ASCL 2 Gene Expression Analysis and Its Association with Carcass Traits in Pigs

Achaete-scute like 2 (ASCL2) gene encodes a member of the basic helix-loop-helix transcription factor which is essential for the maintenance of proliferating trophoblasts during placental development. ASCL2 gene preferentially expresses the maternal allele in the mouse. However, it escapes genomic imprinting in the human. In this study, the complete open reading frame consisting of 193 amino acids of ASCL2 gene was obtained. Sequence analysis indicated that a C-G mutation existed in the 3’ untranslated region between Meishan and Large White pigs. The polymorphism was used to determine the monoallelic or biallelic expression with RT-PCR-RFLP in pigs of Large White×Meishan F1 hybrids. Imprinting analysis indicated that the ASCL2 gene expression was biallelic in all the tested tissues (heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, fat, uterus, ovary and pituitary). PCR-RFLP was used to detect the polymorphism in 270 pigs of the “Large White×Meishan” F2 resource population. The statistical results showed highly significant associations of the genotypes and fat meat percentage (FMP), lean meat percentage (LMP) and ratio of lean to fat (RLF) (p<0.01), and significant associations of the genotypes and loin eye area (LEA) and internal fat rate (IFR) (p<0.05). (

Large White×Meishan F 1 hybrids and their mothers were used for imprinting analysis.Pigs of Large White×Meishan F 2 hybrids used for association analysis were fed as described by Dai et al. (2006).The finishing animals were slaughtered in the year of 2003 and 2004 and measured in the Center of Swine Control of China (Wuhan) according to the method of Xiong and Deng (1999).Genomic DNA was isolated according to the standard phenol-chloroform method.

RNA isolation and cDNA synthesis
Total RNA, from the skeletal muscle tissue of the adult Large White and Meishan pigs and from twelve tissues (heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, fat, uterus, ovary and pituitary) of four heterozygous pigs (based on SNP) of the twelve F 1 hybrids, was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, US) according to the manufacturer's instructions.The first strand cDNAs were synthesized from 3 µg total RNA treated with DNAse I (TaKaRa, Tokyo, Japan) in a 25 µl reaction containing 40 U M-MLV reverse transcriptase, 5 µM oligo(dT) 18 primer, 1×M-MLV first-strand buffer, 1 mM each of dNTP and 8 U RNase inhibitor (Promega, Madison, WI, USA) at 42°C for 60 min.

PCR of DNA and cDNA
Human ASCL2 cDNA sequence (GenBank: NM_005170) was used to identify pig expressed sequence tags (EST) through standard BLAST (http://www.ncbi.nlm.nih.gov/blast/)searches of the 'EST-others' database.Pig ESTs sharing more than 85% sequence identity with the human cDNA sequence were assembled into an EST-contig.The exon-intron structures of the porcine ASCL2 gene were estimated according to the human ASCL2 gene.Primer pair ASF/ASR (ASF: ACAGTGCCAGATCGTCGCT and ASR: TGTCCCTGGGCAGTTCAAG) was designed according to the EST-contig.Primer pair ASDF/ASDR (ASDF: CCTGACCAAGGGCTAGTG and ASDR: GTCCCTGGG CAGTTCAAG) was used to detect SNP clearly.PCR conditions were as follows: 94°C for 4 min, 35 cycles of 94°C for 45 s, 60°C (ASF/ASR) or 56°C (ASDF/ASDR) for 50 s, 72°C for 90 s (ASF/ASR) or 35 s (ASDF/ASDR) and a final extension at 72°C for 7 min.Primers F: ACCA CAGTCCATGCCATCAC and R: TCCACCACCCTGTT GCTGTA, which amplified the fragment spanning intron 8 of the GAPDH gene, were applied to exclude the possibility of DNA contamination during all RT-PCR reactions.

Sequencing and SNP detection
PCR products were purified with the Wizard prep PCR purification system (Promega), cloned with pMD18-T easy vector (TaKaRa) and sequenced commercially.Sequences of Large White and Meishan pigs were compared with DNAStar software to search for SNP.

RFLP of PCR and RT-PCR products
PCR or RT-PCR products (6 µl) amplified by primer pair ASDF/ASDR were incubated at 37°C for 4 h with 3 U restriction enzyme Msp I (TaKaRa), 2.7 µl purified water and 1 µl digestion buffer.Digested products were separated by 2% agarose gel.

Statistical analysis
The association between genotype and carcass traits was performed with the least square method (GLM procedure, SAS version 8.0).According to the method of Liu (1998), both additive and dominance effects were also estimated using REG procedure of SAS version 8.0, where the additive effect was denoted as -1, 0 and 1 for GG, GC and CC, respectively, and the dominance effect represented as 1, -1 and 1 for GG, GC and CC, respectively.The statistical model was assumed to be: T ijk = µ+S i +Y j +G k +b ijk X ijk +e ijk , where T ijk is the observed values of traits; µ is the leastsquare mean; S i is effect of sex (i = 1 for male or 0 for female), Y j is the effect of year (j = 1 for year 2003 or 0 for year 2004), G k is the effect of genotype (K = GG, GC and CC), b ijk is the regression coefficient of the slaughter weight, X ijk is the slaughter weight, and e ijk is the random residual.

