Short Communication: Molecular cloning and expression pattern of the porcine 5-aminolevulinate synthase 1 (ALAS1) gene and its association with reproductive traits.

5-Aminolevulinate synthase 1 (ALAS1) is the first enzyme in the heme biosynthetic pathway and is upregulated in follicular tissue during the early stages of ovulation. In this study, we isolated the complete coding sequence of the porcine ALAS1 gene and its 2-9 intron sequence, identified a single nucleotide polymorphism (SNP; T/C) in intron 9, and developed a PCR-MspI-restriction fragment length polymorphism genotyping assay. Association of the SNP with litter size was assessed in two populations [purebred Large White and the experimental synthetic (DIV) line]. Statistical analysis demonstrated that for total number of piglets born (TNB) in all parities, pigs with the CC genotype had an additional 0.68 and 0.74 piglets compared to the TC and TT animals (P < 0.05) in the DIV line, respectively. Purebred Large White sows inheriting the CC and TC genotypes gave birth to an additional 0.96 and 0.70 piglets compared to the TT animals (P < 0.05) in all parities, respectively. In addition, for TNB in all parities, a significant additive effect of 0.48 ± 0.23 and 0.37 ± 0.17 piglets/ litter was detected in sows of both populations (P < 0.05), respectively. The highest levels of ALAS1 gene expression were observed in isolated ovarian granulosa cells 2 and 12 h after stimulation with pregnant mare serum gonadotropin human chorionic gonadotropin, which represents the time of follicular development and ovulation, respectively. Therefore, the ALAS1 gene was significantly associated with litter size in two populations and could be a useful molecular marker for the selection of increasing litter size in pigs.

Serum hepcidin level and its clinical significance in maintenance hemodialysis patients.

Hepcidin is a key protein of iron metabolism, which may play an important role in the prognosis of patients with chronic renal failure on maintenance hemodialysis. The purpose of this study was to investigate the relationship between the prognosis of maintenance hemodialysis patients and serum hepcidin level. We enrolled 60 patients on maintenance hemodialysis and 30 healthy controls from March 2012 to December 2012 in our hospital. Peripheral blood samples were collected to determine hepcidin by an ELISA method. Hepcidin levels of hemodialysis patients were significantly higher than those of the healthy control group. Hepcidin level was positively correlated with the degree of anemia in the dialysis group. Therefore, we conclude that hepcidin level is significantly increased in patients with chronic renal failure on maintenance hemodialysis and that increased hepcidin seriously affects the prognosis of chronic renal failure.

Correlation between hepcidin level and renal anemia.

Anemia in patients with chronic renal insufficiency (CRI) is related to the chronic inflammatory state, low iron absorption rate, and low utilization rate. As a key protein for iron metabolism, hepcidin plays an important role in CRI anemia. The study aimed to determine the correlation between hepcidin level and renal anemia. Ninety CRI anemia patients treated in our hospital from February 2012 to December 2012 were enrolled in the study to compare with a healthy control group of 40 cases by measuring the hepcidin level and analyzing the correlation between hepcidin level and CRI anemia. The hepcidin level was significantly higher in the CRI anemia group than the control group; there was a positive correlation between hepcidin level and serum ferritin as well as IL-6 level. Hepcidin level was significantly related to degree of anemia, indicating that an increase in hepcidin level will result in anemia.

Differential expression and effect of the porcine ANGPTL4 gene on intramuscular fat.

In a previous study, we investigated differences in gene expression in backfat between Meishan and Large White pigs and their F1 hybrids, Large White x Meishan, and Meishan x Large White pigs. One potential differentially expressed sequence tag from the mRNA differential display was a homolog of the human angiopoietin-like 4 (ANGPTL4) gene, which encodes a protein that is secreted by both liver and white adipose tissues and can inhibit lipoprotein lipase activity and stimulate white adipose tissue lipolysis. Here, ANGPTL4 mRNA was found to be upregulated in the backfat of Large White compared with that in the Meishan pigs and the F1 hybrids, Meishan x Large White and Large White x Meishan, whereas expression was lowest both in the longissimus dorsi and the heart, as shown by the tissue distribution profile. Only one mutation, a G/A transition located in the third intron, was found. The ANGPTL4 G/A polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) showed a significant effect on intramuscular fat (IMF), water moisture of the longissimus dorsi, meat marbling of the longissimus dorsi, and pH of the longissimus dorsi (P < 0.05). This site seemed to be significantly (P < 0.05) additive in its actions on IMF, water moisture, and pH, whereas it showed significant dominance in its action on meat marbling (P < 0.05). This locus can be potentially considered as a marker for IMF improvement.

