Introduction

Genomic imprinting in mammals is an epigenetic gene-marking phenomenon, by which only paternally or maternally inherited genes but not both are expressed, and also is usually described as monoallelic expression. Imprinting has a great impact on normal mammalian development, fetal growth, metabolism and adult behaviors [1]. The human necdin gene (NECD, also called NDN) is maternally imprinted and located in the Prader-Willi syndrome (PWS) chromosomal region [2]; it mediates skeletal muscle regeneration by promoting myoblast survival and differentiation [3] and also plays an important role in regulating cytoskeleton polarization during development [4]. Small nuclear ribonucleoprotein polypeptide N gene (SNRPN) is located within the PWS and Angelman syndrome (AS) region containing multiple imprinted genes that are coordinately regulated by a bipartite imprinting center (IC) [5]. Abnormal imprinting of SNRPN gene was found in day-40 bovine fetuses generated by in vitro culture and somatic cell nuclear transfer (SCNT) [6]. Human SNURF (SNRPN upstream reading frame)-SNRPN transcript extends from SNURF to UBE3A (UBE3A ubiquitin protein ligase E3A) which is antisense to UBE3Aas, spans 460 kb and 148 exons [7, 8] and is exclusively paternally expressed. UBE3A is involved in targeting cellular proteins for degradation, and in human, a defect in the maternal copy of UBE3A produces AS. Human UBE3A is imprinted in the brain, fibroblast, lymphoblast and neural-precursor cells [9, 10] and mice with maternal deficiency for UBE3A resembles human AS with motor dysfunction, inducible seizures, and a context-dependent learning deficit [11].

Most records concerning genomic imprinting were found in human and mouse, less imprinted genes were identified in pigs (http://igc.otago.ac.nz/home.html). Recently, the imprinting of NNAT and DLX5 [12], DIRAS3 [13], MAGEL2 [14], NAP1L5 and PEG3 [15] were reported in pigs. Bischoff et al. [16] characterized the conserved and nonconserved imprinted genes in pigs. However, whether NECD, SNRPN and UBE3A are imprinted in pigs remains unknown. In this study, we first obtained the cDNA sequence of SNRPN and UBE3A genes [NECD sequence is downloaded from the National Center for Biotechnology Information (NCBI)], then analyzed their expression pattern in adult pig tissues and examined their imprinting status in the skeletal muscle of neonate pigs.

Materials and methods

Tissue collection

Tissues from skeletal muscle longissimus dorsi were collected from 10 neonate pigs of Landrace boars × Laiwu sows cross and nine of the reciprocal cross which were reared under the same conditions. The two pig breeds of Landrace and Laiwu (an indigenous pig breed in China) differ in many economic traits such as growth rate, litter size and meat quality, and the pedigree established between crosses of these two breeds are expected to be heterozygous at many sites, therefore are appropriate for imprinting analysis. Tissues from liver, lung, small intestine, skeletal muscle, heart, kidney, spleen, inguinal lymph nodes and fat were gathered from 15 adult Landrace × Laiwu crossbred pigs, which were also reared under the same conditions. All neonate and adult pigs were slaughtered according to the standard of Animal Management of Shandong Agricultural University. After slaughter, the samples were washed briefly with phosphate-buffered saline (PBS) and snap frozen in liquid nitrogen.

Nucleic acid isolation and cDNA synthesis

Genomic DNA was isolated with DNA isolation Kit (TIANGEN, Beijing, PRC) according to the manufacturer’s instructions and stored at −20 °C. Total RNA was isolated from the above mentioned porcine tissues with Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and stored at −80 °C. The quality and concentration of nucleic acids were checked by running electrophoresis with 1 % agarose gel and quantified with BioPhotometer plus UV spectrophotometer (Eppendorf AG, Hamburg, Germany). First-stranded cDNA was synthesized through a two-step procedure in a nuclease free reaction tube: first mix 1 μg of DNaseI(Fermentas, Glen Burnie, USA) treated total RNA, 2 μl of 10 pmol/μl oligo(dT)15 primer and 13 μl nuclease-free H2O, incubate at 65 °C for 10 min and place the tube immediately on ice; then add 0.5 μl of RNase inhibitor (20 U/μl, Fermentas, Glen Burnie, USA), 2 μl of dNTPs (10 mM, TaKaRa, Dalian, PRC), 4 μl of Transcriptor RT reaction buffer and 0.5 μl of Transcriptor Reverse Transcriptase (20 U/μl, Roche, Penzberg, Germany), mix and incubate at 55 °C for 30 min, 85 °C for 5 min. The resultant cDNA was stored at −20 °C.

