272 J.Indonesian Trop.Anim.Agric. 46(4):272-281, December 2021 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 46(4):272-281, December 2021 DOI: 10.14710/jitaa.46.4. 272-281 Association of GHR|AluI gene polymorphism with body weight parameters in Grati-PO cattle H. Hartati 1* , A.A.R. Hapsari 2 , B.D.P. Soewandi 2 , S. Anwar 3 , S. P. Rahmadani 4 , A. Aryogi 1 and D. Pamungkas 1 1 Indonesian Beef Cattle Research Station, Indonesian Agency for Agricultural Research and Development, Ministry of Agriculture of Indonesia, Jl. Pahlawan No. 2 Grati, Pasuruan, East Java 67184 - Indonesia 2 Indonesian Research Institute for Animal Production (IRIAP), Ciawi, Bogor-Indonesia 3 Research Center for Biotechnology, National Research and Innovation Agency (BRIN), Jl. Raya Bogor Km. 46, Cibinong 16911, West Java Indonesia 4 Graduate Student of Animal Husbandry, University of Jambi *Corresponding Email: hartatifakhri16@gmail.com Received February 13, 2021; Accepted August 6, 2021 ABSTRAK Gen growth hormone receptor (GHR) merupakan anggota dari famili reseptor sitokin kelas I yang penting dalam regulasi pertumbuhan termasuk pada sapi. Penelitian ini bertujuan untuk menganalisa asosiasi antara gen GHR|AluI dan parameter bobot badan pada populasi sapi PO Grati. Sebanyak 304 sampel darah sapi PO dikoleksi dari kandang percobaan Loka Penelitian Sapi Potong yang dilengkapi dengan 304 data fenotipiknya. Sampel darah diisolasi menggunakan zymo extraction kit. Deteksi keragaman gen GHR menggunakan metode polymerase chain reaction-restriction frag- ment length polymorphism (PCR-RFLP) dengan enzim pemotong AluI. Indikator polimorfisme meli- puti frekuensi genotipe, frekuensi alel, Hardy-Weinberg Equilibrium, heterozigositas pengamatan (Ho), heterozigositas harapan (He), polymorphism information content (PIC) dan jumlah alel efektif (Ne). Analisis asosiasi dilakukan menggunakan linier mixed model. Performans bobot lahir, bobot sapih, bobot setahun dan bobot umur 18 bulan masing-masing adalah 25,4 ± 0,2kg; 113,8 ± 2,3 kg;159,9 ± 2,7 kg; dan 213,7 ± 3,6kg. Hasil analisis asosiasi signifikan (p<0,05) pada bobot sapih, bo- bot setahun dan bobot umur 18 bulan. Gen GHR berpotensi sebagai penanda genetik untuk sifat per- tumbuhan dan dapat digunakan sebagai kandidat penanda genetik untuk seleksi sapi PO-Grati. Kata kunci: Bobot badan, Growth hormone receptor, Polimorfisme, Sapi PO-Grati ABSTRACT The growth hormone receptor (GHR) gene is member of the class I cytokine receptor family that important in growth regulation including in cattle. This study aimed to analyze the association be- tween GHR|AluI gene with body weight, parameters in Grati-PO cattle population. A total of 304 blood samples were collected from the experimental station at the Beef Cattle Research Station, which Association GHR|AluI gene with Body Weight in Grati-PO Cattle (Hartati et al.) 273 INTRODUCTION Growth traits is one of the most important economic traits in beef cattle production. Body weight, body size measurements and weight gains are several types of growth traits. Growth traits have relationship between growth traits itself or with carcass and meat component (Meyer, 1995; Albertí et al., 2008; Caetano et al., 2013; Coyne et al., 2019), so it is widely used as a selection criterion in beef cattle breeding program. Body weight measurement is the most common type of growth traits used in the selection program, which are taken from birth and throughout an animal’s life (Buzanskas et al., 2014). The growth trait was controlled by many genes and their expression was the accumulation effects of genetic, environmental and interaction (Putra et al., 2014). Several genes that were thought to be the main genes in influencing growth traits were growth hormone (GH) and growth receptor (GHR) genes. The mechanism of action of these two genes has interrelated activi- ties in influencing cell growth and metabolism in vertebrates. The GH gene in influencing growth and metabolism will interact with a specific re- ceptor called the growth hormone receptor (GHR) on the surface of the targeted cell (Di Stasio et al., 2005). Growth hormone (GH) has proven to be the major regulator of postnatal growth and me- tabolism in mammals and thus affects growth rate, body