Procedia Environmental Sciences 17 ( 2013 ) 105 – 108

1878-0296 © 2013 The Authors. Published by Elsevier B.V.Selection and peer-review under responsibility of SUSTAIN conference’s committee and supported by Kyoto University; (OPIR), (GCOE-ES), (GCOE-HSE), (CSEAS), (RISH), (GCOE-ARS) and (GSS) as co-hosts.doi: 10.1016/j.proenv.2013.02.017

Available online at www.sciencedirect.com

The 3rd International Conference on Sustainable Future for Human Security SUSTAIN 2012

Polymorphism leu/val of growth hormone gene identified from limousin cross local cattle in Indonesia

Tety Hartatik*a, Slamet Diah Volkandaria, Mifta Pratiwi Rachmana, Sumadia aLaboratory of Animal Breeding, Faculty of Animal Science, Universitas Gadjah Mada,

Jl Fauna 3 Bulaksumur, Yogyakarta 55281,Indonesia

Abstract

This study attempted to identify Leu/Val polymorphism of growth hormone gene in Limousin cross local cattle. The DNA samples were collected from Madura cattle (65), Limousin-Madura cattle (81), Ongole Crossbred cattle (PO, 52), and Limousin-PO cattle (56), also Limousin bull semen (6). A 211 bp DNA fragment of growth hormone gene spanning from the forth intron (49 bp) to five exon (162 bp) was amplified using PCR method and digested with AluI restriction enzyme to identified polymorphism at this locus. Two genotypes LL and LV were observed in PO (96.15%LL, 3.85%LV), Limousin-Madura (81.46%LL, 18.52%LV), Limousin-PO cattle (78.57%LL, 21.43%LV), and Limousin (66.67%LL, 33.33%LV). Madura cattles were found monomorphic for this locus producing only one genotype LL (100%). The results of this research show that the increasing number of genotype leu/val in Limousin cross local cattle is about 17.58% and 18.52% for Limousin-PO cattle and Limousin-Madura, respectively, compared to the local cattle (PO and Madura cattle). The allelic frequencies are statistically similar as revealed by Chi-Square test. This research concludes that the polymorphic leu/val of growth hormone gene occurs in Limousine-cross local cattle whereas monomorphic leu/leu of growth hormone gene is found in Madura cattle. Thus, the finding of this research would be useful as a control for genetic equilibrium in Madura cattle population and to protect uncontrolled crossbreeding. © 2012 The Authors. Published by Elsevier B.V. Selection and/or peer-review under responsibility of SUSTAIN conferences committee and supported by Kyoto University; (OPIR), (GCOE-ES), (GCOE-HSE), (CSEAS), (RISH), (GCOE-ARS) and (GSS) as co-hosts. Keywords: Polymorphism, Leucine, Valine, Growth Hormone, Madura Cattle

* Corresponding author. Tel.: +62-274-4333373; fax: +62-274-521578. E-mail address: tetyharuta@yahoo.com.

Available online at www.sciencedirect.com

© 2013 The Authors. Published by Elsevier B.V.Selection and peer-review under responsibility of SUSTAIN conference’s committee and supported by Kyoto University; (OPIR), (GCOE-ES), (GCOE-HSE), (CSEAS), (RISH), (GCOE-ARS) and (GSS) as co-hosts.

106 Tety Hartatik et al. / Procedia Environmental Sciences 17 ( 2013 ) 105 – 108

1. Introduction

Crosses among breeds allow the formation of new breeds, selected for favorable traits including adaptation to the environment. Nowadays, Indonesian government has improved the local cattle such as Ongole Crossbreed (PO) and Madura cattle which are generally crossed with Limousin bull by artificial insemination. The purpose of the crosses program is to increase the performance of the local cattle. Hence, a good performance of the cattle is influenced by gene and environment. Growth in animals is controlled mainly by growth hormone (GH) that acts on the growth of bones and muscles mediated by insulin-like growth factor (IGF-1) [1]. Both of the genes are candidates for growth in bovine, since they play a key role in growth regulation and development [2].

