GENETIC POLYMORPHISM OF THE KAPPA CASEIN GENE IN HOLSTEIN-FRIESIAN DAIRY CATTLE IN WEST JAVA PROVINCE Anneke Anggraeni1 Cece Sumantri2 Achmad Farajallah3.

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GENETIC POLYMORPHISM OF THE KAPPA CASEIN GENE IN HOLSTEIN-FRIESIAN DAIRY CATTLE IN WEST JAVA PROVINCE Anneke Anggraeni1 Cece Sumantri2 Achmad Farajallah3 Eryk Andreas4 1) Indonesian Research Institute for Animal Production, Ciawi - Bogor 2) Faculty of Animal Science, Bogor Agriculture University 3) Faculty of Biology Sci., Bogor Agriculture University 4) Postgraduate of Faculty of Animal Sci., Bogor Agriculture University

Introduction DNA polymorphic markers  candidate genes in selection Marker-assisted selection (MAS) + traditional method of selection  an effective way to improving quantitative traits. increase the accuracy of selection reduce interval generation accelarating selection responses

Introduction Milk proteins are mainly divided into two fractions soluble fraction  whey protein (α-lactalbumin and β- lactoglobulin) insoluble fraction  casein (78- 82% milk proteins) κ-Cn approximately 12% of the whole milk casein

Introduction κ-Cn gene there is in the chromosome 6q31, consisting 5 exon and 4 intron two alleles are the most commonly identified in Bos taurus dairy cattles A allele with threonine (136) and aspartic acid (148) B allele with isoleucine (136) and alanine (148)

Introduction Milk protein variants influence of milk composition, proteins and cheese production  Milk protein genes could be useful as genetic markers for selection in dairy cattle breeding Several studies showed association of κ-Cn variants with lactation performance and effect on milk composition and its processing properties

Introduction Relationships between variants κ-Cn genotypes for different milk proteins and yield traits have been reported by several authors κ-Cn BB cows produced milk with a 0.08 percent higher protein content than that of AA cows κ-Cn variants had a significant effect on milk production (p<0.001), with cows of the BB genotype producing 173 kg less milk than AA cows

Introduction The aim of this research was to know the frequencies of both genotype and allele of the κ-CN gene in HF under the two different research locations, both from government dairy breeding stations and a number of small dairy farmers in central dairy region in West Java Province, Indonesia

Material and Method Animal and blood samplings Total of 342 Holstein-Friesian (HF) females, reared under two different managements  intensive and semi intensive management Total of 57 males both actively used (bulls) and candidates (young sires) artificial insemination (AI)  AI Stations in Lembang and in Singosari Blood samplings were collected via vena jugularis/vena cocsigalis

Tabel Total Sample were Used in This Resaerch Material and Method Tabel Total Sample were Used in This Resaerch Location Number of Blood Sample Sex Eco Farm 20 Female BET Cipelang 50 BPPT-SP Cikole 82 Sub Total 152 KPSBU-Cilumber 98 KPSBU-Pasar Kemis 92 190 Total 342 BIB Lembang 25 Male BBIB Singosari 32 57

Material and Method DNA Extraction DNA from blood sample was extracted by method as described by Sambrook et al. (1989) with minor modification Polymerase chain reaction (PCR) PCR with volume 17.5 µl was followed: 2 l DNA sample 1 pmol kappa-casein (-casein) primer 200 µM dNTPs 0.5 unit AmpliTaq gold DNA polymerase

Table Sequence Primer were Used in This Resaerch Material and Method Table Sequence Primer were Used in This Resaerch Primer Sequent PCR Product Forward 5’-AAA TCC CTA CCA TCA ATA CC -3’ 183 bp Reverse 5’ CTT CTT TGA TGT CTC CTT AG-3’ Table PCR Condition were Used in This Resaerch Step Temperature (ºC) Time Cycle First Denaturation 95 10.00 1 Denaturation Annealing Extention 55 72 00.30 01.00 40 Final Extention 05.00