composition, health, milk production, and aging by modulating the expression of many genes (Ge et al., 2003). The growth hormone receptor (GHR) is a member of the class I cyto- kine receptor family with a single transmem- brane domain (Waters and Brooks, 2015; Chia, 2014). GH receptor (GHR) are active in the cen- tral nervous system (CNS) and are crucial in reg- ulating several aspects of metabolism (Cady et al., 2017). Therefore, the essential biological function of GHR, makes the GHR gene proposed as a potential candidate gene for several econom- ically desirable traits including growth traits. The bovine GHR gene has been assigned located on bovine chromosome 20 (Moody et al., 1995). The polymorphism of the bovine GHR gene is relatively high. Waters et al., (2011) have conducted bovine GHR gene resequensing and found a total of 39 SNP entire gene using five different breeds. One of these SNP is g.257A>G (in exon 10) which causes a change in the amino acid serine to glycine (Ge et al., 2000). This SNP can be identified using PCR-RFLP with AluI restiction enzyme (GHR|AluI). The GHR|AluI gene was found to be polymorphic in bovine spe- cies (Di Stasio et al., 2005; Olenski et al., 2010; Zulkarnaim et al., 2010; Deepika and Salar, 2013; Deng et al., 2015). This polymorphism has been reported to be associated with the final weight and some meat quality traits in some Bos taurus cattle (Di Stasio et al., 2005; Reardon et al., 2010). However, has no association with var- ious body weight parameters including calf birth weight (CBW) in Aberdeen Angus cattle (Fedota et al., 2017),as well as with CBW in Grati-PO cattle (Hartati et al., 2019). However, the associ- were also equipped with 304 phenotypic data. DNA was extracted using a zymo extraction kit. Detec- tion of of the polymorphism of GHR gene using PCR-RFLP method with AluI restriction enzymes. polymorphism indicators include genotype frequency, allele frequency, Hardy-Weinberg Equilibrium, observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information content (PIC) and number of effective alleles (Ne). Association analysis was carried out using a linear mixed model. The performance of birth weight, weaning weight, yearling weight and 18 months weight were 25.4 + 0.2 kg; 113.8 + 2.3 kg; 159.9 + 2.7 kg and 213.7 + 3.6 kg, respectively. The result of association analy- sis was significant (P<0.05) on weaning weight, yearling weight and 18 months weight. It could be concluded that the GHR gene has potential to be a candidate genetic marker for growth traits in Grati- PO cattle. Keywords: Body weight, Growth hormone receptor, Polymorphism, PO-Grati cattle 274 J.Indonesian Trop.Anim.Agric. 46(4):272-281, December 2021 ation has not been reported yet for other body weight parameters in Grati-PO cattle. Therefore, the present study was aimed to analyze the asso- ciation between GHR|AluI gene with body weight parameters in Grati-PO cattle. MATERIALS AND METHODS Performans Data of Grati-PO Cattle Grati-PO cattle performance data were collected from 304 animals in the experimental enclosure at the Beef Cattle Research Station including birth, weaning, yearling and 18 months weight were accordance with recording data in breeding activities. Grati-PO cattle blood sam- ples were collected from the jugular vein (3- 5mL) using a vaccutainer tube containing K3 EDTA. DNA was extracted using a Quick-DNA mini prep kit (Zymo Research, USA) and then stored at -20 0 C for futher use. PCR-RFLP Analysis The method used to detect DNA variations was PCR-RFLP. The primers used for the GHR gene refers to Di Stasio et al. (2005). The prima- ry sequences of forward and reverse are shown in Table 1. PCR reaction was performed in a to- tal volume of 20 µL. A total 3 µl DNA contain- ing of approximately (10-100) ng/µL of DNA, 0.2 µl of each primer, 10 µL PCR mix of MyTaqTM HS Red Mix (Bioline, USA), and 6.2 µl ddH2O to a final volume of 20 µl. The PCR conditions were pre-denaturation at 95°C for 1 min, followed by 35 cycles of denaturation at 95°C for 15 s, annealing for 15 s at 53.8°C, ex- tension at 72°C for 10 s, and a final extension at 72°C for 5 min. PCR products were electro- phoresed on 1.5% agarose gels, stained with GelRed ® 10,000X in water (Biotium, USA) and visualized under a