Molecular characterization, expression and association analysis of the porcine CMYA4 gene with carcass traits

Breeds used for testing the allele frequencies of the porcine CMYA4 gene

The allele frequency analysis included 225 unrelated animals from six breeds (Table 2): Qingping pigs (n = 27), Tongcheng pigs (a typical indigenous Chinese breed; n = 42), Yushan Black pigs (n = 50), Dahuabai pigs (n = 41), Erhualian pigs (a kind of Taihu pig; n = 39) and Duroc pigs (n = 26). A chi-squared test on the allele frequencies for the seven pig breeds was performed using SAS V8.0.

Tissue sample collection, cDNA preparation and SMART cDNA synthesis

Seven tissue samples for expression profile analysis collected from a mature Tongcheng (a typical indigenous Chinese breed) pig’s heart, liver, spleen, lung, kidney, longissimus dorsi muscle and fat tissues were harvested, frozen in liquid nitrogen and then stored at −80°C. The total RNA was extracted from 200 mg of each specimen with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to standard protocols, and the reverse transcription was performed based on the method described previously by Pan et al. (2003). SMART cDNA were synthesized from a mature Tongcheng pig’s skeletal muscle total RNA by using the SMART PCR cDNA Synthesis Kit (Clontech Laboratories, Palo Alto, CA, USA) for rapid amplification of cDNA ends (RACE)-polymerase chain reaction (PCR).

Isolation and cDNA sequence analysis of the porcine CMYA4 gene

The human CMYA4 gene sequence (GenBank accession no. is NM_173167.2) was applied to compare with all sequences in the expressed sequence tags (EST)-others database using standard BLAST (http://www.ncbi.nlm.nih/gov/blast/), and the porcine EST which shared at least 80% identity to the corresponding human mRNA were selected to design gene-specific primers (Table 1). The porcine CMYA4 gene mRNA sequence was obtained by reverse transcription-PCR (RT-PCR) and the RACE method (Hirano et al. 1990). RACE was performed according to the instructions of the SMART^TM RACE cDNA Amplification Kit (Clontech Inc.). The PCR profile was 5 min at 95°C, followed by 34 cycles of 40 s at 94°C, 30 s at annealing temperature (Table 1), 1 min at 72°C and a final extension of 5 min at 72°C.

The PCR products of RACE and RT-PCR were purified with Wizard PCR Preps DNA Purification System (Promega, Madison, WI, USA) and cloned into the pMD-18T vector (Promega), and sequenced commercially. The ORF (open reading frame) and the amino acid sequences were deduced with the program Seqman (DNAstar, Madison, WI, USA). The secondary structure was performed by online tools of the ExPASy website (http://cn.expasy.org/tools/).

Spatial expression analysis of the porcine CMYA4 gene

The porcine CMYA4 gene mRNA expression in different tissues was detected by semi-quantitative RT-PCR with the β-actin gene as positive control. CMYA4 gene-specific primers (PE-F and PE-R in Table 1) and β-actin-specific primers (β-actin-F and β-actin-R in Table 1) amplified a product of 248 bp and 233 bp, respectively. PCR amplification was done in a total volume of 20 μl with 50 ng of cDNA, 1× PCR buffer, 0.3 μm of each primer, 75 μm of each dNTP, 1.5 mm of MgCl₂ and 1 U Taq DNA polymerase (Yeli Bioscience Co. Ltd, Shanghai, China) plus a negative control (non-reverse-transcripted RNA). The PCR began with a 95°C incubation temperature for 5 min followed by 27 cycles in total which were performed at a 62°C annealing temperature. The PCR ended with a 5-min extension at 72°C.

