J I T A A Journal of the Indonesian Tropical Animal Agriculture Accredited by Ditjen Penguatan Risbang No. 60/E/KPT/2016 J. Indonesian Trop. Anim. Agric. pISSN 2087-8273 eISSN 2460-6278 http://ejournal.undip.ac.id/index.php/jitaa 42(3):175-184, September 2017 DOI: 10.14710/jitaa.42.3.175-184 Genetic polymorphism analysis of 5' untranslated region of thyroglobulin gene in Bali cattle (Bos javanicus) from three different regions of Indonesia S. Anwar1, A.C. Putra2, A.S. Wulandari1, P. P. Agung1, W.P.B. Putra1 and S. Said1 1Research Center for Biotechnology-Indonesian Institute of Sciences (LIPI), Jl. Raya Bogor KM. 46, Cibinong 16911, West Java - Indonesia 2Department of Biological Science, Faculty of Mathematics and Natural Science, Padjajaran University, Jl. Raya Bandung-Sumedang Km. 21 Jatinangor Sumedang 45363, West Java - Indonesia Corresponding E-mail:saif005@lipi.go.id Received April 06, 2017; Accepted June 05, 2017 ABSTRAK Variasi nukleotida g.422C>T pada gen thyroglobulin dibagian 5’untranslated region (5’UTR) (disebut TG5) telah dilaporkan mempunyai asosiasi dengan tingkat kandungan lemak intramuskuler atau marbling pada sapi potong. Penelitian ini ditujukan untuk mengkonfirmasi keragaman genetik gen TG5 pada sapi Bali dari tiga wilayah sumber bibit yang berbeda di Indonesia. Sebanyak 200 ekor sapi Bali telah dilakukan genotyping pada gen TG5 menggunakan metode polymerase chain reaction-restriction fragment lenght polymorphism (PCR-RFLP) dan analisis sekuen. Hasil penelitian menunjukkan bahwa TG5 terkonfirmasi monomorfik pada sapi Bali dimanapun wilayah asalnya. Selain itu, sebanyak sembilan kandidat SNP ditemukan di dalam gen TG bagian 5’UTR pada sapi Bali dibandingkan dengan sekuens Genbank, meskipun tidak ditemukan variasi SNP di dalam sampel sapi Bali yang diteliti. Marker genetik lain dari gen TG perlu dieksplorasi dan diverifikasi keragamannya pada sapi Bali. Sembilan kandidat SNP juga perlu diverifikasi dan divalidasi lebih lanjut dengan sampel yang lebih besar untuk dapat dikatakan sebagai SNP baru antara sapi Bali dengan referensi sekuen dari GenBank. Kata kunci : gen thyroglobulin, marbling, polimorfisme genetik, sapi Bali, 5’-untranslated region ABSTRACT The g.422C>T nucleotide variations in the 5’ untranslated region (5’UTR) of TG gene (called as TG5) has been reported to be associated with level in intramuscular fat (IMF) content or marbling in beef cattle. The objective of this study was to confirm genetic polymorphism of TG5 gene in Bali cattle populations from three different regions as the main resources of Bali cattle in Indonesia. A total of 200 head of Bali cattle have been performed genotyping on TG5 gene using polymerase chain reaction- restriction fragment lenght polymorphism (PCR-RFLP) method and sequence analysis. Results of the study confirmed that TG5 was monomorphic in Bali cattle wherever their origin regions. Moreover, nine candidate SNPs were detected within 5’UTR of TG gene in Bali cattle compared to Genbank reference sequences, although no SNP variations among Bali cattle sample studied. The new other genetic markers within an entire TG gene suggested to be explored and verified for their polymorphisms in Bali cattle. 5’UTR of Thyroglobulin Gene Polymorphism in Bali Cattle (S. Anwar et al.) 175 The nine candidate SNPs were also required further verification and validation in a larger sample to be regarded as new SNPs between Bali cattle and Genbank reference sequences. Keywords : Bali cattle, genetic polymorphism, marbling, thyroglobulin gene, 5’-untranslated region INTRODUCTION Beef palatability has becoming a major concern of consumers satisfaction in consuming beef. Intramuscular fat (IMF) or also called as marbling has been studied contributed to the beef palatability or eating quality of beef through its attributes such as flavour, juiciness and tenderness (Okumura et al., 2007; Hudson et al., 2015), even though several studies showed that the association between marbling score and beef palatability attributes was a positively small (Wheeler et al., 1994; Platter et al., 2003). However, marbling score has been used as one of major determinants for beef palatability in international markets especially in