A SNP in the miR-27a gene is associated with litter size in pigs.

MicroRNAs (miRNAs) are 20-25 nt, endogenous non-coding RNA molecules that act by binding to the complementary sequence of target messenger RNAs. Many evidences showed that miRNAs were involved in the process of germ proliferation and differentiation. In the present study, miR-27a gene was selected as a candidate gene for litter size due to its biological function, its location near a mummified pigs QTL, and its differentially expressed profile in Large White and Chinese Erhualian PMSG-hCG stimulated preovulatory ovaries. By comparative sequencing of miR-27a gene in Large White and Chinese Meishan pigs, one SNP (T/C) which created an additional HpaII site was detected. Then associations of this SNP with litter size traits were assessed in Large White (n=142) and DIV (n=140) pig populations. The statistical analysis demonstrated that AA differed from AB (P<0.01) and BB (P<0.05) for total number of piglets born in the first parities, and also differed from AB (P<0.01) for the number of piglets born alive in all parities (P<0.05) in DIV pigs. No significant difference was observed between different genotypes in Large White pigs.

A C/T mutation in microRNA target sites in BMP5 gene is potentially associated with fatness in pigs.

Bone morphogenetic protein 5 (BMP5) gene was suggested to be a potential positional candidate gene for fatness trait QTLs on SSC7. Here by comparative sequencing of BMP5 gene in Meishan (MS) and Large White (LW) pigs, one SNP *131C>T in 3' un-translated region was detected. Association analysis results in 322 LW×MS F(2) pigs showed that CC pigs had thinner back fatness and heavier ham than TC (P<0.05 or P<0.1), and had a higher fat percentage and a lower lean meat percentage (P<0.1) than TT. Moreover, this C/T transition was predicted to alter BMP5 interaction with let-7c and miR-184 by using RNA22 and RNAhybrid. The negative expression of BMP5 gene with let-7c and miR-184 detected from miRNAs overexpression in swine fibroblast, indicating these 2 miRNAs might participate in the translational inhibition of BMP5 gene. Overall, SNP *131C>T might be a good marker for fatness traits.

Molecular characterization, expression pattern and association analysis of the porcine BTG2 gene.

B-cell translocation gene 2 (BTG2), a member of the B-cell translocation gene family with anti-proliferative properties, have been characterized to be involved in cell growth, differentiation and survival. In this study, we cloned the full length sequences of cDNA and genomic DNA of BTG2 gene from the porcine skeletal muscle. Spatial expression analysis showed that the porcine BTG2 gene is expressed predominantly in muscle. Temporal expression analysis in longissimus dorsi muscle demonstrated that the expression of BTG2 gene has the highest expression at 60 days old in Large White while with a peak expression at 120 days old in Meishan. Temporal analysis also revealed that the expression of BTG2 gene is generally higher in Large White than in Meishan at all the developmental stages tested (65 days of conception and 3, 35, 60, 120, and 180 days of postnatal). A single nucleotide polymorphism (G417C) in the intron of BTG2 gene was then detected by PCR-RFLP in Large White × Meishan F2 resource population and association analysis suggested that this polymorphic site had significant association (P < 0.05) with the buttock fat thickness, fat percentage, lean muscle percentage, ratio of lean to fat and carcass length.

Real-time reverse transcription-PCR expression profiling of porcine troponin I family in three different types of muscles during development.