Genomic DNA amplification and SNP identification

One pair of primers (PS-F and PS-R in Table 1) on exons 15 and 16 according to the porcine mRNA sequence and aligned with the human DNA sequence (GenBank accession no. is NC_ 000017.9) were designed to amplify the intron 15 and scan the single nucleotide polymorphism (SNP) using the genomic DNA pool from Tongcheng, Erhualian and Duroc pigs. The PCR reactions were performed in a volume of 20 μl containing 50 ng of DNA, 1× PCR buffer, 0.3 μm of each primer, 75 μm of each dNTP, 1.5 mm of MgCl₂ and 1 U of Taq DNA polymerase (Yeli, Bioscience Co. Ltd, Shanghai, China). The PCR reaction conditions were 5 min at 95°C followed by 34 cycles of 40 s at 94°C, 30 s at 66°C, 40 s at 72°C and a final extension of 5 min at 72°C. The PCR products were pooled and sequenced. The polymorphism site was analysed by sequence comparisons using the DNAstar software (DNAstar Inc.) and was further identified by the PCR–restriction fragment length polymorphism (PCR–RFLP) method.

Association analysis of the porcine CMYA4 gene with economic traits

The animals used in the association analysis of CMYA4 gene were 205 pigs, including Yorkshire (26), Landrace (26), Tongcheng (49), Landrace × (Yorkshire × Tongcheng) (54) and Yorkshire × (Landrace × Tongcheng) (50). Nineteen traits were recorded, including average daily gain (ADG) from birth to 90 kg, dressing percentage (DP), percentage of leaf fat (PLF), percentage of leaf and caul fat (PLC), backfat thickness at the shoulder (BTS), last rib backfat thickness (LRB), average backfat thickness (ABT), backfat thickness between sixth and seventh ribs (BTR), longissimus dorsi height (LDH), longissimus dorsi area (LDA), longissimus dorsi width (LDW), percentage of ham in the carcass (PHC), proportion of lean + bone of the ham (PLBH), loin pH, muscle colour score, loin marbling (LM), water loss, loin muscle drip loss (LDL) and intramuscular fat (IMF).

An analysis method using the PROC GLM procedure in SAS software package (SAS Inst. Inc., Cary, NC, USA) was employed to analyse the association between genotypes and traits. The linear model with the fixed effects is:

where Y_ijklmn is the ijklmnth traits’ observation value; μ is the mean; G_i is the effect of the ith genotypes; B_j is the effect of jth batch; S_k is the effect of jth sex; C_l is the effect of lth population; F_m(C) is family effects within breed and ε_ijklmn is the random residual corresponding to the traits observation value with .

Isolation and sequence analysis of the porcine CMYA4 gene

The human mRNA (GenBank accession no. is NM_173167) was used to screen the porcine EST in the EST-others database through BLAST algorithm (http://www.ncbi.nlm.nih.gov/blast). Porcine EST which share more than 80% identity to the corresponding human mRNA were employed to design primers. The RT-PCR and 5′ RACE procedures were applied to obtain the cDNA sequence including the full-length coding sequence of 2790 nucleotides and 97 nucleotides of 5′ untranslated region (UTR) of the porcine CMYA4 gene (GenBank accession no. is DQ_286571). The human (NM_173167) and mouse (NM_178680) homologues () have a 91% and 87% identity with the porcine CMYA4 gene, respectively. The porcine CMYA4 gene encodes 930 amino acids and the amino acid sequences show 91% identity to human UNC-45 protein. In addition, the predicted secondary structures of pig and human CMYA4 were similar to each other. The analysis of CMYA4 amino acid sequences indicated that the protein contains three amino-terminal tetratricopeptide repeats (TPR) and a COOH-terminal UCS (UNC-45/CRO1/ SHE4) domain. The TPR motif was originally identified as a protein interaction module in cell division cycle (CDC) proteins in yeast (Hirano et al. 1990; Sikorski et al. 1990). TPR-containing proteins are involved in a diverse spectrum of cellular functions with the majority of them participating in cell cycle control (Malek et al. 1996), transcription (Moir et al. 1997) and splicing events (Urushiyama et al. 1997), protein transport with special regard to protein import (Gindhart & Goldstein 1996) and regulatory phosphate turnover (Becker et al. 1994). The COOH-terminal UCS domain is required for correct folding and assembly of conventional class II myosin (Barral et al. 2002) and it is not class II myosin-specific factors but general factors that are involved in myosin assembly and localization (Wesche et al. 2003).