Japan, Australia and USA (Watson et al., 2008; Gotoh et al., 2014). Molecular markers could be used to identify the quantitative trait loci (QTLs) that affect desired quantitavive traits (Dekkers, 2004; Van Eenennaam et al., 2007). Several studies have identified QTLs associated with fatness traits in cattle located on bovine chromosome 14 (BTA14) including thyroglobulin gene (TG) (Moore et al., 2003; Casas et al., 2005). Thyroglobulin gene encodes thyroglobulin, a glycoprotein hormone that is sinthesized by thyroid follicular cells and stored in thyroid gland. The TG acts as precursor for the thyroid hormone, the triiodothyronine (T3) and thyroxine (T4) hormones. Thyroid hormones are known to play an important role in influencing the lipid metabolism, adipocyte growth and differentiation (Ailhaud et al., 1992; Casas et al., 2005; Pannier et al., 2010). The nucleotide substitution cytosine (C) to thymine (T) at the position of 422 in the 5’- untranslated region (5’UTR) of TG gene (TG5) has been reported to be associated with level in IMF content or marbling score in beef cattle (Barendse, 1999). Therefore, this finding has been proposed as candidate gene for improving marbling level and eating quality in beef cattle. Recently, TG5 has been used as genetic marker to improve the marbling level and other fatness traits of beef in Bos taurus and Bos indicus cattle (Casas et al., 2005; Van Eenennaam et al., 2007; Gan et al., 2008; Bonilla et al,. 2010; Anton et al., 2011). In previous study, Anwar et al. (2016) have identified that TG5 genetic marker tend to be monomorphic in 100 samples of Bali cattle at Maiwa Breeding Center (MBC), Enrekang District, South Sulawesi Province using PCR- RFLP method. For further confirmation of this result, more sample size from three different purebreeding regions of Bali cattle have been examined to investigate the possibility of changes in the distribution of alleles and genotypes in the sample studied by using PCR-RFLP method and sequence analysis. Therefore, the objective of this study was to confirm genetic variation of TG5 and to explore new candidate SNPs within 5’UTR of TG gene in Bali cattle (Bos javanicus) compared to GenBank reference sequences. MATERIALS AND METHODS Animals and Blood Collection A total of 200 head of Bali cattle were used in this study. The animals were originated from three different regions as the main resources of Bali cattle in Indonesia, including : 1) South Sulawesi Province (SS) represented samples from Barru District (76 heads), 2) Bali Province (Bali) represented samples from island of Nusa Penida (52 heads) and West Nusa Tenggara (NTB) repesented from Banyumulek Sub-district (72 heads). All animals were unkown pedigree data. Blood sample of each animal was collected into vaccutainer tube containing K2EDTA as anticoagulant. The blood samples were kept at 4- 5°C for further use. DNA Isolation and PCR Amplification Genomic DNA isolation was performed using Genomic DNA mini kit (Geneaid Biotech Ltd., Taiwan) according to the the manufacturer’s instructions. DNA products were stored at -20°C for further use. A 545 base pairs (bp) of 5’UTR of TG gene was amplified using a pairs of primer designed by Barendse (1999) based on the GenBank reference sequence (accession no: X05380.1). The PCR reaction and program were performed according to Anwar et al.(2016). 176 J.Indonesian Trop.Anim.Agric. 42(3):175-184, September 2017 PCR-RFLP Genotyping Allele and genotype variations of TG5 gene were detected using PCR-RFLP method. The BstYI restriction enzyme (New England Biolabs, USA) with the restriction site at 5’ R↓GATCY 3’ were used to detect g.422C>T polymorphic site in 5’UTR of TG gene. The digestion mixture and procedure were performed according to Anwar et al. (2016). Gene Sequencing and Sequence Analysis In this study, DNA fragments of all samples have been found only one electrophoretic band pattern (CC genotype), while CT and TT genotype were not observed. However, in sequence analysis, we used seven samples consisting a total of six samples represent of CC genotype (four from SS, one from Bali and one from NTB regions). The three of four samples from SS were taken from our previous study (Enrekang