Genetic Polymorphisms of the Coding Region (Exon 6) of Calpastatin in Indonesian Sheep

Calpastatin (CAST) is an indigenous inhibitor of calpain that involved in regulation of protein turn over and growth. The objective of this research was to identify genetic polymorphisms in the entire exon 6 of calpastatin gene in Indonesian local sheep. A PCR-SSCP method was carried out to identify genetic variation of CAST gene. In total 258 heads of local sheep from 8 populations were investigated, three groups of samples were Thin Tail Sheep (TTS) from Sukabumi, Jonggol, and Kissar. The rest samples were Priangan sheep (PS) from Margawati (Garut meat type) and Wanaraja (Garut fighting type) and Fat Tail Sheep (FTS) from Donggala, Sumbawa, and Rote islands. SSCP analysis revealed that three different SSCP patterns corresponded to three different alleles in the CAST locus (CAST-1, 2, and 3 allele) with five different genotypes. Genetic variation between local sheep populations were calculated based on genotypic and allelic frequencies. Most populations studied were polymorphic, with genotype frequencies of CAST-11, CAST-12, CAST-22, CAST-32, and CAST-33 were 0.286, 0.395, 0.263, 0.046, and 0.007 respectively. CAST-1 and 2 alleles were most commonly found in all populations with total frequency was 0.970, while CAST-3 was a rare allele 0.030 and only found in TTS population. Variation in the CAST gene could be used for the next research as genetic diversity study or to find any association between CAST polymorphism with birth weight, growth trait and carcass quality in Indonesian local sheep.


INTRODUCTION
Calpastatin (CAST) is a member of calpain calpastatin system involving three molecules, µ-calpain, m-calpain and calpastatin as specifi c inhibitor of the two calpain. This system implicated in various physiological and pathological processes (Kidd et al., 2000;Huang et al., 2001;Goll et al., 2003;Raynaud et al., 2004) and involved in regulation of protein turn over and growth (Goll et al., 1992), myoblast migration (Dedieu et al., 2003) and fusion (Temm-Grove et al., 1999). CAST is therefore believed to be an excellent candidate gene for growth and carcass trait in livestocks.
Many studies have demonstrated the association of CAST polymorphism with carcass and meat quality, especially tenderness in several livestocks (Schenkel et al., 2006;Casas et al., 2006;Curi et al., 2009). In the sheep, the polymorphism of CAST gene was reported to have signifi cant association with birth weight (Byun et al., 2008), and body weight (Sumantri et al., 2008,) but did not infl uence lamb tenderness (Zhou et al., 2008a).
Previous study reported the polymorphisms of CAST in Indonesian local sheep but this study only concentrated in intron region (Sumantri et al., 2008), and no investigation yet reported in coding sequences of local sheep. Exon 6 was the largest exon in the ovine calpastatin, and known to be polymorphic in the sheep with fi ve diff erent alleles (Zhou et al., 2007;Byun et al., 2009a). Previous study has shown that sequences coded by exon 6, contained multiple phosporylation sites, and directly involved in determining the cell localization of calpastatin (Tullio et al., 2009). This study suggested that variation in these sequences may impact on the activity of Ca 2+ channels and hence regulate or modulate calpain activity. The objectives of this research were to identify polymorphism of CAST gene in the coding region (Exon 6) in Indonesian local sheep population.

PCR Amplifi cation
A pair of PCR primer, forward: 5'-GTTATGA ATTGCTTTCTACTC-3' and reverse: 5'-ATACGATT GAGAGACTTCAC-3' was designed to amplify part of intron 5 and whole exon 6 of CAST gene, as described by Zhou et al. (2007). PCR amplifi cation was carried out in 25 µl reaction containing 50-100 ng genomic DNA, 0.25 µM of each primer, 200 µM dNTPs (Fermentas), 4.0 µM Mg 2+ , 0.5 U of Toptaq DNA polymerase (Qiagen, Hilden, Germany), and 1x the reaction buff er. The condition of thermal cycling consisted of pradenaturation at 95 o C for 5 min, followed by 35 cycles of denaturation 95 o C for 30 s, annealing 56 o C for 45 s, and extension 72 o C for 45 s. The fi nal extension step was at 72 o C for 5 min. Amplifi cation was carried out in a thermal cycler (Mastercycler Personal 22331, Eppendorf, Germany). The PCR amplicon were checked on 1.5% agarose gels in 0.5 x TBE buff er containing 10% of ethidium bromide at 100 volt for 45 min and visualized by UV transiluminator.