Spatial expression analysis of the porcine CMYA4 gene

The RT-PCR was used to detect the tissue distribution of the porcine CMYA4 gene. Control PCR (the negative control of non-reverse-transcripted RNA material) gave no signal (Figure 1), excluding genomic DNA contamination of the RNA preparations and, thus, confirming the RT-PCR results of expression profile analysis of the porcine CMYA4 gene. Positive control PCR (β-actin) in each tissue displayed a basically identical signal. The gene was only expressed in heart and skeletal muscle tissues (Figure 1). It is reported that there are two different isoforms: a general cell (GC) and an SM type, which is found in mouse and human UNC-45. The SM UNC-45 mRNA is abundant in skeletal muscles and the heart, both of which consist predominantly of SM fibres (Price et al. 2002). Thus, our result was consistent with the observation from humans and mice.

Genomic DNA amplification and SNP identification

One pair of primers (PS-F and PS-R in Table 1) on exons 15 and 16 according to the porcine mRNA sequence and aligned with the human DNA sequence (GenBank accession no. is NC_ 000017.9) were designed to amplify the intron 15. The PCR products were pooled and sequenced, a 776 bp partial genomic DNA sequence was obtained including partial exon 15 – 50 bp, intron 15 – 677 bp and partial exon 16 – 49 bp (GenBank accession no. is EF523777). One A558G SNP in the intron 15 was found and detected by digestion with MspI, resulting in a 776-bp PCR amplicon-produced allele A (776 bp) and allele G (608 bp and 168 bp) (Figure 2).

Allele frequencies of the porcine CMYA4 gene in different breeds

Allele frequencies of the porcine CMYA4 gene indicated that, with the exception of the Erhualian pig, all the breeds are polymorphic (Table 2). The allele distribution revealed that the Chinese indigenous breeds had higher frequencies of the G allele whereas the western breed Duroc pigs had higher frequency of the A allele. The statistical analysis showed a significant difference (p < 0.001) in allele frequencies between Chinese indigenous breeds and Duroc pigs.

Association analysis of the porcine CMYA4 gene with economic traits

The allele distribution showed that there are no GG genotypes in the tested Yorkshire population, so the populations Yorkshire and Yorkshire × (Landrace × Tongcheng) have been excluded from the associated analysis. The CMYA4 gene intron 15 A558G locus polymorphism showed a significant association with BTR (p < 0.05) and BTS (p < 0.05), and the effects of the breed/breed crosses are significant on the traits (p < 0.01) (Table 3). The BTR of pigs with the AA genotype was significantly lower than that of pigs with the AG genotype (p = 0.027) and the BTS of pigs with AA genotypes was significantly lower than that of pigs with the GG genotypes (p = 0.042).

Our previous work assigned the porcine CMYA4 gene to SSC12, which is closely linked to Sw62 (distance 16cR, Logarithm of the odds ratio (LOD) score value 18.07 and retention frequency 27%) (Xu et al. 2005) and to S0090 (distance 45cR, LOD score value 7.89 and retention frequency 22%) (unpublished results), a marker associated with the backfat at shoulder (Ponsuksili et al. 2005), backfat depth at last rib (Malek et al. 2001; Yue et al. 2003), diameter of muscle fibre (Wimmers et al. 2006) and total number of fibres (Wimmers et al. 2006). Combining the association analysis with the chromosome assignment we can predict a function of the porcine CMYA4 gene for meat production. Thus, we may assume that the CMYA4 gene acts as a candidate gene for carcass traits.

In summary, we isolated a novel porcine CMYA4 gene, obtained its entire coding sequence and predicted its amino acid sequence. The gene is specially expressed in the skeletal muscle and heart tissues. Meanwhile, we have provided evidence that a polymorphism (A558G) in the CMYA4 gene intron 15 is associated with BTR and BTS. Hence, it could simply be acting as a genetic marker for a linked quantitative trait loci with effects on backfat thickness. However, its involvement in the muscle production needs to be further studied.