Material and Method PCR-RFLP Analysis of PCR-RFLP with volume 10 µl was followed: 4 l PCR Product 2.5 unit PstI Data Analysis The genotype frequencies were calculated by The allelic frequencies were calculated by Nei (1987)

Result and Discussion Amplification The existing technology of PCR makes possible to amplify segments of the genome containing genotypic (allelic) variations The DNA fragment amplified from the A and B allel were characterized by the presence the fragment lenghts of 152 bp and 183 bp respectively

Result and Discussion a b Figure PCR Product 183 bp (a). The RFLP patterns of the k-casein after digestion with PstI on the 6% PAGE AA= 152 bp, AB=183 and 152 bp, and BB= 183 bp

Genotype frequency (Sample) Result and Discussion Table Frequencies of Genotypes and Alleles of the -Casein Gene in Holstein-Friesian by Location Location and Blood Sample (Number) Genotype frequency (Sample) Allele frequency AA AB BB A B Eco Farm Fapet IPB (20) 0,45 (9) 0,55 (11) 0,00 (0) 0,73 0,27 BET-Cipelang (50) 0,54 (27) 0,46 (23) 0,77 0,23 BPPT-SP Cikole (82) 0,10 (8) 0,85 (70) 0,05 (4) 0,52 0,48 Sub total (152) 0,29 (44) 0,68 (104) 0,03 (4) 0,63 0,37 KPSBU - Cilumber (98) 0,32 (31) 0,62 (61) 0,06 (6) KPSBU - Pasar Kemis (92) 0,34 (31) 0,64 (59) 0,02 (2) 0,66 0,34 Sub total (190) 0,33 (62) 0,63 (120) 0,04 (8) 0,64 0,36 Total (342) 0,31 (106) 0,65 (224) 0,04 (12)

Result and Discussion The results showed that those animals with the BB genotype were very low, at a range of 0 - 6 % with the average of 4 % Samples at the Eco Farm Anim Sci. IPB and BET Cipelang showed that none of these animals had the BB genotype Frequency BB genotype in BPPT-SP Cikole is 5 % Frequency BB genotype in Pasar Kemis and Cilumber villages are 6% and 2% respectively.

Result and Discussion Table Frequencies of Genotypes and Alleles of the -Casein Gene at BIB Lembang and BBIB Singosari

Result and Discussion BBIB Singosari have the only one bull with the BB genotype BIB Lembang have none active bull with the BB genotype Total BB frequency of the κ-Cn gene both at BIB Lembang (0,26) and Singosari (0,45) was sufficient non active AI (young) males not gave any contribution to increase the BB genotype females

Result and Discussion at KPSBU Lembang, it was generally found that the A allele was higher compared to the B allele, both in Cilumber (0,63 vs 0,37) and Pasar Kemis (0,66 vs 0,34) villages at BPPT-SP Cikole had the frequencies of the A and B alleles almost similar (0,52 vs 0,48). However, all of observated HF females had a higher frequency for the A allele over the B (0,64 vs 0,36)

The occurence of more higher the B allele than the A allele was also found for the active and non active AI males at the two AI stations of BIB Lembang (0,74 vs 0,26) and BBIB Singosari (0,63 vs 0,37) With regarding to the lower BB genotype females at Ecofarm Fapet IPB, the reason for the lower frequency of the B allele over the A allele (0.27 vs 0.73) was that only one bull continously used for mating females and this bull was identified for the AA genotype

Result and Discussion The A allele frequency in this study was still lower than USA, Europe and Japan (0,8-0,9) This was due to the main purpose to develope dairy cattle for high milk yield, the A allele is more related to higher milk yield, while the B allele closely associated with high protein yield This was can be associated with decision for the imported HF bull policy mainly based on high values of Estimated Transmitting Ability (ETA) of their milk yield

Conclusion Genotyping к-casein gene in HF dairy cattle resulted two alleles were identified (A and B)  three genotypes (AA, AB, and BB) The BB genotype HF females in this study were identified at a very low frequency in the range of 0-6 % All of this finding seemingly be a possible indication for a small number of the BB genotype HF females in some central dairy region in West Java

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