G-BOX Gel Documentation System (Syngene, UK). The PCR products of GHR were digested with AluI restriction enzyme (New England Biolabs, USA). The digested fragments were electrophorezed on 2% agarose gels, stained with GelRed ® 10,000X in water (Biotium, USA) and visualized under a G-BOX Gel Documentation System (Infiity VX2). The sequencing analysis was performed in this study using two sample of PCR product (20 μl) to confirm the findings.The sequencing anal- ysis was performed through commercial labora- tory service (PT Genetika Science). Statistical Analysis Polymorphism indexes such as genotype frequency and allele frequency were calculated according to Nei and Kumar (2000). Expected heterozygosity (He) and observed heterozygosi- ty (Ho) were calculated according to Weir (1991). Polymorphism Information Content (PIC) was calculated according to Botstein et al., (1980) by direct counting. The genotypic fre- quency distribution for its deviation to Har- dyWeinberg Equilibrium (HWE) was analyzed by chi-square test was calculated according to Kaps and Lamberson (2004). The number of effective allele was calculated according to for- mula Nei and Tajima (1981). Association between GHR|AluI genotype and body weight parameters in Grati-PO cattle population were analyzed applying a random- ized block design model as follows : Table 1. GenBank Accession Number, Amplicon Size (bp), Location of Polymorphic Site and Primer Sequences GHR |AluI Gene of Grati-PO Cattle Gene GenBank Accession number Amplicon size (bp) Location of polymorphic site Primer Sequences (5’-3’) GHR|AluI AF140284.1 342 Exon 10 F:5'-GCT AAC TTC ATC GTG GAC AAC-3' R : 5’-CTA TGG CAT GAT TTT GTT CAG-3’ Di Stasio et al. (2005) Association GHR|AluI gene with Body Weight in Grati-PO Cattle (Hartati et al.) 275 RESULTS AND DISCUSSION Body Weight Performance of Grati-PO Cattle Descriptive analysis of the body weight performance of Grati-PO cattle in the GHR|AluI gene validation material was shown in Table 2. In this study, the mean of birth weight, weaning weight, yearling weight and 18 months weight of Grati-PO cattle were 25.4 + 0.2 kg; 113.8 + 2.3 kg; 159.9 + 2.7 kg and 213.7 + 3.6 kg, respec- tively. The results of statistical analysis show that sex has a significant effect (P<0.05) on birth weight of Grati-PO cattle. The birth weight male calves was higher than female calves were 26.0 + 0.3 kg and 24.8 + 0.2 kg, respectively. The performance of Grati-PO cattle birth weight in this study was not different from the birth weight in the previous study that was 25.58 ± 3.31 kg (Hartati et al., 2015). The birth weight of Grati- PO cattle in this study was higher than the birth weight of PO cattle in Napis village, Tambakare- jo, Bojonegoro district that was 11.07 + 3.92 kg (Nonok and Darmawan, 2014). However, the birth weight of PO cattle in this study was much lower than those of PO Kebumen which reached 31.88 ± 3.78 kg (Maharani et al. 2018) or com- pared to the same Bos indicus cattle in other country, such as Brahman cattle in Columbia which reach to 33.06 ± 3.60 kg (Martínez et al., 2017), and Nellore cattle in Brazil which reach to 32.30 ± 3.80 kg (Chud et al., 2014). In this study showed insignificant results between sex on weaning weight and yearling weight in Grati-PO cattle although during this period, growth was strongly influenced by the environment because at the age of 7 months the calves have begun to wean and learn to consume finished feed until mature. Meanwhile, at 18 months weight there was a very significantly (P<0.05) between sex, where the body weight of the 18 months PO male cattle : as higher than the female PO cattle (225.2 ± 5.8 kg and 203.2 ±4.2 kg, respectively). This probably because the age of yearling to 18 months was the optimal growth period for beef cattle, male and female cattle growth was influenced by sex steroid hor- mones. The estrogen hormone can inhibit bone growth, especially at the ends of the bones, so that female growth was stunted after puberty begins, whereas male after puberty will continue to grow for a longer period which was stimulat- ed by androgen hormones (testosterone) espe- cially bone growth and other parts, so that : males were always heavier than females at the same age (Djagra et al., 