A novel porcine mRNA differentially expressed in the Longissimus muscle tissues from Meishan and Large White pigs are associated with carcass traits and meat quality

The mRNA differential display technique was performed to investigate the differences of gene expression in the Longissimus muscle tissues from Meishan and Large White pigs. One novel mRNA that was differentially expressed was identifi ed through semi-quantitative RT-PCR and the cDNA complete sequence was then obtained using the rapid amplifi cation of cDNA ends (RACE) method. Sequence analysis revealed that this mRNA is not homologous to any known porcine gene. Sequence prediction analysis revealed that the this mRNA is not protein-coding mRNA. Polymorphism analyses revealed that there was a A-G mutation on the position of 490bp and PCRTaqI-RFLP analyses revealed that Chinese indigenous pig breeds and European pig breeds displayed obvious genotype and allele frequency differences at this locus. Association analyses revealed that this polymorphic locus was signifi cantly associated with the meat pH of M. longissimus dorsi (LD), M. biceps femoris (BF), M. semispinalis capitis (SC), water holding capacity, drip loss rate, meat colour value M. longissimus dorsi (LD), and estimated lean meat percentage (P<0.05).


INTRODUCTION
The mRNA differential display fi rst described by Liang and Pardee (1992) is a fast, and effi cient method for isolating and characterizing altered gene expression in different cell types.It was statistically shown that 80-120 primer combinations would be suffi cient to cover all the transcript populations in the cell (Liang et al., 1993).This technique possesses the following advantages over other similar techniques: it is based on simple and established methods, more than two samples can be compared simultaneously and only a small amount of starting material is needed (Yamazaki and Saito, 2002) .
Chinese indigenous pig breeds such as Meishan, Erhualian and Tongcheng often have valuable traits such as disease resistance, high fertility, good maternal qualities, unique product qualities, longevity and adaptation to harsh conditions.Pig breeds in Europe such as Large White, Landrace, Duroc possess the good achievements in growth rate, high lean rate, especially, the introduced pigs have higher lean meat rate and feed conversion effi ciency, whereas Chinese indigenous pigs have more fat deposition and superior meat quality (Pan et al., 2003) .Phenotypic variances are mainly determined by the genetic differences.So that detecting the genetic differences between Chinese indigenous pig breeds and European pig breeds or fi nding out the differentially expressed genes between Chinese indigenous pig breeds and European pig breeds which determine these phenotypic variances is necessary for pig breeders.
Our present study was carried out with the mRNA differential display technique to isolate the differentially expressed genes or mRNAs in the Longissimus dorsi muscle tissues from one Chinese indigenous pig breed -Meishan and one European pig breed -Large White pigs.

Animals and sample preparation
For mRNA differential display Large White and Meishan pigs, the two purebred populations were constructed in December, 2002.The Longissimus dorsi muscle samples were collected from 180-day-old pigs (for each breed, fi ve male and fi ve female) slaughtered in March, 2003 and frozen in liquid nitrogen.These Longissimus dorsi muscle samples would be used to perform the mRNA differential display.
For polymorphism analyses blood samples were collected from 225 unrelated animals belonging to six swine populations presented in Table 1.Genomic DNA isolated from the blood samples would be used to perform the polymorphism analyses.
For association analyses 300 F2 pigs from Large White×Meishan were slaughtered in Autumn, 2000 and2003, when these pigs were 180-day-old.The growth, carcass and meat quality traits presented in Table 2 were recorded according to the method of Xiong and Deng (1999).Genomic DNA was isolated from blood white cells and DNA extraction procedure was described by Sambrook et al. (1989).The growth, carcass and meat quality traits data and Genomic DNA would be used to perform association analyses.mRNA differential display The mRNA differential display was performed as that described previously (Liu et al., 2004).

Statistical analysis
The association analysis between genotypes and traits was performed with the least square method (GLM procedure, SAS version 8.0) (Liu, 1998).Both additive and dominance effects were estimated using REG procedure of SAS version 8.0, where the additive effect was denoted as -1, 0 and 1 for AA, AB and BB, respectively, and the dominance effect was denoted as 1, -1 and 1 for AA, AB and BB, respectively.The model used to analyse the data was assumed to be: Yijk = μ + Si + Fj + Gk + bijkXijk + eijk where: Yijk -the observation of the trait; μ -the least square mean; Si -the effect of its sex (i = 1 for male or 2 for female); Fj -the effect of its family (j = 2000 or 2003); Gk -the effect of its genotype (k= AA, AB, BB); bijk -the regression coeffi cient of the slaughter weight; Xijk -the slaughter weight; eijk -the random residual.