Single Strand Conformational Polimorphism (SSCP) Analysis
A SSCP procedure was used to identify variation in the amplicon of CAST locus. A 5 µl aliquot of each amplimer was mixed with 5 µl of loading dye (98% formamide, 10 mM EDTA, 0.025% bromophenol blue, 0.025% xylene cyanol). After denaturation at 95 o C for 5 min, samples were rapidly cooled on ice bath and then loaded on 12% acrylamide : bisacrylamide (29 : 1) gels. Electrophoresis was performed by using Protean II xi cells (Bio-Rad), at 300 V for 18 h at refrigerator condition in 0.5 x TBE buff er. Gels were silver stained based on the method of Byun et al. (2009b) with modifi cation in staining solution (0.1% AgNO 3 , 0.04% NaOH 10 N, and 0.4% NH 3 ).

DNA Sequencing and Analysis
be confi rmed as homozygous were used directly as templates for DNA sequencing. Prior to sequencing, one of the unique bands representing each allele was cut out of the polyacrylamide gel (PAGE) and purifi ed by the method as described by Hu et al. (2010). This was then used as the DNA template for reamplifi cation and sequencing. To ensure these templates were similar to original sequences and not the result of amplifi cation error, the identity of the templates cut from PAGE generated from the templates and the corresponding genomic DNA. Sequence alignments, translations and comparisons were carried out using MEGA software version 4.0 (Tamura et al., 2008). The BLAST (basic local alignment search tool) program was used to search the NCBI GenBank ( /BLAST) databases for homologous sequences.

Statistical Analysis
The genotype and allele frequencies were calculated based on Nei & Kumar (2000) formulation.
where Xi= ii th genotype frequency, X i = i th allele frequency, n ii = number of sample of ii genotype, n ĳ = number of sample of ij genotype, and N= total sample.
where χ²= chi-square , Obs= number of observation of ii th genotype, and Exp= number of expected of ii th genotype.
where H o = observed within-population heterozygocity, H e = expected within-population heterozygocity, w k = relative population size, X kij (i≠j)= the frequency of A i A j in the k th population.

PCR-SSCP Analysis of CAST Gene
Part of intron 5 and entire exon 6 of CAST gene were amplifi ed by PCR using forward and reverse primer, with predicted amplicons 254 bp in length ( Figure 1). PCR-SSCP analysis showed polymorphism three diff erent alleles, CAST-1, CAST-2, and CAST-3 found in each individual sample that consistent with either homozygous or heterozygous with fi ve diff erent genotypes, CAST-11, CAST-12, CAST-22, CAST-32, and CAST-33. The CAST-31 genotype was not observed in this study. Figure 2 shows the electrophoresis of CAST genotypes after SSCP.
The level of polymorphisms that found in this study was lower than reported by Zhou et al. (2007) with fi ve diff erent allelic in Merino, Corriedale, Poll Dorset and NZ crossbreed sheep. However, it was higher than previously reported in Indonesian local sheep (using PCR-RFLP; Sumantri et al., 2008), Iranian Karakul sheep (Shahroudi et al., 2006), and Kurdi sheep .