Reverse transcription (RT)-PCR amplification, cDNA cloning and mRNA expression

The cDNA of porcine SNRPN and UBE3A genes were obtained by RT-PCR, sequenced and assembled with DNAMAN software (Version 5.2.2, Lynnon Biosoft, Canada). First, we used human SNRPN (GenBank: NM_022807) and UBE3A cDNA sequences (GenBank: NM_130838) as seeds to search ‘EST-others’ database with standard BLAST (http://www.ncbi.nlm.nih.gov/blast/) and obtained porcine ESTs. Then, we assembled the corresponding porcine genes with DNAMAN software (Version 5.2.2, Lynnon Biosoft, Canada) that are used for designing primers (Table 1). RT-PCR was performed in 20 μl volume containing 4 μl 5× PrimeSTAR™ Buffer (Mg2+ plus), 1.6 μl of dNTPs (10 mM, TaKaRa, Dalian, PRC), 0.5 μl of each forward and reverse primers (10 μM), 100 ng of cDNA and 0.2 μl of PrimeSTAR™ HS DNA polymerase (2.5 U/μl, TaKaRa, Dalian, PRC). The PCR amplification was performed with normal PCR or Touchdown PCR procedure (marked with an asterisk in Table 1) at the annealing temperature shown in Table 1. The blunt-end RT-PCR amplicons with PrimeSTAR™ HS DNA polymerase were purified and ligated into pJET1.2/blunt cloning vector (Fermentas, Glen Burnie, USA), then transformed into the competent Escherichia coli DH5α cells. For each RT-PCR amplicon, at least four positive clones were sequenced with ABI 3730 sequencer (Applied Biosystems, CA, USA).

Table 1 Primer sequence, annealing temperature and fragment size of NECD, NRPN, UBE3A and GAPDH genes in pigs
Full size table

The mRNA expression of porcine NECD gene was analyzed with primers cND263F and cND263R which were designed according to the sequences of porcine NECD gene (GenBank: NM_001123144) in a 20 μl volume containing 2 μl of 10× PCR buffer (200 mM Tris–HCl pH 8.4, 500 mM KCl), 1.6 μl of dNTPs (10 mM, TaKaRa, Dalian, PRC), 0.6 μl of 50 mM MgCl2, 0.5 μl of each forward and reverse primers (10 μM), 100 ng of cDNA and 0.15 μl of Platinum® Taq DNA polymerase (5 U/μl, Invitrogen, Carlsbad, CA, USA). The thermal profile was as follows: 94 °C 5 min, 94 °C 30 s, 58 °C 30 s, 72 °C 20 s, for 34cycles; and 72 °C 5 min. For mRNA expression analysis of porcine SNRPN and UBE3A genes, primer pairs SNR3F/SNR4R and cUB2F/cUB3R for RT-PCR were designed according to sequences obtained above and performed using the same condition as above except that different annealing temperatures (Table 1) were used. The housekeeping gene GAPDH was used as internal control for mRNA expression analysis with primers and annealing temperature listed in Table 1.

Homology analysis

The cDNA sequences of porcine NECD, SNRPN and UBE3A genes were translated into amino acids sequences with DNAMAN software (Version 5.2.2, Lynnon Biosoft, Canada), then, with the three genes’ amino acid sequences of other species retrieved from the GenBank and Ensembl database, the phylogenetic trees were constructed with MEGA 4.0 software [17]. The neighbor joining method was used in bootstrap, and the replication was 500. The ‘Complete Deletion’ for the gaps/miss data and amino ‘Poisson Correction’ for the substitution model were chosen. The numbers of each branch (Fig. 1) indicates percentage of reality in the process of bootstrap.

Fig. 1
figure 1

Cladogram tree of the NECD (a), SNRPN (b) and UBE3A (c) amino acid sequences of several species retrieved from the GenBank or Ensembl database

Full size image

Single nucleotide polymorphism identification and imprinting analysis

In order to distinguish paternal and maternal alleles of each gene, at least one single nucleotide polymorphism (SNP) located in the transcript is required. The SNPs in the transcript of porcine NECD and SNRPN genes were identified by sequencing and alignment of PCR amplicons from porcine genomic DNA of neonate pigs with primer pairs NDF/NDR and SNR3F/SNR3R, respectively. The SNP in the coding region of UBE3A was identified through alignment of porcine UBE3A cDNA sequences and confirmed by sequencing the fragment amplified from porcine genomic DNA with primer pair cUB2F/cUB3R (Table 1). PCR amplification, sequencing and alignment were performed as above, the primer sequence and annealing temperature used for amplification were shown in Table 1.