RESULTS AND DISCUSSION
mRNA differential display.From the mRNA differential display, one mRNA, nominated as mRNA2, was found to be almost not expressed in the Longissimus dorsi muscle of Meishan pigs, while it was over-expressed in the Longissimus dorsi muscle of Large White pigs as shown in Figure 1.Semi-quantitative RT-PCR.The differentially expressed gene band was recovered from gel and used as the template for the re-amplifi cation, which was performed with the respective oligo(dT) primer and the arbitrary primers used in the mRNA differential display.The resulting PCR product was 343bp.This was in agreement with the result of the mRNA differential display.The purifi ed PCR product was then cloned into the T-vector and the recombinant plasmid was sequenced.Semi-quantitative RT-PCR was then conducted using the EST specifi c primers and the results are presented in Figure 2.
Semi-quantitative RT-PCR results indicated that mRNA2 was over-expressed in the Longissimus dorsi muscle of Large White pigs and weakly expressed in the Longissimus dorsi muscle of Meishan pigs.This also coincided with the result of mRNA differential display.
RACE and sequence analysis.Through 5'-RACE, one PCR product of 1546 bp was obtained.The 3'-RACE product was 249 bp.These products were then cloned to T-vector and sequenced.Taken together, a 1684-bp cDNA complete sequence was fi nally obtained.The nucleotide sequence analysis using the BLAST software at NCBI server (http://www.ncbi.nlm.nih.gov/BLAST)revealed that this mRNA was not homologous to any of the known porcine genes and it was then deposited into the GenBank database (Accession number: AY864618).The sequence prediction was carried out using the GenScan software and results revealed that the this mRNA is a no-coding mRNA.
Polymorphism.Based on the sequence of mRNA2, primers (forward primer2 and reverse primer2)were used to amplify the DNA of Large White and Meishan and the products were then cloned into PMD18-T vector and sequenced bidirectionally with the commercial fl uorometric method.One A-G mutation was found at the position of 490 bp.This leaded to mutation of one TaqI restriction site.This was confi rmed by PCR -TaqI-RFLP (Figure 3).Subsequently, PCR -Dra I-RFLP were performed using the DNA from 225 unrelated animals belonging to six swine populations including Large White, Landrace, Meishan, Bamei, Tongcheng and Huainan.Results revealed that frequency of G allele is predominant both in Chinese indigenous and European pig breeds: Large White ( 0.90), Landrace (1.00), Meishan (0.85), Bamei (0.88), Tongcheng (0.63) and Huainan (0.68), but Chinese indigenous pig breeds have the obvious higher frequency of A allele than European pig breeds: Large White (0.10), Landrace (0.00), Meishan (0.15), Bamei (0.12), Tongcheng (0.37) and Huainan (0.32).This indicated that Chinese indigenous pig breeds and European pig breeds displayed obvious genotype and allele frequency differences at this A-G mutation locus (Table 3).Association analyses.From the association analysis results it can be seen that pigs with GG genotype have higher pH (LD) (+0.088), pH (BF) (+0.060), pH (SC)(+0.040),but have lower MCV (LD) (-0.813%) than pigs with AG genotype (P<0.05).The pigs with GG genotype also have greater DLR (+0.985%) than pigs with AA genotype (P<0.05).On the contrary, pigs with AA genotype have more WHC (+1.379%) than pigs with GG genotype (P<0.05).The pigs with AG and GG genotypes have signifi cantly higher ELMP (+0.527% and +0.529%) than pigs with AA genotype (P<0.05).From the polymorphism analysis (Table 4), it can be seen that Chinese indigenous pig breeds and European pig breeds displayed obvious genotype and allele frequency differences at this A-G mutation locus.Results revealed that frequency of A allele is predominant in Chinese indigenous pig breeds than European pig breeds.Combining to the results of association analysis: GG genotype have signifi cantly higher DLR, signifi cantly lower WHC and signifi cantly greater ELMP than pigs with AA genotype.These results agree to the differences of two kinds of pigs: Chinese indigenous pig breeds often have low lean rate, but have better meat quality.European pig breeds possess the good achievements in high lean rate, worse meat quality.The pig industry is now actively using this information to improve the pig production by marker assisted selection (MAS).So it is of outmost importance to fi nd more candidate genes or DNA molecular markers in order to improve pig production.From our results, we found the polymorphism of the TaqI locus of this porcine mRNA signifi cantly affected some important traits which were related to carcass traits and meat quality traits.This indicates that this polymorphic locus of porcine mRNA is a valuable marker deserved to be applied to the marker assistant selection (MAS) in pig breeding.
In spite that gene prediction has show that this novel porcine mRNA is not protein-coding gene,we still fi nd some ORFs in this novel porcine mRNA using the ORF Finder (Open Reading Frame Finder) (http://www.ncbi.nlm.nih.gov/gorf/ gorf.html).We do not know which ORF represents its protein-coding region.

Figure 1 .
Figure 1.The differential expression analysis of mRNA2.The arrow indicates the cDNA profi le for the mRNA2 on a polyacrilamide gel of 8%, stained with silver nitrate.MS -Meishan sample; LW -Large White sample

Figure 2 .
Figure 2. The Semi-quantitative RT-PCR analysis of mRNA2 on the agarose gel of 1% stained with ethidium bromide.MS -Meishan sample; LW -Large White sample

Table 2 .
Abbreviations of names for the productive traits in pig resource family No.

Table 1 .
The information of 225 unrelated pigs from 6 populations

Table 3 .
Allele frequency and genotype of TaqI polymorphic locus in different pig breeds

Table 4 .
Association analyses between the different phenotypes of mRNA2 at the TaqI polymorphic locus and the traits in the table are least square means ± standard error; values in each line with lower case superscripts are signifi cantly different at P<0.05; additive effect = (GG-AA)/2; dominance effect = AB-(AA+GG)/2; * means signifi cant at P<0.05; ** means signifi cant at P<0.01