Polymorphism of GHRH Gene in Local Buffalo Using PCR-RFLP Method

1. IDENTIFICATION THE POLYMORPHISM OF GROWTH HORMONE RELEASING HORMONE(GHRH) GENE IN LOCAL BUFFALO (Bubalusbubalis) 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

2. BACKGROUND Improved management of maintenance and repair of genetic Buffalo great potential to complement beef 8% meatcattle SELECTION

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

4. GHRH (Growth Hormone Releasing Hormone) GHRH: 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.

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

6. 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 CyclerMachinesSentrifuge Deep Freezer (-40º C)Electrophoresis device Refrigerator (4º C)Micropipette Incubator (37 º - 120 º C)Digital Scales AutoclaveVortex™

7. materials • Blood samples320 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 enzymeHaeIII

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

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

10. In Vitro amplification ≈ PCR Procedure 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 Annealing60 0C; 2 min Elongation 72 0C; 7 min

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

12. Data analysis 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): • The frequency of alleles at locus calculated using the formula Nei (1987)

13. 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

14. 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

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

16. 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 4801aggcagcagg 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).

17. 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

18. M AA AB BB 500 pb 400 pb 300 pb 200 pb 194 pb 100 pb 45 pb Detection ofDNA Polymorphism 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. Alellefrequency equal and less 0,99 (Nei, 1987) Polymorphism

19. Table 1. Frequencies of GHRH/HaeIII genotypes and alleles in local buffaloes based on location

20. Heterozygosis ĥlocal buffaloes 0,037 – 0,485 ĥ total 0,461

21. Table 4. Heterozygosis values (ĥ) and average heterozygosis (Ĥ) GHRH gene in Local Buffaloes

22. not fixed into one gene type Fixation Index Fixation index GHRH gene≠ 0 Table 5. Fixation index values of Local Buffaloes GHRH gene

23. Genetic Distance Table 6. Genetic distance values of GHRH Gene in Local Buffaloes 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)

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

25. 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).

26. 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.

27. 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).

28. ThankS Bogor, Nov 2009