District) while one of four samples was taken from Barru District. The one heterozygous CT animal found in Enrekang District was used as control to detect g.422C>T mutation. The two GenBank accession numbers (X05380.1 and AY615525) of TG gene of Bos taurus species were used to compare the different SNP (g.422C>T). The accession number of AY615525 was an updated sequence data from X05380.1 which contained two newly SNPs found in Korean cattle populations (g.257C>T and g.335A>G) and has been submitted to GenBank by Shin and Chung (2007). DNA fragments of each sample were sequenced for both directions using PCR primers forward and reverse performed by commercial laboratory service at First BASE Laboratories Sdn. Bhd. (Selangor, Malaysia) using ABI PRISM 96-capillary 3730xl DNA Analyzer (Applied Biosystems, USA). Sequence analysis begins with a contig analysis which combines both the sequencing results (forward and reverse) to obtain a complete single sequence of each sample. The nucleotide sequence obtained were then aligned with the GenBank reference sequences (Accession number : X05380.1 and AY615525) and analyzed by using Bioedit sequence alignment editor (Hall, 1999) and ClustalW alignment method in Molecular Evolutionary Genetic Analysis (MEGA6) software (Tamura et al., 2013). RESULTS AND DISCUSSION PCR Product and Genotype Variants The TG5 is code used for genetic marker found in the 5’UTR of TG gene which has nucleotide substitution (C>T) at the position of 422. In this study, a 545 bp targeted DNA sequence of 5’UTR of TG gene was succesfully amplified using a pairs of primer designed by Barendse (1999) as shown by the result of a single clear band (Figure 1). Detection of genotype variants of TG5 was performed using PCR-RFLP method and then confirmed using sequence analysis. The genotype found in this study only CC genotype whereas CT and TT genotypes were not detected in 200 sample studied. Therefore, in order to know the position of g.422C>T mutation, one DNA sample represent CT genotype in previous study was used 5’UTR of Thyroglobulin Gene Polymorphism in Bali Cattle (S. Anwar et al.) 177 Figure 1. Electrophoretic PCR Product Visualization of TG5 Gene (545 bp) in a 1.2% Agarose Gel (w/v). Lane M: marker with 100 pb DNA ladder; lane 1,2,3,4,5: number of sample. to be sequenced (Anwar et al., 2016) as a control in sequence analysis. The genotype variants (CC and CT) corresponding to g.422C>T mutation of TG5 were succesfully confirmed through DNA sequencing. On the C allele, the base C at the position of 422 were not changed so that the BstYI restriction enzyme recognize the restriction site and cuts the DNA into three fragments (72, 295 dan 178 pb). While on the T allele, BstYI could not recognize the restriction site at the position of 422 because the base C was replaced by base T and generates only two fragments (72 dan 473 pb). The position of TG5 gene within GenBank reference sequence (accession no : X05380.1), its restriction map and chromatogram visual representation of SNP (g.422C>T) are shown in Figure 2. Genetic Polymorphism Analysis Genetic polymorphism could be measured at various levels, e.g. allelic and genotypic variation at certain locus or loci within a populations. In previous study, Anwar et al. (2016), showed that the frequency of C allele and CC genotype of TG5 gene from 100 samples derived from Maiwa Breeding Center (MBC), Enrekang District, South Sulawesi (SS) were 0.995 and 0.990, respectively, and suggested to be monomorphic. In this study, the different sample locations have been used. A total of 200 head of Bali cattle were taken from three different regions that designated by Indonesian government as purebreeding locations for Bali cattle including South Sulawesi (SS), Bali and West Nusa Tenggara (NTB) Province to confirms the polymorphism of TG5 gene. The allelic and genotypic frequencies for the TG5 gene studied in Bali cattle from three regions are shown in Table 1. The C allele and CC genotype were found to be fixed in all three region samples (1.000 and 1.000, respectively), whereas the T allele did not observed. As the consequent of the absence of T allele in total samples, CT and TT genotypes were not