2002) GHR|AluI Gene Polymorphism Visualization of GHR restriction with AluI enzyme, sequence of GHR gene and chro- matogram of GHR gene are presented in Figure 1, 2 and 3. In Figure 3 was shown the chromatogram sequence of the GHR gene from individual PO cattle with genotypes AA and GG genotypes. In the GG genotype, there was a substitution of A to G at exon 10 causing a change in the amino acid serine (AGC) to glycine (GGC). Morihito et al. (2017) stated that the mutation in DNA in organisms was substitutions, deletion or addi- tion of one or more parts of the DNA molecule. The genotype frequency, allele and heter- ozygosity of the GHR|AluI gene in Grati-PO cattle were shown in Table 3. The genotype fre- quency in Grati-PO cattle were detected to have three genotypes, namely AA, AG and GG (Figure 1 and Figure 2). The AA and AG Geno- types of the GHR|AluI gene in Grati-PO cattle were found at almost the same frequency, that were 42.1% and 44.4%, while the GG genotype was found in the lowest frequency that was 13.5%. The A allele was a common allele found in Grati-PO cattle with a frequency of 0.643 meanwhile the : the G allele was 0.357. Where, 𝑌𝑖𝑗𝑘 = dependent variable (BW, WW, YW or 18 MW) µ = overall mean 𝜎𝑖 = the effect of i th sex (male and female) 𝛽𝑗 = fixed effect of the j th genotype (AA, AG, GG) 𝑒𝑖𝑗𝑘 = random residual effect 276 J.Indonesian Trop.Anim.Agric. 46(4):272-281, December 2021 The previous research on several local Indonesian beef cattle, the A allele was also a common allele, such as on Grati-Bali Cattle 0.579 (Hartati, et al., 2021), Kebumen cattle 0.58 (Soewandi et al., 2021), Bali cattle 0.991 and Pesisir cattle 0.615 (Zulkharnaim et al., 2010). Likewise with other exotic cattle such as Simmental cattle, the most common genotype Figure 1. Visualization of GHR restriction with AluI restriction enzyme Figure 2. Sequence alignment of the AA and GG genotype sequenced samples with Genbank AF140284.1 of bovine GHR gene. The g.257A>G are targeted SNP of bovine GHR gene in present study. The grey highlighted sequences are flanking primers (forward and reverse). The restriction site of AluI (5' AG^CT 3’) are shown in the yellow highlighted sequences. The three novel SNPs found between the PO cattle sequences (present study) compared with Genbank (AF140284.1) are indicated by red letters. Association GHR|AluI gene with Body Weight in Grati-PO Cattle (Hartati et al.) 277 was the AA genotype (64.19%) and the A allele was the common alel (0.720) (Ardicli et al., 2017). In contrast, the G allele was the common allele found in Simmental and Limousin cattle raised at Singosari National Artificial Insemina- tion Center BBIB Singosari Malang were 0.714 and 0.735, respectively (Zulkharnaim et al., 2010). The results of statistical analysis showed that the genotype frequency of the GHR|AluI gene in Grati-PO cattle was still in Hardy- Weinberg Equilibrium (HWE). This explains that the frequency of alleles and genotype in the Grati-PO cattle population will remain constant from generation to generation. The PO cattle population was large enough to have the chance to mate randomly, which causes the gene fre- quency to remain the same. The factors that can change the frequency of genes in a population were selection, mutation, migration, genetic drift and evolution (Falconer and Mackay, 1996). The genetic diversity of a gene can be evaluated by the heterozygosity value (Ho, He, Ne) and polymorphism information content (PIC) value. The statistical analysis result showed that the expected heterozygosity (He) and the observed heterozygosity (Ho) values in the GHR|AluI gene were 0.459 and 0.460. This Table 2. Performance of Grati-PO cattle Sex N BW + SE WW + SE YW + SE 18 MW + SE Male Female Total 146 158 304 26,0 + 0,3ª 24,8 + 0,2ᵇ 25,4 + 0,2 113,2 + 3,2 114,3 + 3,2 113,8 + 2,3 162,1 + 4,1 157,9 + 3,5 159,9 + 2,7 225,2 + 5,8ª 203,2 + 4,2ᵇ 213,7 + 3,6 N : number of observation; BW : birth weight; WW : weaning weight; YW : yearling weight; 18 MW : 18 month weight; SE: standard error; means in the same row with different superscripts differ significantly (P<0.05). Table 3. Genotype Frequency, Allele and Heterozygosity GHR|AluI Gene of Grati-PO Cattle. Gene Sex N Genotype Frequency Allele Frequency χ2test He Ho PIC Ne AA AG GG A G GHR Male 14 6 0.418 0.459 0.123 0.647 0.353 0.004 0.457 0.458 0.352 1.840 Female 15 8 0.424 0.430 0.146 0.639 0.361 0.707 0.461 0.463 0.355 1.856 Total 30 4 0.421 0.444 0.135 0.643 0.357 0.323 0.459 0.460 0.354 1.849 N : number of observation; X 2 test : chi-square value; He : heterozigosity expected; Ho: heterozigosity observed; PIC: polymorphic information content; Ne: number of effective allele. (a) (b) Figure 3. Chromatogram sequences of GHR gene in individual of (a) PO with AA genotype (b) PO with GG genotype. Substitution of A to G at exon 10 causing a change in the amino acid serine (AGC) to glycine (GGC). 