In this study, the expression profiling of three troponin I isoforms (TNNI1, TNNI2 and TNNI3) was investigated in two pig breeds differing in muscularity (Yorkshire and Meishan) at six stages (fetal 60 days and postnatal 3, 35, 60, 120, and 180 days) and three types of muscles (longissimus dorsi muscle, LD; semitendinosus, ST; cardiac muscle, CM) using relative real-time quantitative PCR. Significant differences of troponin I expression in three muscles were found between Yorkshire and Meishan breeds at some stages. The expression peak of TNNI1 and TNNI2 in LD and ST was at postnatal 35 or 60 days in Yorkshire and at postnatal 120 or 180 days in Meishan pigs, while it occurred in CM at postnatal 3 days in two pig breeds. The relative expression values of TNNI1 and TNNI2 were significantly higher in LD than ST at most of stages after birth. The expression ratio of TNNI2 versus TNNI1 favoured TNNI2 expression in ST and LD, but on the contrary in CM. The expression peak of TNNI3 occurred at postnatal 60 and 120 days in Yorkshire and Meishan pigs, respectively. TNNI1 and TNNI3 were co-expressed in CM during the fetal and earlier stages after birth.

Molecular cloning, tissue expression and association of porcine NR4A1 gene with reproductive traits.

Nuclear receptor subfamily 4, group A, member 1 (NR4A1), other aliase NGFI-B, is an immediate-early gene that encodes an orphan nuclear receptor, which play a potential role in the ovulatory process. In this study, a 4,870 bp fragment covered the complete coding region (CDS) and its unique intron sequences of porcine NR4A1 gene was obtained. The reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that NR4A1 was highly expressed in ovary, uterus, kidney, heart but at very low level in oviduct and not expressed in other tissues. Compared the sequence of CDS and its unique intron of Large White and Meishan pigs, a A/G mutation in intron 5 was found and a PCR-Dde1-RFLP genotyping assay was developed. Association of the SNP and litter size was assessed in two populations [purebred Large White and an experimental synthetic Line (DIV) sows]. Statistical analysis demonstrated that, in the first parity, AG animals in experimental synthetic Line (DIV) sows had 1.805 more piglets born compared to the GG animals (P<0.05). For all parities, in the purebred Large White pigs, those with the GG genotype had an additional 0.877 piglets born and 0.780 piglets born alive compared to the AA animals (P<0.05), those with the AG genotype had additional 0.780 piglets born compared to the AA animals (P<0.05). In addition, significant additive effect of 0.438±0.182 piglets/litter and 0.368±0.165 piglets/litter on piglets born and piglets born alive were detected in the purebred Large White lines (P<0.05), respectively. Therefore, NR4A1 gene was significantly associated with litter size in two populations and could be a useful molecular marker in selection for increasing litter size in pigs.

Association of 3 polymorphisms in porcine troponin I genes (TNNI1 and TNNI2) with meat quality traits.

The contractile protein troponin I (TnI), a constituent protein of the troponin complex located on the thin filaments of striated muscle, is involved in inhibition of calcium-induced myosin ATPase activity (and thus contraction). TnI-slow (slow-twitch skeletal muscle isoform, named TNNI1) and TnI-fast (fast-twitch skeletal muscle isoform, named TNNI2) are muscle-fiber-type-specific proteins, and expression of their genes may affect the composition of muscle fiber, thereby influencing the meat quality traits. Thus, the TnI genes are potential candidate genes for traits related to meat quality in animals. Association of 2 SNPs (EU743939:g.5174T>C in intron 4, and EU743939:g.8350C>A in intron 7) of the TNNI1 gene and a SNP (EU696779:g.1167C>T in intron 3) of the TNNI2 gene with 11 meat quality traits were studied on 334 Large White x Meishan F(2) pigs. In the TNNI1 gene, g.5174T>C and g.8350C>A were found to be significantly associated with intramuscular fat content and meat color value of biceps femoris. The g.5174T>C also showed significant effects on meat color value and marbling score of longissimus dorsi, as well as pH of longissimus dorsi and semispinalis capitis. The g.1167C>T polymorphism in the TNNI2 gene affected significantly the pH of longissimus dorsi, meat color value of longissimus dorsi and semispinalis capitis, marbling score of longissimus dorsi, and intramuscular fat.

Identification of three novel SNPs and association with carcass traits in porcine TNNI1 and TNNI2.