observed in this study (0.000 and 0.000, respectively). The results in this study indicate that C allele and CC genotype of TG5 gene were found to be fixed in Bali cattle (Bos javanicus) wherever their origin region. According to Nei (1987), Frankham et al. (2004) and Allendorf and Luikart (2007), when a locus in a population found only one allele or if the most common allele is known to be a high frequency (more than 95% or 99%), the locus was considered as monomorphic. This finding confirms on Anwar et al. (2016) study, that the TG5 gene was monomorphic in Bali cattle in which the most common allele (C allele) was 0.995 or 99.5%. The TG5 marker was located in the 5’ leader sequence or 5’ UTR of the TG gene. Barendse et al. (2004) stated that TG5 was chosen for marker 178 J.Indonesian Trop.Anim.Agric. 42(3):175-184, September 2017 Figure 2. Schematic Representation of the Position of TG5 Gene, Restriction Map and Chromatogram Visual Representation of SNP within GenBank Reference Sequence (accession no : X05380.1). The arrows indicate a pairs of primer position (forward and reverse) to initiated a process of amplification. The restriction site of BstYI (5' R↓GATCY 3’) are indicated with an asterisk. In the T allele, nucleotide subtitution (C>T) at the position of 422 causing BstYI restriction enzyme do not recognize the restriction site at this position. genetic test, due to the known low level of polymorphism in the TG coding sequence and more importantly to the features present in that DNA sequence. Furthermore, TG5 is in the leader sequence of the gene affects for the RNA polymerase III binding site which important to initiate transcription in regulation of gene expression, although its functional effect to circulating level of thyroid hormones (T3 and T4) were not undertaken yet. The T allele in TG5 marker has been proposed by Barendse et al. (2004), Casas et al. (2005), Bonilla et al. (2010) and Anton et al. (2011), as the favourable allele because the homozygous TT cattle tend to be higher in IMF fat deposition or marbling score than heterozygous CT and homozygous CC cattle. This TG5 marker was then launched as a DNA marker test for commercial use and has been performed a validation studies although genetic association analysis with marbling traits still varied results in some cattle breeds (Casas et al., 2005; Rincker et al., 2006; Shin dan Chung, 2007; Van Eenennaam et al., 2007; Johnston and Graser, 2010 and Pannier et al., 2010). The allelic and genotypic frequencies of TG5 gene has been reported in several studies in Bos taurus, Bos indicus and its crossbreeds cattle as presented in Table 2. It showed that C allele of TG5 was the most common allele found in all cattle breeds. However, C allele was more frequent found in Bos indicus breeds such as Brahman cattle (Casas et al., 2005) and Nelore cattle (Fortes et al., 2009) and tend to be fixed as well as in Bos javanicus than in Bos taurus breeds. Conversely, the T allele was mostly frequent in Bos taurus breeds (0.229) compared to Bos indicus (0.020) and Bos javanicus (0.002), although the frequency of T allele in Bos taurus was varied (0.016-0.487). Wagyu cattle breeds were regarded as Bos taurus type that carrying T allele, and the consequently on its inheritances (Casas et al., 2007; Van Eenennaam et al., 2007). This occurance may caused by strong selection in Wagyu breed for marbling traits as well as on alleles of other candidate genes associated with high marbling (Watanabe et al., 2011). In this study, the C alel was fixed in Bali cattle populations represented from three selected regions (SS, Bali and NTB). These regions were established by Indonesian government for Bali cattle purebreeding since 1976 as well as in East Nusa Tenggara (NTT) (Handiwirawan and Subandriyo, 2004). Then, in 2013, Barru District, South Sulawesi Province, designated as a new location for Bali cattle purebreeding through Ministerial Decree No. 4437/Kpts/SR.120/7/2013 (Ministry of Agriculture of the Republic of Indonesia, 2013). This results also indicate that if C allele was fixed in Bali cattle, additional samples of Bali cattle should not be increasing the frequency of T allele with the exception in case of