Imprinting assay was performed by deciding which allele was expressed in the heterozygous offspring. The genotype at each SNP for genomic and cDNA of porcine NECD, SNRPN and UBE3A genes was determined by PCR/RT-PCR amplification and direct sequenced using the BigDye v3.1 kit (Applied Biosystem). Imprinting assay was performed by comparing the genotype of the heterozygous offspring, its expressed transcript in the skeletal muscle longissimus dorsi of neonate pigs and the genotype of the dam to determine whether the maternally inherited allele is expressed in the offspring. Both pedigrees of Landrace × Laiwu and Laiwu × Landrace were used for determining imprinting status of each gene.

Results

cDNA cloning and homology

The cDNA sequence of porcine SNRPN and UBE3A genes was obtained by RT-PCR from skeletal muscle tissues. The cDNA sequence of 1304 bp including the complete coding region (CDS, 723 bp) of porcine SNRPN gene was obtained, which shared 95.02 % in nucleotide and 99.17 % in amino acid with the cattle SNRPN gene, respectively; for porcine UBE3A gene, the cDNA sequence of 2,740 bp including full-length of CDS (2,628 bp) was obtained, which shared 96.46 % in nucleotide and 98.63 % in amino acid with the cattle UBE3A gene, respectively. The cDNA sequence of porcine SNRPN and UBE3A genes was deposited in GenBank with accession numbers JQ316195 and JQ316194, respectively. Phylogenetic trees (Fig. 1) indicated the NECD, SNRPN and UBE3A genes were highly conserved among mammalian species, such as pig, cattle, mouse, orangutan, monkey and human; while evolutionarily distal from fish, reptiles and amphibians.

mRNA expression in porcine tissues

The mRNA expression in nine porcine tissues of liver, lung, small intestine, skeletal muscle, heart, kidney, spleen, inguinal lymph nodes and fat of adult pigs was analyzed by RT-PCR with GAPDH as housekeeping gene. The amplified fragment size was 221, 593 and 533 bp for porcine NECD, SNRPN and UBE3A genes, respectively (Fig. 2). The results showed that porcine NECD, SNRPN and UBE3A genes were expressed in all of the nine tissues investigated. The expression levels were not different among tissues for NECD and UBE3A genes; however, for SNRPN gene, the expression in liver, skeletal muscle and fat was slightly lower compared with those expressed in other tissues.

Fig. 2
figure 2

The mRNA expression of NECD, SNRPN and UBE3A genes in adult pigs. Porcine GAPDH was used as housekeeping gene

Full size image

SNPs in the transcript

With primer pair NDF/NDR, a fragment of 1983 bp was amplified and sequenced from porcine genomic DNA, alignment of sequences amplified from 19 neonate pigs revealed one SNP named as g.263G>C, existing in the coding region of porcine NECD gene. With primer pair SNR3F/SNR3R, a fragment of 360 bp was amplified and sequenced from porcine genomic DNA, alignment of sequences amplified from 19 neonate pigs revealed that one SNP named as g.402T>C in the coding region of porcine SNRPN gene exists. A SNP (g.340A>G) in the coding region of UBE3A was identified through alignment of porcine UBE3A cDNA sequence and confirmed by PCR from porcine genomic DNA with primer pair cUB2F/cUB3R (Fig. 3). The g.263 G>C and g.402 T>C mutations do not change the encoded amino acids, while the g.340A>G mutation of porcine UBE3A gene results in amino acid substitution of lysine with glutamic acid.

Fig. 3
figure 3

Single nucleotide polymorphism (SNP) detection and imprinting analysis of NECD, SNRPN and UBE3A genes in the skeletal muscle of neonate pigs. The arrows point to SNP sites. a, d Sequence at SNP sites of genomic DNA from the hybrid pigs, showing heterozygosity at the polymorphic sites of the three genes. b, e cDNA sequence at SNP sites of the three genes expressed in the skeletal muscle of the heterozygous pigs. c, f Sequence at SNP sites of genomic DNA from the skeletal muscle of the mother. For both NECD and SNRPN genes, the maternal alleles are not expressed in the heterozygous pigs, so they are maternally imprinted; whereas for the UBE3A gene, both alleles are expressed

Full size image

Imprinting in the skeletal muscle of neonate pigs

Imprinting assay in the skeletal muscle longissimus dorsi of neonate pigs was carried out in both Landrace × Laiwu and Laiwu × Landrace pedigrees. For NECD gene, five Landrace × Laiwu and three Laiwu × Landrace neonate pigs were used for imprinting analysis; for SNRPN gene, three Landrace × Laiwu and seven Laiwu × Landrace neonate pigs; and for UBE3A gene, two Landrace × Laiwu and four Laiwu × Landrace neonate pigs, were used. The results indicated that the maternally inherited allele of both NECD and SNRPN genes was not expressed in the skeletal muscle longissimus dorsi of neonate pigs, suggesting that these two genes were maternally imprinted in skeletal muscle. For UBE3A gene, however, both alleles were detected in the offspring, which suggest that UBE3A was not imprinted in the skeletal muscle of neonate pigs (Fig. 3).