In this study, two novel SNPs (EU743939:g.5174T>C in intron 4 and EU743939:g.8350C>A in intron 7) in TNNI1 and one SNP (EU696779:g.1167C>T in intron 3) in TNNI2 were identified by PCR-RFLP (PCR restriction fragment length polymorphism) using XbaI, MspI and SmaI restriction enzyme, respectively. The allele frequencies of three novel SNPs were determined in the genetically diverse pig breeds including ten Chinese indigenous pigs and three Western commercial pig breeds. Association analysis of the SNPs with the carcass traits were conducted in a Large White × Meishan F(2) pig population. The linkage of two SNPs (g.5174T>C and g.8350C>A) in TNNI1 gene had significant effect on fat percentage. Besides these, the g.5174T>C polymorphism was also significantly associated with skin percentage (P < 0.05), shoulder fat thickness (P < 0.05) and backfat thickness between sixth and seventh ribs (P < 0.05). The significant effects of g.1167C>T polymorphism in TNNI2 gene on fat percentage (P < 0.01), lean meat percentage (P < 0.05), lion eye area (P < 0.05), thorax-waist backfat thickness (P < 0.01) and average backfat thickness (P < 0.05) were also found.

Differential proteome analysis of porcine skeletal muscles between Meishan and Large White.

Western and indigenous Chinese pig breeds show obvious differences in muscle growth and meat quality; however, the underlying molecular mechanism remains unclear. In this study, proteome analysis of LM between purebred Meishan and Large White pigs was performed by 2-dimensional gel electrophoresis and mass spectrometry. A total of 25 protein spots were differentially expressed in the 2 breeds. The 14 identified proteins could be divided into 4 groups: energy metabolism, defense and stress, myofibrillar filaments, and other unclassified proteins. Quantitative real-time PCR was used to analyze the partly differentially expressed proteins in mRNA level, which revealed a positive correlation between the content of the proteins and their mRNA levels. We also analyzed the mRNA levels of myosin heavy chain isoforms using quantitative real-time PCR. The results indicated that IIa and IIx fibers were elevated in Meishan pigs, whereas the IIb fiber was more highly expressed in Large White pigs. To the best of our knowledge, this was the first proteomics-based investigation of total skeletal muscle protein in different pig breeds, and these results may provide valuable information for understanding the molecular mechanism responsible for breed-specific differences in growth performance and meat quality.

Genetic polymorphisms and preliminary association analysis with production traits of the porcine SLC27A4 gene.

Solute carrier family 27 (fatty acid transporter), member 4 (SLC27A4) is a fatty acyl-CoA synthetase producing very long chain fatty acid-CoA for lipid metabolic pathways, suggesting that the SLC27A4 gene is a potential candidate gene for traits related to fat deposition in animals. This study was conducted to sequence the genomic region from exon 6 to 12 of porcine SLC27A4 and detect polymorphisms by comparative sequencing. In silico mapping assigned SLC27A4 gene between gene COQ4 (coenzyme Q4 homolog) and URM1 (ubiquitin related modifier 1 homolog) on pig chromosome 1q24-q2.12 where significant QTL affecting backfat depth had previously been identified. Thirty six putative sites of variation were detected, of which 31 polymorphisms including 28 SNPs and 3 indels were located in the intronic region, and 5 in the exonic regions. The g.1777G>A (EU703769) in intron 8 was confirmed by PCR-RFLP using HpaII restriction enzyme and further genotyped in four Chinese native pig breeds (Meishan, Erhualian, Tongcheng and Qingping) and three western meat-type pig breeds (Duroc, Large White and Landrace). Allele G was exclusively present in Tongcheng and Qingping pigs and predominant in the other pig populations analyzed. Significant differences of backfat at rump, body weight at birth and average daily gain on weaning between the AG and GG genotype were observed in Landrace pig population (P < 0.05).

Polymorphism of the pig 17beta-hydroxysteroid dehydrogenase type1 (HSD17B1) gene and its association with reproductive traits.

