Association between PCR-RFLP Polymorphism of the Fifth Intron in Lipoprotein Lipase Gene and Productive Traits in Pig Resource Family

The study was aimed at detecting polymorphism of the fifth intron in lipoprotein lipase (LPL) gene and analyzing association between the polymorphism and productive traits. A pair of primers was designed for amplifying the fifth intron. Sequence analysis indicated that a G1171C substitution existed in Large White breed. The mutation was detected by PCR-AfaI-RFLP. Polymorphism analysis in a pig resource family showed that there existed significant effects on carcass and meat quality traits. Thoraxwaist fat thickness of BB genotype was significantly higher (14.2%, p<0.05) than that of AA on carcass traits, while BB genotype was significantly lower (3.6% p<0.01, 4.1% p<0.01; 2.3% p<0.01, 1.9% p<0.01; 1.8% p<0.01, 1.4% p<0.05) than AA and AB genotype in pH of m. Longissimus Dorsi (LD), m. Biceps Femoris (BF), m. Semipinali Capitis (SC). The allelic frequencies were also significantly different between indigenous Chinese breeds and exotic breeds. Data analyzed revealed that the mutation locus affected production traits mostly by additive effects. Based on these results, it is necessary to do more studies on LPL gene before making the LPL locus into the application of marker-assisted selection (MAS) programs. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 4 : 458-462)


INTRODUCTION
Lipoprotein lipase (LPL) is a major enzyme responsible for the hydrolysis of triglycerides in chylomicrons and very low density lipoproteins (VLDL) (Chamberlain et al., 1989;Ahn et al., 1993;Zhang et al., 1996).It also promotes the exchange of lipids between VLDL and high density lipoproteins (HDL) and is an essential enzyme in the formation of low density lipoprotein (LDL) particles (Hokanson et al., 1999).So LPL plays a key role in lipid metabolism.The chylomicrons are not broken down properly if the lipoprotein lipase enzyme is deficient, which would result in a fat-laden chylomicrons in blood.Many studies have demonstrated significant associations between changes in coronary heart disease (CHD) risk factors and allelic variants in the LPL gene in human (Dichek et al., 1991;Hokanson et al., 1997).However, studies on the association of productive characteristics with LPL gene polymorphism are scanty.Hence, the purpose of this study was to determine whether there was any evidence of genetic correlation between productive traits in pig resource family and the polymorphisms of the fifth intron in porcine LPL gene.

Experimental animals
The pig resource family used in this study was established by mating three Large White boars to seven Meishan sows.Five males and twenty-three females in the F 1 generation were selected for intercrossing randomly.One hundred and forty F 2 individuals were slaughtered for test.Carcass traits and meat quality traits were recorded according to the method of Xiong and Deng (1999).For each trait (Table 1), one hundred and forty phenotypic records were available.

DNA preparation
Genomic DNA was isolated from blood white cells and DNA extraction procedure was described by Sambrook et al. (1989).After isolation, DNA pellet was dissolved in TE buffer and was stored at -20°C.

Cloning and sequencing of the amplified fragment of intron 5
The intron 5 was amplified with genomic DNA from two pig breeds (Qingping and Large White).The primer pair 1 (forward: 5`-CCAACCAGGGTGTAACAT-3`; reverse: 5`-GGATTTTCTTCATTCAACAGA-3`) was designed according to cDNA sequence of Landrace breed in Genbank (X62984) for cloning and sequencing.Polymerase chain reaction (PCR) was performed in 25 µl reaction volume containing: 1×PCR buffer, 1.5 mM MgCl 2 , 250 µmol/l dNTP, 5 pm of each PCR primer, 2 units Taq DNA polymerase (Biostar International, Canada), 200 ng genomic DNA as template.PCR was run in the GeneAmp PCR system 9600 (Perkin-Elmer Co., USA) thermocycler as follows: initial denaturation at 94°C for 4 min, 35 cycles of 94°C for 1 min, 63°C for 1 min, 72°C for 1.5 min, and a final extension for 10 min at 72°C.The purified PCR products were cloned into the pGEM-T vector and were sequenced by Sangon Company.The sequencing results of different pig breeds were compared by using BLAST (http://www.ncbi.nlm.nih.gov).

Detection of PCR-AfaI-RFLP
According to the BLAST results of intron 5 in Large White and Qingping breeds, Primer pair 2(forward: 5'-CAGGGAACTTTCCATACTTTGTG-3'; reverse: 5'-AGC ATGTAATACTTCCAGAGGCT-3') was designed for detection of PCR-AfaI-RFLP.PCR amplification (20 µl final volume) was performed using 250 ng genomic DNA, 200 µmol/l dNTP, 5 pm of each primer, and 1 U Taq DNA polymerase with the reaction buffer supplied by the manufacturer.The thermocycler profile was 94°C for 4 min, 35 cycles of 94°C for 45 s, 56°C for 45 s, and 72°C for 45 s, followed by a final extension step at 72°C for 10 min.For the PCR-RFLP assays, PCR products were digested with 3 units AfaI (TaKaRa Company) in 1×buffer with 1×BSA added in a total volume of 10 µl, following digestion for 4 h at 37°C.Digested products were separated by electrophoresis on a 3% agarose gel in 1×TAE buffer and stained with 0.5 µg/mL ethidium bromide.

Statistical analysis
The association between genotype and production traits was performed with the least square method (GLM procedure, SAS version 8.0).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 AA, AB and BB, respectively, and the dominance effect represented as 1, -1 and 1 for AA, AB and BB, respectively (Liu, 1998).The model used to analyze the data was assumed to be: Where, Y ijk is the observation of the trait; µ is the least square mean; S i is the effect of its sex; F j is the effect of its family; G k is the effect of its genotype; b ijk is the regression coefficient of the slaughter age and e ijk is the random residual.