Sequence analysis and SNP discovery
Primer pair ASF/ASR amplified a 1,654 bp fragment (Genbank accession number DQ666420) in genomic DNA.Aligning the sequence with human ASCL2 gene cDNA sequence showed it including 117 bp of 5' untranslated sequence, 582 bp of coding sequence and 955 bp of 3' untranslated sequence.Sequence analysis indicated that it had 84% and 81% sequence similarity in nucleotides or 84% and 79% sequence similarity in amino acids compared to the homology of human and mouse, respectively.Compared sequences of Large White and Meishan breeds revealed one SNP (C/G) which existed in the 3' untranslated region at position 1,556 (DQ666420).

Screening for heterozygous pigs
The C-G mutation could be detected by restriction enzyme Msp I. Primer pair ASDF/ASDR which amplified fragment covered the SNP was designed to distinguish the genotypes well.Allele C was 98 bp and 99 bp, and allele G was 197bp (Figure 1).Twelve F 1 hybrids of Large White and Meishan pigs were used to choose heterozygous individuals at the SNP.Amplification of primer pair ASDF/ASDR was conducted with the genomic DNA of the twelve pigs and their mothers.PCR products were digested by Msp I. Results of PCR-Msp I -RFLP indicated that four pigs of Large White and Meishan F 1 hybrids were heterozygous at the SNP.

Imprinting analysis of ASCL2 gene
RT-PCR of heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, fat, uterus, ovary and pituitary from the four pigs with primer pair ASDF/ ASDR showed that the ASCL2 gene was expressed in all tissues.RFLP analysis of the RT-PCR products showed that the gene expression was biallelic in all the examined tissues (Figure 2).

Association analysis of genotypes and carcass traits
Two hundred and seventy pigs of the Large White×Meishan F 2 resource family were used to estimate the association between the polymorphism and carcass traits.At this locus, the number of genotypes GG, GC and CC were 32, 110 and 128 respectively, and the genotype distribution conformed to the Hardy Weinberg Equilibrium (χ 2 = 1.222<χ 2 (0.05) , d.f.= 2).The detailed results of association analysis are listed in Table 1.Statistical results   showed that the PCR-Msp I -RFLP genotypes had highly significant association with fat meat percentage (FMP), lean meat percentage (LMP) and ratio of lean to fat (RLF) (p<0.01), and significant associations with internal fat rate (IFR) and loin eye area (LEA) (p<0.05).At this locus, additive and dominance both seemed to be highly significant (p<0.01) and allele C was associated with increases in the trait value.Pigs with the CC genotypes had higher lean meat percentage (4%) and more loin eye area (3.122 cm 2 ).

DISCUSSION
The ASCL2 gene was maternally expressed in the mouse (Guillemot et al., 1995), but Miyamoto et al. (2002) reported biallelic expression in the human.In pigs, expression was also biallelic in various tissues of the four heterozygous pigs.Similar imprinted status between pigs and humans showed that the two species were adjacent in genomic imprinting, which also confirms the conservation of genomic imprinting between the two species.Sequence analysis showed 84% and 81% sequence similarity in nucleotides and 84% and 79% sequence similarity in amino acids compared to human and mouse homology, respectively.The high sequence similarity in nucleotides and amino acids between humans and pigs showed they may have similar biological function.The different imprinted status in the human, mouse and pig indicated that different imprinting mechanism or imprinting regulation may exist among them.
Imprinted genes have an important function in the regulation of fetal growth, development, function of the placenta and postnatal behavior in mammals (Reik et al., 2003).In previous studies, many imprinted QTLs affecting porcine carcass composition and growth traits were found in several chromosomes (Andersson et al., 1994;Alexander et al., 1996;Nezer et al., 1999;Koning et al., 2000).IGF2, which was identified as the first imprinted gene in pigs, has important effects on porcine growth, meat quality and carcass composition, especially fat deposition (Jeon et al., 1999;Jungerius et al., 2004;Estelle et al., 2005).Zhang et al. (2006) reported that the PLAGL1 gene which was imprinted in the human and mouse had important effects on shoulder backfat thickness (SFT) and internal fat rate (IFR).In addition, ASCL2, H19 and IGF2 were associated with an imprinted QTL in the human and mouse (Lee et al., 1999).Therefore, the porcine ASCL2 gene was selected as a candidate gene affecting fat deposition and carcass traits in our study.The polymorphism had highly significant associations with fat meat percentage (FMP), lean meat percentage (LMP) and ratio of lean to fat (RLF) (p<0.01), and significant associations with internal fat rate (IFR) and loin eye area (LEA) (p<0.05).Our statistical results confirmed that the ASCL2 gene had an important effect on porcine carcass traits like IGF2.Although the ASCL2 gene was not imprinted in pigs in our study, its effect on porcine carcass traits was not affected by its imprinted status, which is consistent with the results that IGF2 and IGF2R were not imprinted, but they also had important effects on body growth and carcass traits in chickens (Nolan et al., 2001;Wang et al., 2005).Pigs with the CC genotype had higher lean meat percentage (4%) and more loin eye area (3.122 cm 2 ).Increasing the allele C may be favorable for carcass traits in pig breeding.The polymorphism site could be a useful molecular marker for lean meat percentage and carcass traits.

Figure 2 .
Figure 2. Tissue expression of porcine ASCL2 gene.Allelic expression of ASCL2 by PCR-RFLP.The cDNA samples were from 12 different tissues of a heterozygous pig (lanes 3-14), and genomic DNA samples were from the pig (lane 2) and its mother (lane 1) Digestion with MspI revealed that expression of the ASCL2 gene was biallelic in all the tested tissues.M: Mark DL-2000 (2,000 bp, 1,000 bp, 750 bp, 500 bp, 250 bp, 100 bp).

Table 1 .
Association of ASCL2 genotypes and carcass traits