17beta-Hydroxysteroid dehydrogenase type 1 (HSD17B1) is a key enzyme of 17beta-estradiol biosynthesis, which might play an important role in follicular development of the ovary. In this study, we isolated the complete coding sequence of porcine HSD17B1 gene and its unique intron sequences of porcine HSD17B1 gene, identified a single nucleotide polymorphism (SNP: A/C) in intron 4, and developed a PCR-MvaI-RFLP genotyping assay. Association of the SNP and litter size was assessed in two populations (purebred Large White and a experimental synthetic Line (DIV) sows). Statistical analysis demonstrated that, in the first parity, AC animals in experimental synthetic Line (DIV) sows had 0.52 more piglets born compared to the CC animals (P<0.05). In the all parities, pigs with the AA genotype had an additional 1.11 and 0.96 piglets born alive compared to the CC animals (P<0.05) in both experimental synthetic Line (DIV) and purebred Large White, respectively. Experimental synthetic Line (DIV) sows inheriting the AC genotype had additional 0.84 piglets born alive compared to the CC animals (P<0.01) in all parities. In addition, significant additive effect of -0.55+/-0.24 piglets/litter and -0.48+/-0.22 piglets/litter on piglet born alive was detected in both experimental synthetic Line (DIV) sows and purebred Large White lines (P<0.05), respectively. Therefore, HSD17B1 gene was significantly associated with litter size in two populations and could be a useful molecular marker in selection for increasing litter size in pigs.

Identification of polymorphism and association analysis with reproductive traits in the porcine RNF4 gene.

The ring finger protein 4 gene (RNF4), which might play a role in fetal germ cell development as well as in oocyte and granulosa cell maturation, was one of the potential candidate genes for reproductive traits. In the present work, we isolated the complete coding sequence of porcine RNF4 gene, identified a single nucleotide polymorphism (SNP: T/C) in intron5, and developed a PCR-SacII-RFLP genotyping assay. Association of this SNP with reproductive traits was assessed in three populations with diverse genetic backgrounds. One was Chinese Qingping sows. Another was consisted of crossbred sows derived from Landrace, Large White, Chinese Tongcheng and/or Chinese Meishan (Line DIV). The third is Large White x Meishan (LW x M) F(2) slaughtered population. Statistical analysis demonstrated that, in the first parity, the difference between RNF4 genotypes and reproductive traits of both Qingping and Line DIV sows was not significant. In the second and subsequent litters, CC animals in Qingping population had more piglets born (+1.74 piglets) and piglets born alive (+2.02 piglets) than sows with the TT genotype (P<0.05). Line DIV sows inheriting the CC genotype had additional 0.69 piglets born compared to the TC animals (P<0.05) in second and subsequent litters. No significant difference was observed between genotypes and reproductive tracts components in F(2) animals. In addition, we found RNF4 gene has a significant additive effect on both piglet born and piglet born alive in Qingping animals (P<0.05). Results here suggested that the RNF4 SNP was significantly associated with litter size in two populations and could be useful in selection for increasing litter size in pigs. Further studies were needed to confirm these preliminary researches.

Molecular cloning and polymorphism of the porcine H2AFZ gene.

H2A histone family, member Z (H2A.Z) is required for early mammalian development. In the present study, the 932 bp of full-length cDNA encoding a 128 amino-acid protein and the sequences of intron 2 to 4 of the porcine H2A histone family, member Z (pH2AFZ) gene were obtained. By comparative sequencing of pH2AFZ gene in Large White and Meishan pigs, a 4 bp deletion/insertion in intron 2 was detected and a PCR-Bsu15I-RFLP was established to detect this variation. In DIV (4th Dam line of Chinese lean-type new lines) pigs, the first-parity females with AA genotype had fewer piglets born alive (-2.64 and -1.83 piglets per litter) than those with AB (P < 0.01) and BB (P < 0.05) genotype. The additive allelic and dominance effect were estimated to be 0.92 (P < 0.05) and -0.87 piglets per litter (P < 0.01) for number of piglets born alive, respectively. This result suggests that the pH2AFZ gene might be a good candidate gene of litter-size trait and provides some marker information for marker-assisted selection.

Molecular characterization and SNPs analysis of the porcine Deleted in AZoospermia Like (pDAZL) gene.

