Association of a Single Nucleotide Polymorphism in the 5 ’-Flanking Region of Porcine HSP 70 . 2 with Backfat Thickness in Duroc Breed

Higher environmental temperature affects the economic performance of pigs. Heat shock protein 70 has been shown to play an important role in thermoresistance. The purpose of this study was to assess the effect of a singlenucleotide polymorphism in the 5'-flanking region of porcine HSP70.2 on growth performance in Taiwanese Duroc. The genotype of this nt 393 polymorphic site could be verified by digestion with Bsa WI restriction enzyme of a PCR product. Pigs with TT and TC genotypes have thinner backfats than those with CC type (p<0.05). The result suggested that the polymorphic Bsa WI site in the 5'-flanking region of porcine HSP70.2 may be used as a marker for the early selection of ultrasonic backfat thickness in Duroc pigs.


INTRODUCTION
Higher ambient temperature is important for pigs because it affects their growth and carcasses (Holme et al., 1967).Generally, food intake and growth rate both decrease markedly at high temperature (Heitman et al., 1949;Heitman et al., 1958).Backfat depths measured above the eye muscle were significantly increased by hot treatment (Holmes, 1971).Living organisms are capable of producing heat shock proteins (HSPs) in response to environmental insults such as temperature elevation (Lindquist et al., 1988).The 70 kDa HSP (HSP70) has been demonstrated to be related to thermoresistance (Johnston et al., 1988;Riabowol et al., 1988).
A complete nucleotide sequence of porcine HSP70.2 (heat inducible form) was reported by Peelman et al. (1992).The gene is located on chromosome 7 cen-p1.1 within the major histocompatibility complex (Nunes et al., 1993;Dezeure et al., 1993).Restriction fragment length polymorphism analysis with Pst I and Pvu II (Ruohonen-Lehto et al., 1993;Dezeure et al., 1993) have shown limited polymorphisms of HSP70.2.In addition, five polymorphisms (nt 44, 232, 250, 345, and 393) have been detected in the 5'-flanking region of porcine HSP70.2 (Schwerin et al., 1999;Chen et al., 2000).Two of these sites appeared to be associated with meat quality (Schwerin et al., 1999) and birth weight (Maak et al., 1998).A recent report suggested that inactivation of the GC box site in the porcine HSP70.2 promoter resulted in a significant decrease of heatinduced transcription compared to the corresponding wildtype promoter (Schwerin et al., 2001).The objective of the present study is to assess the effect of a single nucleotide polymorphism in the 5'-flanking region of porcine HSP70.2 on growth performance in Taiwanese Duroc.

Animals
The animals used in this study included 88 unrelated purebred Duroc pigs which were from private seedstocks located in Taiwan.Genomic DNA was extracted from blood using a DNA Isolation Kit for mammalian blood (Boehringer Mannheim, IN, USA).The isolated genomic DNA was stored at -20°C until used for polymerase chain reaction (PCR) amplification.

Analysis of restriction fragment length polymorphism
To investigate the polymorphism in the 5'-flanking region of HSP70.2 one fragment was amplified and digested with Bsa WI restriction enzyme.The PCR amplification was performed in a final volume of 50 µl containing 100 ng of genomic DNA, 2 µM of each primer, 200 µM of each dNTP, 1×PCR reaction buffer, and 1unit of Taq polymerase (Viogene, CA, USA).The primers were as follows: 5'-GGG GCT TGA GGA AAA AAA-3'; 5'-GGC GAT GAT CTC CAC CTT GC-3'.The reaction was loaded onto a Perkin-Elmer 9600 thermal cycler (Norwalk, CT, USA) under the following conditions: 1 cycle at 94°C for 10 min; 40 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 1 min; 1 cycle at 72°C for 10 min.The amplified fragment (227 bp) was purified and then digested with restriction enzyme Bsa WI in a total volume of 15 µl containing 10 µl purified PCR product, 1.5 µl BSA (1 mg/ml), 1.5 µl buffer and 4 units of enzyme (New England Biolabs, MA, USA) at 65°C for 3 h and then examined by electrophoresis on 2% agarose gel.The gels were stained with ethidium bromide and photographed.

