1. 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

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

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

4. 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.

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 Cycler Machines Sentrifuge
Deep Freezer (-40º C) Electrophoresis device
Refrigerator (4º C) Micropipette
Incubator (37 º º C) Digital Scales
Autoclave Vortex™

7. 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

8. DNA amplification (PCR) Restriction enzyme applications
Procedure
Blood Samples
DNA extraction
DNA amplification (PCR)
Restriction enzyme applications
Genotyping
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
In Vitro amplification ≈ PCR
PCR component
DNA sample 2 ul
primer 0,2 ul
10x buffer ul
MgCl mM
dNTPs 0.24 mM
Taq 0.75 unit
DW ul
amplification process
Denaturation C; 1 min
Annealing C; 2 min
Elongation C; 7 min

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

12. 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):

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 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

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 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).

17. 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

18. Detection of DNA PolymorphismAA 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

19. 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

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

21. 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

22. 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

23. 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)

24. 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

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 to with a total of ĥ and Ĥ
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 Restriction fragment length polymorphism in amplification products of the bovine growth hormone releasing hormone gene. J. Anim. Sci. 73 : 3789.
Nei, M Molecular Evolutionary Genetics. Columbia University Press. New York.
Zhou, P., G. W. Kazmer, dan X. Yang Bos taurus growth hormone releasing hormone gene, complete cds. GenBank, AF (2000).

28. ThankS
Bogor, Nov 2009