of Asian mtDNA haplotypes was 38.3% across the investigated European breeds but varied within each breeds (13.3∼76.7%). Phylogenetic analyses were performed also considering some published sequences in the databases; the sequences were divided into three distinct groups, denoted A, E1, and E2. The Asian AC haplotype existed among the European domestic pigs was fully consistent with the results of previous molecular studies and well-documented history. This study will help us to better understand the genetic variations of mitochondrial genes among different Chinese pig breeds.

ssc-miR-7134-3p regulates fat accumulation in castrated male pigs by targeting MARK4 gene

Castration of male pigs is a common practice used to reduce boar taint in commercial pork production, but it also significantly results in fat accumulation in carcass. Our previous study revealed a miRNA gene, ssc-miR-7134-3p that was implicated in adipogenesis. However, the relationship between ssc-miR-7134-3p and fat deposition due to castration is unknown. In the present study, we observed that ssc-miR-7134-3p targets the coding sequence (CDS) region of MARK4 based on bioinformatics analysis and dual-luciferase assays. Experiments using silent mutations and sub-cloning showed that ssc-miR-7134-3p binds independently to two adjacent sites in the MARK4 CDS. Subsequently, ssc-miR-7134-3p inhibits MARK4 protein expression in pig fibroblast cells, being consistent with the targeting demonstrated in vitro. We found higher MARK4 protein levels in the back fat of castrated pigs than in intact pigs, providing further evidence that MARK4 is involved in regulation of fat deposition. In addition, one SNP (g.2581A>G) in MARK4 was significantly associated with the back fat trait in Chinese and European pig populations. Taken together, we would conclude that ssc-miR-7134-3p targets the MARK4 gene for fat accumulation in the castrated male pigs.