Genetic Diversity of CAST Gene
The results of this study may indicate that the CAST gene in the local sheep is polymorphic in all populations. Genotype frequency and allele frequency of the CAST gene is presented in Table 1. The study observed only three alleles (CAST-1, 2 and 3) and fi ve genotypes 12,22,32,and 33) in Indonesian local sheep populations, but not genotype of CAST-31. The most frequent alleles were CAST-1 and CAST-2 that contribute 48.4% for each allele and both counted 97%, while CAST-3 was rare allele (3%). The most frequent genotype was CAST-12 (39.5%). Zhou et al. (2007) also found that CAST-1 and CAST-2 were most common alleles, and both counted for 82% of the allele population in Merino, Corriedale, Romney, Poll Dorset and NZ cross-breed, while the rare allele were CAST-3 (13%), CAST-4 (2%), and CAST-5 (3%).
CAST-11 genotype frequencies in FTS group from Sumbawa, Rote, and Donggala population ranged from 0.08 to 0.166. Those values were lower than the PS group from Margawati (0.850) or TTS group from Jonggol (0.423) population. CAST-12 genotype frequencies in FTS group with the range of 0.103-0.833 was higher than PS group from Wanaraja (0.571) and TTS group from Kissar (0.531) population. While the CAST-22 frequencies in FTS with the range of 0.083-0.351 were higher than MTS and TTS group. CAST-32 genotype only found in TTS group (Sukabumi, Kissar, and Jonggol), with the highest frequencies in Kissar population (0.156). Frequency of CAST-33 genotype was 0.040 and only found in Sukabumi population.
Genotype and allele frequency diff erences in populations studies demonstrated the high diversity of local sheep. Local sheep population in Indonesia has CAST-1 and 2 alleles in the same frequency (48.4%) and spread throughout population, while the CAST-3 was rare Another study using PCR-RFLP reported the polymorphisms of calpastatin (CAST-Msp1 locus) in Indonesian local sheep. With two types of alleles (M and N), but only found two types of genotypes (MN and NN) with the frequency of 25% and 75% for MN and NN genotypes, and 13% and 87% for M and N alleles (Sumantri et al., 2008).
The result of chi-square (X 2 ) test showed the distribution of six genotypes in the population were not in Hardy-Weinberg Equilibrium (Tabel 2). CAST-31 genotype was not found, probably due to a non-random mating system or because of direct selection (Bourdon, 2000). According to Nei & Kumar (2000), genetic diversity can be measured by using heterozygosity value. Observed heterozygosity (44.2%), expected heterozygosity (53.0%), Nei's expected heterozygosity (52.9%),

Sequences Analysis
Sequence analysis revealed that amplicons varied from 253 to 254 bp in length. These were the expected size based on previously reported by Zhou et al. (2007). All of the sequences identifi ed were shared high similarity or identical to the published ovine and bovine CAST gene sequences (Figure 3). Based on homology of bovine CAST gene sequences with GenBank Accession Nos. EF443057 and AY834770 (Zhou et al., 2008c& Raynaud et al., 2005, exon 6 was the largest exon of ovine CAST gene, with 114 bp in length and coding around 38 amino acid residues. Based on sequence analysis, it identifi ed that three ovine CAST, and identifi ed four single nucleotide polymorphisms (SNPs). All of the nucleotide variation identifi ed in this study was similar to that reported previously by Zhou et al. (2007). were GenBank accession nos. of published ovine and bovine CAST sequences (Zhou et al., 2007(Zhou et al., & 2008c Levene (1949) and Nei's (1973).
and it functional signifi cance was unknown (Zhou et al., 2007). However, it has been suggested that calpastatin has a Ca2+ channel regulating function located in the L domain (Hao et al., 2000), and reported by Tullio et al. (2009) that sequences were coded by exon 6, containing multiple phosporylation sites, and directly involved in determining the cell localization of calpastatin. This suggests that variation in this sequences may impact on the activity of Ca2+ channels and hence regulate or modulate calpain activity. All variation in the intron region may aff ect RNA processing and consequently the function and level of expression of calpastatin (Zhou et al., 2007). Byun et al. (2008) reported that allele A (CAST-1) and C (CAST-3) had a signifi cant eff ect on birth weight, but did not signifi cantly aff ect the growth rate to weaning, while allele B (CAST-2) did not signifi cantly aff ect the birth weight and growth rate. All of these three alleles (CAST-1, 2, and 3) or genotypes variation in the CAST locus did not signifi cantly aff ect lamb tenderness (Zhou et al., 2008a).
The polymorphism of CAST exon 6 in goat also reported by Zhou et al. (2008b), who identifi ed a nonsynonymous amino acid variation in the caprine CAST which would result in a Ser/Arg amino acid change in the domain L of the protein. A synonymous SNP (T>C) mutation was also indentifi ed in bovine CAST exon 6 sequences (Zhou et al., 2008c).

CONCLUSION
Calpastatin (CAST) in the intron 5-exon 6 regions show polymorphism in Indonesian local sheep. Five genotypes were observed in this study, i.e CAST-11, CAST-12, CAST-22, CAST-32, and CAST-33 with the genotype frequencies were 0.286, 0.395, 0.263, 0.046, and 0.007 respectively. CAST-1 and CAST-2 were the most common alleles with total frequency in population 0.970, while the rarest allele was CAST-3 (0.030). Variation in the CAST gene could be used for the next research as genetic diversity study or to fi nd any association between CAST polymorphism with birth weight, growth trait and carcass quality in Indonesian local sheep.