Growth performance
The boars were performance tested in cool season, hot season, and season between the two seasons (intermediate) season.The procedures of performance test were describe by Huang et al. (1995)  BF: ultrasonic backfat thickness: Backfat thickness was measured on both sides of the following position: behind the scapula, last rib, and lumbar vertebra.BF was corrected for weight as follows (Chyr, 1980)

Statistical analysis
The effect of genotypes of the 5'-flanking region of porcine HSP70.2 on growth performance traits was analyzed using the following model: Where µ is the mean value common to all records; G i is the effect of the ith genotype; S ij is the effect of the jth season; and e ijk is the random error.
The analysis was performed by the GLM procedure of SAS (SAS institute, 1989).The significance of differences among genotypes was determined by the least-squares means method using season-corrected data.

Effect of polymorphism in the 5'-flanking region of porcine HSP70.2 on performance measures
The effect of genotypes of the 5'-flanking region of porcine HSP70.2 on growth performance traits was evaluated.Since the season effect was significant on the performance trait, the effect of genotypes on growth performance was determined based-on season-corrected data.Table 1 summarized the association of porcine HSP70.2 with ADG, BF, AGE and FE.Statistical analyses of animals from purebred farms in Taiwan revealed significant differences between genotypes with regards to the growth performance of backfat thickness.The A deletion homozygotes at nt 250 and TC heterozygotes and TT homozygotes at nt 393 displayed the thinner backfat thickness (p<0.05).The polymorphism did not have significant effect on the other performance traits.
A PCR-RFLP in the 5'-flanking region of porcine HSP70.2Although all five polymorphic sites were associated with growth traits, only the nt 393 could be detected quickly by RFLP. Figure 1 shows the PCR-RFLP at nt 393.After Bsa WI digestion, the 227 bp long PCR product was cleaved into two fragments of 157 and 70 bp (variant T) each or was still the original fragment (variant C).

DISCUSSION
The development of genetic maps of swine has allowed identification of quantitative trait loci (QTL) affecting a variety of traits of economic importance.While these results are promising with respect to potential improvement of pork production through marker-assisted selection procedures, it would be of significant interest to identify the genes and specific allelic polymorphisms underlying the observed variation.This achievement would allow development of specific DNA-based genetic tests to evaluate individual animals for merit and genetic potential, as well as to increase understanding of the genetic basis underlying non-disease producing variability among populations.Several QTL were identified on the distal portion of the long arm of SSC1, representing correlated traits of early growth rate (from 8 to 18 weeks), weight at 26 weeks, backfat thickness at 14 and 26 weeks of age, and age at puberty.However, backfat thickness QTL on SSC2 and SSC7 in Meishan × Large White and Landrace (F2) cross which was reported by Rattink et al. (2000) is in the same locus as this study indicated.
The results of the present study confirmed the presence of nt 393 polymorphism in the 5'-flanking region of HSP70.2.Our previous study (Chen et al., 2000) indicated the presence of this polymorphism in three pig breeds.We found that the "T" allele was mostly in the heterozygous form.Homozygotes TT appeared only in Duroc pigs, not in Landrace and Yorkshire.Therefore, in the present study the relationship of this polymorphic site with growth performance was examined only in Duroc pigs.Promoter variants differing in potential cis-acting elements and showing impaired binding of corresponding transcription factors were described in the inducible porcine HSP70.2 (Schwerin et al., 2001).Mutation of the GC box in the HSP70.2promoter resulted in a significant decrease of heatinduced transcription compared to the corresponding wildtype promoter.Therefore, other variants in the 5'-flanking region of porcine HSP70.2 could be associated with mRNA abundance and protein translation level.
The results of this study suggest that the nt 393 single nucleotide polymorphism in the 5'-flanking region of porcine HSP70.2 may be used as a marker for early selection of ultrasonic backfat thickness in Duroc pigs.

Figure 1 .
Figure 1.Characterization of Bsa WI polymorphism in the 5'flanking region of porcine HSP70.2.Amplification products of genomic DNA from three different individuals with the three possible genotypes are shown.The PCR products were digested with Bsa WI at nt 393 as indicated.Lane 1: DNA molecular marker, 2: homozygous CC, 3: heterozygous TC, and 4: homozygous TT.

Table 1 .
Effect of the nt 393C/T polymorphism in the 5'-flanking region of HSP70.2 on growth performance in Duroc pigs a,b Least-squares means differ significantly (p<0.05) for genotypes at the same site with different superscripts.