Vohra et al.

MATERIALS AND METHODS

using Mlucl restriction enzyme at 37 °C for 6 to 8

hrs. Genotyping was evaluated by running a small aliquot

Population studied and sample size

of PCR-RFLP product on 2.5% agarose gel. Genotype

and allele frequencies were calculated by gene

Gojri buffalo in Punjab and Himachal Pradesh and

computing method (Falconer and Mackay, 1996).

Chhattisgarhi buffalo from Chhattisgarh.These germplasm

have their own socio-economic utility and are generally

better adapted to their regions, overall buffalo milk is

RESULTS AND DISCUSSION

well known for its higher fat content compared to cow

A 473 bp fragment of exon 40 of FASN gene was

milk. Random blood samples (approximately 8 to 10 mL)

successfully amplified in all samples of Gojri and

were collected from 80 genetically unrelated buffaloes

Chhattisgarhi buffalo. RFLP test using Mlucl restriction

representative of the Gojri (n = 40) and Chhattisgarhi (n

enzyme indicated that exon-40 region of FASN gene is

= 40) breeds. The blood samples of Gojri and Chhattisgarhi

highly polymorphic in all the three breeds of buffalo, with

buffaloes were collected from their respective breeding

the presence of three genotypes namely, AA, AG & GG,

tracts.

withA and G alleles (Table). Three types of genotypes, viz.

AA,AG and GG were having almost same allele frequency

DNA isolation and Primers used

in all three populations studied. GG was rare andAG being

most common genotype. Heterozygous AG was found to

Genomic DNA was isolated from aseptically collected

be most frequent with highest genotype frequency of 0.60

venous blood using the standard phenol/chloroform

and GG allele was found to be rare with least frequency

method with minor modifications (Sambrook and Russel,

of 0.025 in Gojri buffalo were as in Chhattisgarhi buffalo

2001). Quality check and quantification were done by

Heterozygous AG was found to be most frequent with

nanodrop spectrophotometer and electrophoresis on

0.56 and 0.55 respectively. Homozygous GG was found to

0.8% agarose gel. DNA concentration was determined

be rare genotype in Chhattisgarhi buffalo as 0.125.

and samples were diluted 10-40 times (approx. 50-

80 ng/ μl ) with MiliQ water. F orward and reverse

Our results were similar to that (0.31) obtained by

Morris et al . (2007) for Holstein- Friesian cattle

primers (P _F 5’-CTCGCACACCTTCGTGATG-3’ and P

R

where they reported frequency of AA genotype 0.31.

5’-CACGTTGCCGTGGTAGGTAG-3’) with T _M of 57.5

°C and 57.4 °C respectively, were designed using Primer

Higher FASN (AA) frequencies were also reported

3 software to amplify exon-40 region of FASN gene from

by researchers in the following breeds: Holstein-

published NCBI (National Centre of Bioinformatics,

Friesian 0.53 (Schennink et al ., 2009) andAngus 0.62

USA) sequences.

(Zhang et al ., 2008). Significantly lower frequencies

were obtained in Jersey breed 0.13 (Morris et al .,

2007) and Korean breed 0.15 (Oh et al ., 2011) which

PCR amplification and genotyping conditions

is indicative of extensive genetic variability of this

DNA amplification of the exon 40 of FASN gene are

fat related marker in different breeds however, within

achieved by PCR . The optimization of PCR was done to

Gojri and Chhattisgarhi breeds of buffalo there seems

get the best possible amplification of the product, PCR was

to be similar trend. Schennink et al . (2009) and Zhang

carried out in 25 m l reaction volume consisting of 200 µM

et al . (2008) reported similar frequencies (0.50 and

of each dNTP, 5pM of each primer, 1.5mM MgCl ₂ and

0.51, respectively), whereas that showed by Oh et al .

1.0U Taq polymerase (Invitrogen, CA). Amplification

(2011) was 0.25. A very high FASN (GG) frequency

was performed using MASTERCYCLER EP (Eppendorf,

was observed in Korean cattle 0.73 (Oh et al ., 2011).

Germany) with an initial denaturation at 95˚C for 4 min

Zhang et al . (2008) have observed more frequent

followed by 30 cycles of 94˚C for 60 sec, annealing

occurrence of the FASN (AA) homozygote 0.36,

temperature 61 ºC for 60 sec and 72˚C for 60 sec, with a

compared to the FASN (GG) homozygote 0.13. The

final extension for 10 min at 72 ° C. All the buffaloes were

activity of the KR domain is essential for the FASN protein

screened for the presence of FASN gene polymorphism

(Vázquez et al ., 2008). The T1950A and W1955R may

using PCR-RFLP technique. Genotyping was carried out

influence the KR domain, resulting in the alteration of the

326

Journal of Animal Research: v.5 n.2. June 2015