IN LOCAL BUFFALO (Bubalus bubalis) IDENTIFICATION THE POLYMORPHISM OF GROWTH HORMONE RELEASING HORMONE (GHRH) GENE IN LOCAL BUFFALO (Bubalus bubalis) USING PCR-RFLP METHOD A. Primasari 1, C. Sumantri 1 and A. Farajallah2 1) Department of Animal Production and Technology, Fact of Anim. Sci. Bogor. Agric. University. 2) Department of Biology Fact of Mat. And Nat. Sci. Bogor. Agric. University

BACKGROUND Buffalo SELECTION great potential to complement beef Improved management of maintenance and repair of genetic 8% meat cattle SELECTION

Knowing the genetic markers for the selection of livestock Genetic improvement Knowing the genetic markers for the selection of livestock GHRH gene

GHRH (Growth Hormone Releasing Hormone) one of the role of growth factors stimulate the synthesis and secretion of Growth Hormone in an additive effect on growth Important for tissue growth, fat metabolism, reproduction, lactation and weight growth.

Purpose Identifying the polymorphism of GHRH gene in the local buffalo (Bubalus bubalis)

Method Time and Location September-Desember 2008 Laboratory of Zoology Department of Biology, Faculty of Mathematic and Natural Sci. Bogor Agricultural University The main tool Thermo Cycler Machines Sentrifuge Deep Freezer (-40º C) Electrophoresis device Refrigerator (4º C) Micropipette Incubator (37 º - 120 º C) Digital Scales Autoclave Vortex™

materials Blood samples 320 samples : 75 from Semarang (Central Java), 103 from Mataram (West Nusa Tenggara), 65 from Siborong-borong (North Sumatera), and 77 from Banten Genomic DNA Mini Kit Geneaid Primer GHRH F (5’- GTA AGG ATG CCA GCT CTG GGT -3’) GHRH R (5’- TGC CTG CTC ATG ATG TCC TGG A -3’) (Moody et al., 1995) Restriction enzyme Hae III

DNA amplification (PCR) Restriction enzyme applications Procedure Blood Samples DNA extraction DNA amplification (PCR) Restriction enzyme applications Genotyping DNA visualization

Procedure Blood Sample DNA Extraction Genomic DNA Mini Kit Geneaid DNA extraction of blood sample using Genomic DNA mini kit Geneaid

In Vitro amplification ≈ PCR Procedure In Vitro amplification ≈ PCR PCR component DNA sample 2 ul primer 0,2 ul 10x buffer 2.5 ul MgCl2 2 mM dNTPs 0.24 mM Taq 0.75 unit DW 17.85 ul amplification process Denaturation 940 C; 1 min Annealing 60 0C; 2 min Elongation 72 0C; 7 min

+ HaeIII Restriction Enzymes PCR-RFLP Restriction Enzyme Applications + HaeIII Restriction Enzymes 370 C Incubation Elektroforesis Silver staining PCR Product

Data analysis The frequency of alleles at locus calculated using the formula Nei (1987) where: Xi = allele frequency of -i nii = number of individu with genotipe ii Nij = number of individu with genotipe ij N = total individu sample Degree of heterozygosis (ĥ) is calculated based on allele frequencies at each locus DNA using the formula of Nei (1987):

Where ĥ = heterozygosis locus Xi = allele frequency of GHRH gene type-i n = total of individu sample Variance of heterozygosis in each population can be calculated by the following formula:   Average heterozygosis(Ĥ) is calculated by the following formula:   where ĥj= degrees of heterozygosity for the locus of -j r = number of loci tested Ĥ = average heterozygosity

Fixation index in each population derived from the equation: where Xkii = Frequency of allele homozygous genotype in the population of the i-k Xki = Allele frequencies i Genetic distance (D) calculated using the formula: Where D = Genetic distance Pix = i allele to the population X Piy = Frequency of allele i in population Y

RESULTS GHRH gene Amplification ekson 2 - ekson 3 451 bp Figure 1. Results GHRH Gene Amplification Using PCR Method at 6% Poliacrilamida Gel GHRH gene Amplification ekson 2 - ekson 3 451 bp

4321 cctgtctgtc atttcccagg taccagcaca ggggtgaagg atgctgctct gggtgttctt 4381 cctcgtgacc ctcaccctca gcagcggctc ccacggttcc ctgccttccc agcctctcag 4441 gtaagcagtt ctgagaagag aagcaagaga gg|ccctttga ggatgcgact cgagctggtc 4501 cccagctggg tcctcaggca gcctcccttg ctcatctctg ggagggtggc agactgagcc 4561 ccagagaggt caccacccag ccctggttcc agccctctct ggggacgagc agggcaagag 4621 gcgacagaaa gacctcacag agaccaagtg agcacagtcc cctggg|cctc ccaccccacc 4681 ctttgacctc tgactccttc tactaggatt ccacggtacg cagatgccat cttcactaac 4741 agctaccgga aggttctggg|ccagctgtct gcccgcaagc tactccagga tatcatgaac 4801 aggcagcagg ggtgagccgg cgttctcgtg acttctccct gcaccctcgg ttcatcatga Figure 2. Primary attachment position and the site HaeIII Restriction Enzyme based on the GHRH gene sequences in diary cattle (GenBank Access No. AF242855) (Zhou et al., 2000).