Discussion

More and more studies reveal the importance of genomic imprinting in mammalian development and growth. Although the imprinted genes reported in pigs were growing in recent years, the imprinted genes identified are still very less compared with those in human and mice. Some imprinted genes like IGF2 play important roles in controlling skeletal muscle growth and lean mass in pigs [18], therefore, identifying more imprinted genes will be helpful for explaining the parental-specific-effect in affecting economic traits such as meat mass and meat quality. The NECD, SNRPN and UBE3A genes were located in an imprinted region affecting human PWS and AS, and NECD mediates skeletal muscle regeneration by promoting myoblast survival and differentiation [3]. In this study, the homology, expression and imprinting status of these three genes were investigated in pigs.

Phylogenetic trees constructed based on amino acid sequence indicated that NECD, SNRPN and UBE3A genes were highly conserved in mammals, and the highest homology was found between pigs and cattle, which is consistent with the species evolutionary relationship between them, suggesting that NECD, SNRPN and UBE3A genes were essential for development. With GAPDH as housekeeping gene, we further characterized the mRNA expression of these genes in nine tissues including liver, lung, small intestine, skeletal muscle, heart, kidney, spleen, inguinal lymph nodes and fat of adult pigs, and found that they were expressed in all the tissues investigated (Fig. 2). Previous studies have shown that the SNRPN gene was abundantly expressed in rodent brain and heart and relatively lower in lung, liver, spleen, kidney, skeletal muscle and gonads [19], which is consistent with the results of the present study revealing that, in adult pigs, its expression in liver and skeletal muscle was slightly lower compared with other tissues. A previous study on 180-day-old Wujing pig showed that only in muscle and kidney the NECD was expressed, and was not detected in small intestine, large intestine, liver, muscle, backfat, lung, spleen and kidney [20]. We found that, in adult Landrace × Laiwu crossbred pigs, NECD gene was expressed in all of the nine tissues including liver, lung, small intestine, skeletal muscle, heart, kidney, spleen, inguinal lymph nodes and fat. The difference is likely due to that pigs with different age, breed, or genotypes were used.

Previous studies on the imprinting status of NECD and SNRPN genes that were carried out by quantitative allelic pyrosequencing after comparing biparental (BP) and parthenogenetic (PRT) fetal tissues showed that both genes were predominantly paternally expressed in brain, fibroblast, liver, and placenta of day 30 porcine fetuses [16]. In this study, the polymorphism-based approach was used to detect the imprinting status of NECD, SNRPN and UBE3A genes in reciprocal pedigrees constructed with Landrace and Laiwu pigs. With the three SNPs identified in the coding region, the maternal alleles of NECD and SNRPN were found not to be expressed in the skeletal muscle of neonate pigs. These results indicated that the NECD and SNRPN genes are maternally imprinted in both porcine fetuses and the skeletal muscle of neonate pigs. As for other mammals, the human and mouse NECD genes are maternally imprinted [2], and SNRPN is also maternally imprinted in mouse [21] and human [22], suggesting that the imprinting status of NECD and SNRPN was conserved among mammals. In cattle, the methylation profile of CpG island of SNRPN gene are comparable with mouse and human [23], and artificial reproductive techniques, such as prolonged in vitro culture and SCNT, may lead to abnormal reprogramming of imprinting of SNRPN gene by altering methylation levels at this locus [23, 24]. These results imply that the imprinting status of SNRPN is prone to environmental changes such as nutrition. In human and rodents neurons, the paternal allele of UBE3A is intact but epigenetically silenced, while in most tissues UBE3A is biallelically expressed [25, 26].We also found that UBE3A gene was not imprinted in the skeletal muscle of neonate pigs, consistent with the results obtained in human and rodents.

In conclusion, porcine NECD, SNRPN, and UBE3A genes were highly conserved among mammals and were expressed in most tissues. Both SNRPN and NECD genes are maternally imprinted genes while UBE3A is not imprinted in the skeletal muscle of neonate pigs. The role of these three genes in porcine skeletal muscle development requires further investigations.