278 J.Indonesian Trop.Anim.Agric. 46(4):272-281, December 2021 result shows that Grati-PO cattle have a high enough value on genetic diversity. The Ne valu in this study was 1.849 which means that there were differences in allele frequencies in the GHR|AluI gene for Grati-PO cattle. Determina- tion of the informative level of a marker can be seen from the PIC value with with three catego- ries, namely the low category (≤0.25), the mod- erate category (0.250.05) with birth weight of Grati-PO cattle. The GHR gene was also reported to be significantly associated with weaning weight and daily weight gain in Pasun- dan cattle (Said et al., 2016), weight gain and carcass weight in Nellore cattle (Bos indicus) and it cross breed (Curi et al., 2006) but it has no ef- fect on reproductive performance in Holstein- Friesian cattle (Hadi et al., 2015). In Bos taurus cattle, the GHR|AluI was found to be associated with birth weight, final wieght and several char- acteristics of meat quality (Di Stasio et al., 2005; Reardon et al., 2010). This research found that the AG genotype in Grati-PO cattle has the highest body weight performance both at weaning weight, yearling weight and 18 months weight. Based on the re- sults of this study, genotype-based selection can be carried out on individuals with the AG geno- type with the selection criteria for weaning weight or yearling weight. Weaning weight and yearling weight were two growth characteristics that has high economic value. The high weaning weight illustrates the mother's good mothering ability in raising calves and having good milk production (Falconer and Mackay 1996). In addi- tion, the heritability value obtained for the two traits where the highest heritability was obtained at the weaning and yearling weight of 0.47+0.15 Table 4. Association between GHR|AluI Gene Polymorphism and Body Weight in Grati-PO Cattle Genotype n BW +SE n WW + SE n WY + SE n 18 MW+SE Male (M) AA 61 25.8±0.4 60 108.0±4.5 60 147.6±5.2 58 202.1±7.1 AG 67 26.1±0.4 67 117.8±5.2 67 174.4±6.5 66 242.6±9.1 GG 18 26.4±1.0 17 113.4±7.5 17 164.9±11.8 17 236.3±17.7 Female (F) AA 67 25.3±0.4 67 104.4±3.5 67 146.9±4.9 67 179.8±4.0 AG 68 24.6±0.3 64 122.4±5.7 64 170.3±5.3 64 224.9±7.3 GG 23 23.9±0.7 23 120.6±9.9 23 155.4±9.8 23 211.0±11.6 M and F AA 128 25.5±0.3 127 106.1±2.8 a 127 147.2±3.5 a 125 190.2±4.1 a AG 135 25.3±0.3 131 120.0±3.8 b 131 172.4±4.2 b 130 233.9±5.9 b GG 41 25.0±0.6 40 117.5±6.5 ab 40 159.4±7.5 ab 40 221.8±10.1 b n = number of observation; BW : birth weight; WW : weaning weight; YW : yearling weight; 18 MW : 18 month weight; SE: standard error; means in the same row with different superscripts differ significantly (P<0.05). Association GHR|AluI gene with Body Weight in Grati-PO Cattle (Hartati et al.) 279 and 0.63+0.17, respectively (Hartati et al., 2015). Thus it could be proposed that the GHR gene is very potential and could be used as a ge- netic marker on genetic improvement and selec- tion for superior of Grati-PO cattle . CONCLUSION The GHR|AluI gene was found to be mod- erately polymorphic in Grati-PO cattle. There was no association between GHR|AluI gene gen- otype and birth weight, but it was significant with weaning weight, yearling weight and 18 month weight. Therefore, GHR|AluI gene may be a potential genetic marker for those three body weight parameters in Grati-PO cattle. However, this association needs to be validated to the sub- sequent offspring so that it can optimistically be used as a genetic marker in improving body weight performance in the breeding program for Grati-PO cattle. 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