The Deleted in AZoospermia Like (DAZL) gene is expressed in prenatal and postnatal germ cells. In this study, we cloned and characterized the porcine Deleted in AZoospermia Like (pDAZL) gene. We found the full-length coding sequence of the pDAZL encoded a protein of 295 amino acids with a RNA recognition motif (amino acids 41-111) and a DAZ repeat (amino acids 167-120). The deduced protein sequence of pDAZL is 92.5% and 91.5% similar to those of human and bovine, respectively. PCR-MspI-RFLP and PCR-TaqI-RFLP were established to detect an A/G mutation in intron 7 and a C/A mutation in intron 9, respectively. Associations of two SNPs with litter size traits were assessed in Large White (n=275) and DIV (n=128) pig populations, and the statistical analysis demonstrated that CC produced 0.716 more (P<0.05) piglets born alive than CD genotypes in Large White pigs at TaqI locus (C/A mutation in intron 9), and the dominance effect was 0.304 pig per litter (P<0.05). This result suggests that the pDAZL gene might be a good candidate gene of litter size trait and provides some marker information for marker-assisted selection (MAS).

Isolation and imprinting analysis of the porcine DLX5 gene and its association with carcass traits.

Imprinted genes play important roles in mammalian growth and development. However, reports on imprinted genes are limited in livestock. In this study, the complete ORF containing 289 amino acids of the porcine DLX5 gene was obtained. A C-to-T SNP mutation in exon 1 of the DLX5 gene was used to detect imprinting status with an RT-PCR/RFLP test (using HhaI) in eight heterozygous pigs from a population of Large White x Meishan F(1) hybrids. Imprinting analysis showed that the porcine DLX5 gene was maternally expressed in skeletal muscle, fat, lung, spleen, stomach and small intestine, but not imprinted in heart, liver, kidney, uterus, ovary, testicle or pituitary. A PCR-RFLP test was also used to detect the polymorphism in 310 pigs of a Large White x Meishan F(2) resource population. The statistical results showed significant association (P < 0.01) of the genotypes and fat meat percentage, carcass length, bone percentage, 6-7 rib fat thickness, average backfat thickness, thorax-waist fat thickness and buttock fat thickness.

Association of the polymorphism in GYS1 and ACOX1 genes with meat quality traits in pigs.

Phenotypic information about several pig meat quality traits on 334 Large White × Meishan F2 pigs was collected. Effects of the association of the FokI variants in the seventh intron of the skeletal muscle glycogen synthase (GYS1) gene and the PstI variants in the ninth intron of the palmitoyl acyl-CoA oxidase 1 (ACOX1) gene on the meat quality traits were examined on all pigs. The FokI variants of the GYS1 gene showed significant effects on pH of m. semipinalis capitis (P < 0.05). Linkage analysis indicated that the peak of the quantitative trait loci (QTL) curve was located around this marker for pH, but it did not reach significance (P > 0.05). The results may be due to several reasons such as linkage disequilibrium to the causal mutations, the limited number of animals or balance of another QTL or marker with negative effects. Significant effects of PstI variants of ACOX1 gene were also found on meat colour value and meat marbling score of both m. longissimus dorsi and m. biceps femoris (P < 0.05). Dominant effects for the affected traits at those two loci were significant except for meat marbling score of m. biceps femoris (P < 0.05). The results of this study give us some evidence for the potential of those dominant markers used in the marker-assisted selection of crossbreeding of the Large White pig sire lines and Meishan-derived synthetic dam lines.

Imprinted status of pleomorphic adenoma gene-like I and paternal expression gene 10 genes in pigs.

Genomic imprinting is theorized to exist in all placental mammals and some marsupials. Imprinted genes play important roles in the regulation of fetal growth, development, and postnatal behavior, but the study of imprinted genes has been limited in livestock. In this study, the polymorphism-based approach was used to detect the expression patterns of the porcine pleomorphic adenoma gene-like I (PLAGL1) and paternal expression gene 10 (PEG10) genes. Single nucleotide polymorphisms in the exons were detected between the Meishan and Large White breeds in the PLAGL1 and PEG10 genes. The polymorphisms were used to determine the monoallelic or biallelic expression with reverse transcription-PCR-RFLP in 44 tissues from 4 heterozygous pigs (based on SNP). Imprinting analysis indicated that the PLAGL1 and PEG10 genes were both paternally expressed in all tissues tested (heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, fat, uterus, and ovary). Our study showed that the method of identifying polymorphic transcripts with reverse transcription-PCR-RFLP may be beneficial for detecting the imprinting status of some candidate imprinted genes.