Growth hormone gene has been assigned to 19q26q-ter position of bovine chromosome [3], with five exons and four introns. Fifth exon of the growth hormone gene at 127 amino acid position was found to be polymorphic with substitution of cytosine (C) to guanine (G) at growth hormone gene. The previous researches [4,5] reported that polymorphism of growth hormone gene occurred at 2141 of the sequence. Substitution of C for G nucleotide at that position caused an amino acid change from leucine to valine of the growth hormone polypeptide that can be identified by AluI restriction enzyme. Several polymorphisms were identified in the growth hormone gene and detected by restriction fragment length polymorphism (RFLP) with the recognition site of AluI restriction enzyme [6,7,8]. This study aims to identify polymorphism Leu/Val of growth hormone gene in Limousin cross local cattle.

2. Materials and Methods

2.1. Sample and DNA Extraction

Blood Samples were collected from 262 cattles consisting of Madura (65), Limousin-Madura (81), Ongole Crossbreed (PO) (52), and Limousin-PO cattle (56). Blood sample was taken through the vena jugularis and preserved in the K3EDTA tube, and then stored in the freezer until the next process. Six semens of Limousin bulls were used as a control. DNA sample was extracted from blood sample using SDS/ProteinaseK modified method from [9].

2.2. Polymerase Chain Reaction

A 211 bp fragment of growth hormone was amplified by polymerase chain reaction (PCR) using forward ( - and reverse ( -

The PCR reaction contains 1 ìl DNA sample, 1 ìl each primer (10 pmol), 10 ìl PCR KIT (Fastart, Roche) and 7 ìl aquabidest in total solution 20 ìl. The steps to conduct amplification in vitro were as follow: pre-denaturation at 95 ºC for 5 minutes, denaturation at 95 ºC for 30 seconds, annealing primers at 65 ºC for 30 seconds and extension of new DNA at 72 ºC for 30 seconds, and the final extension at 72 ºC for 5 minutes [8]. The cycle of PCR was repeated for 35 cycles using GeneAmp® PCR Sys The PCR product was analyzed using horizontal electrophoresis (Mupid) in 1% agarose gel, 1XTBE buffer, 50 Voltage and running for 30 minutes. The PCR product was visualized using UV and the size of PCR product was compared with DNA marker (HaeIII)).

2.3. Restriction Fragment Length Polymorphism

The PCR product was digested with 10 units AluI enzyme (Fermentas) in a total volume 20 l and incubated at 37ºC for 3 hours. The polymorphism of growth hormone gene was analyzed by separating restriction product in vertical electrophoresis (BioRad) with 12% polyacrilamide gel (PAGE), 1X TBE

107 Tety Hartatik et al. / Procedia Environmental Sciences 17 ( 2013 ) 105 – 108

buffer and running at 50 Voltage for 3 hour 30 minutes. The gel was stained gently by ethidium bromide for 15 minutes and washed in aquadest for 5 minutes. Visualization of the fragmentation growth hormone was performed under the UV light.

2.4. Genotyping and Allele Frequency

The polymorphic leu/val of growth hormone gene frequencies was calculated by simple allele counting according to the Hardy-Weinberg equilibrium [11], the possible deviations of genotype frequencies from expectation were tested by chi-square. Genotype frequency represents the ratio of a genotype to total population. Allele frequency is a ratio of an allele to the overall allele at a locus in the population. The allele frequencies (1) and expected genotype frequencies (2) were calculated as follows:

NLVVVqNLVLLp 2/2;2/2 (1)

qpnLVqnVVpnLL 2;; 22 (2)

where: p and q are the frequencies of L and V allele in observed population; LL, LV and VV are the number of genotypes animals ; N is the number of observed animals

3. Results and Discussion

The PCR amplification generated a 211 bp segment of growth hormone gene. The digestion with AluI restriction enzyme showed two types of restriction patterns. The first pattern was assigned as genotype LL which produced 52 bp and 159 bp fragments (Figure 1, lane 3-5 and 7-10), and the second pattern was assigned as genotype LV which produced 52 bp, 159 bp, and 211 bp fragments (Figure 1, lane 6 and 11).