But were found only two type genotype The restriction analysis of 451 bp PCR products of the GHRH gene indicated the presence of three type of restriction pattern : AA Genotype : 312, 94 dan 45 bp. AB Genotype : 312, 194, 118, 94 dan 45 bp. BB Genotype : 194, 118, 94 dan 45 bp. But were found only two type genotype (AB and BB) 312 pb 194 pb 118 pb 94 pb

Detection of DNA Polymorphism AA AB BB 100 pb 200 pb 300 pb 400 pb 500 pb 45 pb 194 pb 312 pb 312 pb 194 pb 118 pb 118 pb 94 pb 94 pb 94 pb 45 pb 45 pb Figure 4. Zymogram of electrophoretic pattern showing genotype AA,AB and BB of GHRH gene. Alelle frequency equal and less 0,99 (Nei, 1987) Polymorphism

Table 1. Frequencies of GHRH/HaeIII genotypes and alleles in local buffaloes based on location Sample Total (n) Genotype Genotype Frequency Allele Frequency Semarang 61 AA (0) 0,000 A = 0,147 AB (18) 0,705 B = 0,853 BB (43) 0,295 Mataram 86 A = 0,186 AB (32) 0,372 B = 0,814 BB (54) 0,628 Medan 53 0, 000 A = 0,019 AB (2) 0,038 B = 0,981 BB (51) 0,962 Banten 45 A = 0,400 AB (36) 0,800 B = 0,600 BB (9) 0,200 Total 245 A = 0,180 AB (88) 0,360 B = 0,820 BB (157) 0,640

Genetic diversity in population Heterozygosis Heterozygosis Value Genetic diversity in population ĥ local buffaloes 0,037 – 0,485 ĥ total 0,461

Table 4. Heterozygosis values (ĥ) and average heterozygosis (Ĥ) GHRH gene in Local Buffaloes Location ĥ ± SE Ĥ Semarang 0,252 ± 0,045 0,270 ± 0,024 Mataram 0,305 ± 0,003 Medan 0,037 ± 0,026 Banten 0,485 ± 0,022 Total 0,461 ± 0,022

not fixed into one gene type Fixation Index not fixed into one gene type Fixation index GHRH gene ≠ 0 Table 5. Fixation index values of Local Buffaloes GHRH gene Location Allele FIski Semarang A -0,175 B -3,450 Mataram -0,229 -0,228 Medan -0,019 Banten -0,666 Total -0,218 -0,197

Genetic Distance Location Semarang Mataram Medan Banten 0,000 0,001 Table 6. Genetic distance values of GHRH Gene in Local Buffaloes Location Semarang Mataram Medan Banten 0,000 0,001 0,012 0,023 0,089 0,064 0,171 The value of genetic distance GHRH gene is the smallest among the local buffaloes population of Semarang and local buffalos Mataram (0.001) and the highest among the local buffaloes population of Medan and Banten (0.171)

Figure 5. Dendogram Based on GHRH Gene in Local Buffaloes Population Semarang 0,001 Mataram 0,017 0,108 Medan Banten Figure 5. Dendogram Based on GHRH Gene in Local Buffaloes Population

CONCLUSIONS GHRH gene in this study are polymorphic with two types of allele A allele (18%) and B allele (82%) Genotypes obtained by the AA (0%), AB (36%) and BB (64%) with Ĥ values of 46%. B allele has a greater frequency than A allele in the buffalo livestock population of the four regions in Indonesia ĥ local buffalo from 0.037 to 0.485 with a total of ĥ 0.461 and Ĥ 0.270 Fixation index value indicates that the GHRH gene in the four local buffalo population of Indonesia is not fixation The value of genetic distance GHRH gene is the smallest among the local buffalo population of Semarang and local buffalo Mataram (0.001) and the highest among the local buffalo population of Medan and Banten (0.171).

ADVICES Further research needs to be done to analyze the relationship between GHRH gene diversity with quantitative properties of Indonesian local buffalo. The data obtained can be used as with guidance in selecting cattle to obtain seeds.

REFERENCES Moody, D. E., D. Pomp, dan W. Barendse. 1995. Restriction fragment length polymorphism in amplification products of the bovine growth hormone releasing hormone gene. J. Anim. Sci. 73 : 3789. Nei, M. 1987. Molecular Evolutionary Genetics. Columbia University Press. New York. Zhou, P., G. W. Kazmer, dan X. Yang. 2000. Bos taurus growth hormone releasing hormone gene, complete cds. GenBank, AF 242855 (2000).

ThankS Bogor, Nov 2009