mutation or when it crossed with other breeds confirmed the T allele carriers. Morever, crosbreeding may still be difficult to increase the frequency of T allele because the action of TG5 suggested as recessive mode of inheritance (Casas et al., 2007). This is the possible reason for previous study by Anwar et al. (2016), that has found only one heterozygous cattle (CT genotype) in Enrekang District, which is not included as location of purebreeding of Bali cattle. However, it needs to be further in-depth investigation. Frankham et al. (2004), stated that the other possible factors that allow allele or genotype to be fixed were inbreeding, genetic drift, small, isolated and sustained reduction population size. In the sequence analysis between six 5’UTR of Thyroglobulin Gene Polymorphism in Bali Cattle (S. Anwar et al.) 179 Table 1. Allelic and Genotypic Frequencies of TG5 Gene in Bali Cattle ( Bos javanicus) from Three Different Regions Marker Region Total(n) Genotypic Frequencies Allelic Frequencies CC (n) CT (n) TT (n) C T TG5 SS 76 1.000 (76) 0.000 (0) 0.000 (0) 1.000 0.000 Bali 52 1.000 (52) 0.000 (0) 0.000 (0) 1.000 0.000 NTB 72 1.000 (72) 0.000 (0) 0.000 (0) 1.000 0.000 Total 200 1.000 (200) 0.000 (0) 0.000 (0) 1.000 0.000 SS = South Sulawesi Province, Bali = Bali Province, NTB = West Nusa Tenggara Province 180 J.Indonesian Trop.Anim.Agric. 42(3):175-184, September 2017 Table 2. Allelic and Genotypic Frequencies of TG5 Gene in Numerous Cattle Breeds Species Breeds n Genotypic Frequencies Allelic Frequencies References CC (n) CT (n) TT (n) C T Bos taurus Limousin 123 0.667 (82) 0.317 (39) 0.016 (2) 0.825 0.175 1 Charolais 80 0.563 (45) 0.350 (28) 0.088 (7) 0.738 0.263 1 Simmental 58 0.397 (23) 0.552 (32) 0.052 (3) 0.672 0.328 1 Simmental 438 0.534 (234) 0.390 (171) 0.075 (33) 0.729 0.271 2 Angus 819 0.643 (527) 0.32 (265) 0.033 (27) 0.805 0.195 3 Angus 39 0.205 (8) 0.615 (24) 0.179 (7) 0.513 0.487 1 Angus 173 0.457 (79) 0.410 (71) 0.133 (23) 0.662 0.338 2 Hereford 32 0.969 (31) 0.031 (1) 0.000 (0) 0.984 0.016 1 Belgian blue 19 0.737 (14) 0.158 (3) 0.105 (2) 0.816 0.184 1 Friesian Holstein 76 0.737 (56) 0.263 (20) 0.000 (0) 0.868 0.132 1 Blonde d'Aquitaine 13 0.615 (8) 0.308 (4) 0.077 (1) 0.769 0.231 1 Friesian Holstein 415 0.745 (309) 0.241 (100) 0.014 (6) 0.865 0.135 2 Friesian Holstein 1279 0.580 (742) 0.390 (499) 0.030 (38) 0.775 0.225 4 Jersey 283 0.601 (170) 0.350 (99) 0.049 (14) 0.776 0.224 2 Korean 309 0.411 (127) 0.460 (142) 0.129 (40) 0.641 0.359 5 TOTAL 4156 0.59 (2455) 0.360(1498) 0.049(203) 0.771 0.229 Bos indicus Brahman 467 0.946 (442) 0.039 (18) 0.015 (7) 0.966 0.034 6 Brahman 383 0.992 (380) 0.008 (3) 0.000 (0) 0.996 0.004 7 Nellore 46 1.000 (46) 0.000 (0) 0.000 (0) 1.000 0.000 8 TOTAL 896 0.969 (868) 0.023 (21) 0.008 (7) 0.980 0.020 Bos javanicus Bali 100 0.990 (99) 0.010 (1) 0.000 (0) 0.995 0.005 9 Bali 200 1.000 (200) 0.000 (0) 0.000 (0) 1.000 0.000 * TOTAL 300 0.997 (299) 0.003 (1) 0.000 (0) 0.998 0.002 Crossbreed British-GPE6 211 0.564 (119) 0.341 (72) 0.095 (20) 0.735 0.265 10 Norwegian-GPE6 72 0.653 (47) 0.306 (22) 0.042 (3) 0.806 0.194 10 Swedish-GPE6 68 0.574 (39) 0.324 (22) 0.103 (7) 0.735 0.265 10 Friesian- GPE6 149 0.631 (94) 0.349 (52) 0.020 (3) 0.805 0.195 10 Wagyu-GPE6 153 0.484 (74) 0.405 (62) 0.111 (17) 0.686 0.314 10 Angus cross 40 0.650 (26) 0.275 (11) 0.075 (3) 0.788 0.213 11 RGxN 26 0.962 (25) 0.038 (1) 0.000 (0) 0.981 0.019 8 Canchim 41 0.610 (25) 0.341 (14) 0.049 (2) 0.780 0.220 8 B3x 19 0.684 (13) 0.316 (6) 0.000 (0) 0.842 0.158 8 BV3x 15 0.467 (7) 0.400 (6) 0.133 (2) 0.667 0.333 8 TOTAL 794 0.591 (469) 0.338 (268) 0.072 (57) 0.759 0.241 n: number of sample; 1: Pannier et al. (2010); 2: Anton et al. (2012); 3: Barendse et al. (2004); 4: Khatib et al. (2007); 5: Shin and Chung (2007); 6: Casas et al. (2005); 7: Smith et al. (2009); 8: Fortes et al. (2009); 9: Anwar et al. (2016); 10: Casas et al. (2007); 11: Anton et al. (2013); *: this study. GPE6 = crossbred genetic group by maternal grandsire. RGxN = Rubia Gallega X Nelore,Canchim =(5/8 Bos taurus + 3/8 Bos indicus), B3x = (9/16 Bos taurus + 7/16 Bos indicus), BV3x =(3/4 Bos taurus + 1/4 Bos indicus). consensus sequence samples in this study compared to