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Study on Genetic Diversity of BLRI Cattle Breed-1 Using Microsattelite Markers

G. K. Deb
Biotechnology Division, Bangladesh Livestock Research Institute, Savar, Dhaka.

S. R. Dey
Animal Production Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka.

N. R. Sarker
Animal Production Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka.

M. R. Mufti
Animal Production Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka.

M. R. Rahman
Biotechnology Division, Bangladesh Livestock Research Institute, Savar, Dhaka.

Abstract/ Executive Summary:

This study aimed to characterize BLRI Cattle Breed I (BCB-1) at genetic level and to find out their genetic distances with other native cattle (Red Chittagong cattle, RCC and Progenitor cattle of BCB-1 of Pabna and Sirajgonj districts, Progenitor) of Bangladesh using three pairs of bovine microsatellite markers (ETH 152, TNRA32 and TNRA63). Blood samples were collected randomly from fifteen animals of each group (BCB-12, RCC and Progenitor). Genomic DNA was extracted using BLRI developed DNA extraction kit. All the primer pairs were successfully amplified microsatellite loci. The PCR products were checked on 3% agarose gel for verification of quantity and size of amplicon. Results showed the highest and lowest gene frequency values (0.850 and 0.150) were observed in allele A and allele B with ETH152 in RCC and BCB-1 cattle and TNRA63 in BCB-1 cattle. All the three cattle populations showed significant deviation from Hardy-Weinberg equilibrium for at least one locus (RCC for ETH152 and TNRA32, BCB-1 for ETH152 and TNRA63 and Progenitor for TNRA63). The estimated mean observed (Ho) and expected heterozygosity (He) were 0.167±0.134, and 0.377±0.134 in RCC vs. 0.167±0.116 and 0.339±0.122 in BCB-1 vs. 0.800± 0.200 and 0.393±0.097, respectively. However, mean heterozygosity of all three loci was same (0.351±0.111) in the studied cattle populations. All the studied microsattelite showed two polymorphic loci each. Average effective number of alleles was 1.604±0.349, 1.507±0.349 and 1.621±O.261 in RCC, BCB-1 and Progenitor cattle population respectively. The mean SI was 0.539±0.139 in RCC vs. 0.498±0.130 in BCB-1 vs. 0.558±0.100 in Progenitor cattle. The mean heterozygote deficiency (FIS) values were 0.528 in RCC vs. 0.519 in BCB-1 vs. 0.514 in Progenitor cattle. Among the three cattle populations genetic distances were low between BCB-1 and its progenitor. Dendrogram indicated segregation of the 3 cattle populations in to two main clusters (cluster I and 2). Cluster I consisted of RCC and that of cluster 2 consisted of BCB-1 and its progenitor. Our preliminary data inferred that BCB-1 is genetically separated from RCC and it has close relation with progenitor. This study is further conducting with numbers of news microsattelite markers for increasing accuracy of our results.

Keywords: BCB-1, dandogram, Genetic distances, Heterozygosity, Fixation index

Location: RCC, BLRI research farm and Farmer's community at Chittagong district

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Cattle

Objectives:

To identify the genetic diversity of BCB-1 with other indigenous stock of Bangladesh and to find out marker genes for selection of breeding animals.

Methodology:

Blood samples were collected from BCB-1, Red Chittagong (RCC, BLRI research farm and Farmer's community at Chittagong district) and BCB-1 progenitor (Sahzadpur upazilla of Sirajgonj and Bhangura, Faridpur and Sathia upazilla of Pabna district) cattle populations in EDTA containing 10 mL collection vials using 21G needle attached with a disposable 10 ml plastic syringe. The vials were gently tilted for mixing the blood with anticoagulant. The vial was marked with the date of collection, sex and genotype. The blood samples were carried into lab in ice box and stored at -20°C until extraction of genomic DNA (gDNA). gDNA was extracted using BLRI developed DNA extraction kit from frozen thawed blood samples. About 400 µL. blood sample was taken in a 1.5 mL eppendorf tube and lysed with 1 mL lysis buffer through vortexing. The lysed sample was centrifuged one minute at 12000 rpm. After centrifugation, supernatant were discarded and the precipitant were re-Iysed with lysis buffer. The precipitant was dilute in MRB buffer and digested by adding proteniase k for 10 minutes at 55°C. After incubation with proteniase K, the sample was poured into a spin column and centrifuged 2 minutes at 10000 rpm. Then the DNA sample was washed with MRW buffer and DNA was eluted in 1.5 mL eppendorf tube. The sample was stored in -20°C until used for PCR analysis. The purity of gDNA was tested by electrophoresis of DNA sample on 1.5% agarose gel using Ix TBE buffer. Six primers were tested for specificity and PCR analyses with three set microsattelite markers (ETH152, INRA32 and INRA63). Amplification was performed in 20 µI reaction volume (200 µm dNTPs, 1X PCR buffer, 500 µm forward and 500 µm reverse primer and 2 µL DNA templates, 1U Taq polymeras). The PCR reactions were started with an initial heating at 94^ûC for 5 min, followed by 35 cycles of 30s at 94^ûC, 40s at 56-60^ûC and 40s at 72^ûC and ended with a final extension at 72^ûC for 10 min. PCR products from each sample were confirmed by running on 3% high resolution agarose gel at 60V, 300 mA and 300 W for 90 m in and visualized by ethidium bromide staining in 1 x TBE buffer.

Source of Information: Proceedings of Annual Research Review Workshop, 2011-2012

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Our preliminary data inferred that BCB-1 is genetically separated from RCC and it has close relation with its progenitor cattle.

Knowledge Management: Report/Proceedings