Amplification and cloning of intron 5
The primer pair 1 produced two fragments with genomic DNA from two pig breeds.Product of PCR (5 µl) was observed through 1% agarose gel electrophoresis.The results were displayed in Figure 1.

Sequencing analysis
Sequence analysis of PCR products revealed that a new AfaI polymorphism was identified at 1,171 th position where the nucleotide G was substituted by nucleotide C. The sequence has been deposited in the GenBank database under the accession number AY 566861.

Restriction site analysis
A 336 bp fragment was amplified with primer pair 2. PCR was performed on DNA of nine pig breeds and one hundred and forty F 2 offspring.Results of amplification were shown in Figure 2.

Frequencies of allele and genotype of different pig breeds
Allele frequencies for the PCR-AfaI-RFLP were studied in 9 different populations (Table 2).Allele A was the predominant allele in Chinese indigenous pig breeds, such as, frequencies of allele A was 1.00 in Huainan pigs, while in Western pig breeds, allele B was predominant.There were three genotypes (AA, BB and AB) existed in all pig populations.Standard t testing showed highly significant difference in genotype frequencies between Chinese indigenous pig populations and Western pig breeds (p<0.01)(Table 3), indicating clearly genetic differentiation at the locus between Chinese and Western pigs, whereas the differences among Chinese indigenous pig populations were slighter.

Analysis of phenotype value about carcass traits and meat quality traits
SAS package was conducted for statistical analyses on carcass traits and meat quality traits on F 2 offspring (Large White×Meishan).Here significant tests and multicomparison were performed.The results of tests for the genotypes and carcass traits were given in Table 4. Pigs with BB genotype had more TFT (+0.28 mm), RFT (+0.09 mm), BFT (+0.21 mm) and ABT (+0.15 mm) than pigs with AA genotype, however, pigs with BB genotype had fewer DR (1.94%) than pigs with AA genotype.Statistically significant associations with only DR and TFT were found.P-value of DR and TFT was 0.026 and 0.014, respectively.No significant differences were observed in RFT, BFT and ABT.Data (Table 5) revealed that an allele (A) was associated with a desirable effect on meat quality traits.Pigs with AA genotype had more pH (LD) (+0.23, p<0.01), pH (BF) (+0.15, p<0.01), pH (SC) (+0.12, p<0.01), and WHC (+5.35, p<0.01), than pigs with BB genotype.Another trend that homozygotes (BB) had significantly higher (p<0.01)WLR and MC 1 than that of homozygotes (AA) and heterozygotes (AB) was observed.

DISCUSSION
In the present study, frequencies of allele A and B were distributed in nine pig breeds.Significant difference was revealed (Tables 2 and 3).Higher frequencies of allele A were found in indigenous Chinese breeds, suggesting that allele A could be associated with fat deposition.It is also in agreement to the fact that Chinese pig populations have a strong tendency to accumulate fat.Therefore, it is a strong possibility that enzymatic activity of lipoprotein lipase changed because of the substitution from G to C.
Backfat thickness would be an important factor for carcass traits (Archibald, 1994;Zuo et al., 2003).It should also be closely correlated with fat deposition.A mutation was also found in 5'-untranslated regions (UTR) of LPL gene, which could be with backfat thickness (Wu, 1998).However, no significant effect on backfat thickness was observed except for TFT in present study, which might be due to the limited numbers of individuals analyzed.More individuals are necessary to confirm the present results.
Ultimate pH of pork is most commonly used to assess pork quality.A higher level of acidity within the muscle (lower pH) causes muscle protein to denature and lose their ability to hold water.Therefore, meat with relatively higher pH will tend to have more desirable characteristics (Simpson et al., 1989;Gu et al., 1992;Zuo et al., 2003).Furthermore, pigs with BB genotype presented greater drip loss and meat color values, which indicated the pigs with BB genotype have a worse meat quality compared to pigs with AA genotype, and eventually resulted in pale, yellow meat, similar to PSE meat rejected by consumers (Minkema et al., 1976).
The polymorphism analyzed in porcine LPL gene was located in intron 5.Although the single nucleotide polymorphism (SNP) in introns do not directly alter any functionally important amino acid residue, they may play a role in regulating gene expression and thus their constituent SNPs may be directly related to functional variation.Other researchers have also reported that introns may play a role in splicing of mRNA and its stability (Padma et al., 2004).
It has not been reported that association exits between the polymorphisms in LPL gene and production traits.The polymorphism could be a potential genetic marker for production traits, especially for meat quality.In addition, data analyzed revealed that the mutation locus controlled production traits mostly by additive effects (Tables 4 and 5).The porcine LPL gene has been assigned to chromosome 14 q12-q14 (Gu et al., 1992).Other closely linked genes on porcine chromosome 14 might affect the observed results.Further investigation is required among more populations of pigs to verify whether the substitution could be utilized as a genetic marker in MAS programs.

ACKNOWLEDGMENTS
We wish to express our thanks to anonymous reviewers

Table 1 .
Abbreviations of names for traits in pig resource family No.

Table 4 .
The effects of AfaI locus in intron 5 of LPL gene on carcass traits Data in the table are least square means±standard error.Values in each line in different lower case superscripts are significantly different at p<0.05.Additive effect (a) = (BB-AA)/2; Dominance effect (d) = AB-(AA+BB)/2.

Table 5 .
Effect of different genotype in intron 5 of LPL gene on meat quality traits