The two types of alleles differed only in terms of

restriction site of AluI restriction enzyme. The L allele indicated the presence of restriction site while its absence was assigned as alles V. In L allele the restriction site contained the nucleotide C while a transition with G at the same site indicated the absence of AluI restriction site. The presence of the nucleotide C in the total length of growth hormone gene (191) occurred in 127th position of the polypeptide. The presence of that nucleotide (C) at triplet codon encoded the amino acid Leusine while nucleotide G encoded the amino acid Valine. This Leusine/Valine substitution indicated polymorphism. In this study polymorphism has been identified in one local cattle and all of Limousine cross local cattle. Since the crossbreed animals were the crosses of local and exotic cattle,

polymorphism of this locus was expected. In this study (see Table 1) polymorphism leu/val of growth hormone gene has been identified in

Limousin bull, PO cattle and Limousin cross local cattle. Madura cattle were monomorphic for this locus producing only one genotype LL (100%). Two genotypes LL and LV were observed in PO (96.15%LL, 3.85%LV), Limousin-Madura (81.46%LL, 18.52%LV), Limousin-PO cattle (78.57%LL, 21.43%LV), and Limousin (66.67%LL, 33.33%LV). The results showed the increasing number of genotype LV in Limousin cross local cattle about 17.58% and 18.52% for Limousin-PO cattle and Limousin-Madura,

Figure 1. Representative results of GH-AluI analysis detected by Polyacrylamid gel (PAGE) 12% and

no.1 DNA Genome, lane no.2 PCR product, lane no.3-11 LL/LV genotype.

108 Tety Hartatik et al. / Procedia Environmental Sciences 17 ( 2013 ) 105 – 108

respectively, compared to the local cattle (PO and Madura cattle). However, the genotypes LV in Limousin-PO (21.43%) were higher than that of Limousin-Madura (18.52%). The allelic frequencies were statistically similar as revealed by Chi-Square test.

Table 1 . Allele frequencies and result of X2 test for comparison of proportions between cattle

Cattle N GH genotype Alleles Frequencies X2 LL LV VV L V

Limousin 6 Observed 4 (66,67%) 2 (33,33%) - 0.83 0.17 0.23 Expected 4.13 (68.95%) 1.69 (28.21%) 0.17 (2.84%)

Madura 65 Observed 100 (100%) - - 1.00 - - Expected - - -

Ongole Crossbreed (PO ) 52 Observed 50 (96.15%) 2 (3.85%) - 0.98 0.02

0.02 Expected 49.94 (96.04%) 2.04 (3.92%) 0.02 (0.04%)

Limousin-Madura 81 Observed 66 (81.48%) 15 (18.52%) - 0.91 0.09 0.90 Expected 67.08(82.81%) 13.27 (16.38%) 0.66 (0.81%)

Limousin-PO 56 Observed 44 (78.57%) 12 (21.43%) - 0.89 0.11 0.78 Expected 44.4 (79.21%) 10.96 (19.58%) 0.68 (1.21%)

X2 0,05;2 = 5.99; GH: Growth Hormone

4. Conclusion

In conclusion, it is noted that growth hormone gene is polymorphic in Limousine-cross local cattle and PO cattle and monomorphic in Madura cattle. The result of this research, then, would be useful as a control for genetic equilibrium in Madura cattle population and to protect uncontrolled crossbreeding. The molecular study of Madura cattle could be improved on the basis of information which has been obtained, and it would be of interest to extend the molecular study to include more populations of Madura cattle in different locations.

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