GenBank reference sequences (X05380.1 and AY615525), obtained only 523 bp of 545 bp targeted sequence which is located on the base pair of 63 to 585 (Figure 3). Individual sample represent CT genotype that found by Anwar et al. (2016) was not regarded as polymorphic, thus it could not be included in the sequence analysis. In alignment analysis, besides g.422C>T mutation, the total of nine new candidate SNPs were also identified in Bali cattle compared to Genbank reference sequences (X05380.1 and AY615525). The two SNPs were known as transversion nucleotide substitutions, while seven SNPs were known as transitions nucleotide substitutions. This findings supports the statement of Nei and Kumar (2000), in which transitionals were more frequent than transversions nucleotide substitutions in most DNA segments. However, nine new candidate SNPs found in this study could not regarded as novel SNPs because the sequence analysis conducted only in small studied samples. The candidate SNPs, its locations and the type of nucleotide subtitutions are shown in Table 3. In this study, the six of nine candidate SNPs compared to both the X05380.1 and AY615525 were located in the base position of 240, 274, 387, 416, 537 and 552, respectively. The two of nine candidate SNPs compared only with X05380.1 were in the base position of 257 and 335, while one candidate SNP compared only with AY615525 was in the base position of 404 (Table 3). However, there are no SNP variations in all those nine candidate SNPs among six samples studied and were not considered as polymorphic site within Bali cattle or Bos javanicus populations. As the results in this study, the other new genetic markers may be explored as well as Fortes et al. (2009) suggestion from their study in Nelore cattle. Gan et al. (2008) and Zang et al. (2015) have reported any SNPs within TG gene in beef cattle. Gan et al. (2008), have found six SNPs in the 3’-flanking region and one of six SNPs significantly associated with marbling score in Bos taurus breeds. Zang et al. (2015), have identified four SNPs in the 5'-flanking region of the TG gene and two of four SNPs significanlty associated with some carcass and meat quality traits in Chinese steers. For further studies, those SNPs entire TG gene can be verified for their polymorphisms in Bali cattle. Furthermore, to ensure the nine nucleotide variations that found in this study to be regarded as SNPs between Bali cattle and Genbank reference sequences, those required further verification and validation in a larger sample. 5’UTR of Thyroglobulin Gene Polymorphism in Bali Cattle (S. Anwar et al.) 181 Figure 3. Schematic representation of the position of nine candidate SNPs within (a) a 523 bp of consensus sequence of 5’UTR of TG gene in Bali cattle compared to b) AY615525 and c) X05380.1 of GenBank reference sequences. The g.422C>T indicated with an asterisk. CONCLUSION It can be concluded that TG5 was confirmed to be monomorphic in Bali cattle (Bos javanicus). Nine candidate SNPs has been found within 5’UTR of TG gene in Bali cattle compared to Genbank reference sequences, although there were no SNP variations within Bali cattle studied. The new other genetic markers within TG gene may be explored and verified for their polymorphisms on Bali cattle. The nine candidate SNPs were also required further verification and validation in a larger sample to be regarded as SNPs between Bali cattle and Genbank reference sequences. ACKNOWLEDGMENTS This research was supported by Enrekang Technopark Program, funded by Center for Plant Conservation-Botanic Gardens, Indonesian Institute of Sciences (LIPI). Part of the samples were from Banyumulek Technopark Program. The authors are grateful to Edy Sophian, Ilham Syarif, Citra and Lusiana for their blood sampling assistance. The authors appreciate Hasanuddin University, Livestock and Fisheries Office of Enrekang District, and Livestock Office of Barru District, South Sulawesi Province, and the farmers for the cooperation and providing the samples of animal. REFERENCES Ailhaud, G., P. Grimaldi and R. Negrel. 1992. Cellular and molecular aspects of adipose tissue development. Annu. Rev. 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