Journal of Pharmacognosy and Phytochemistry 2018; 7(2): 1940-1942

E-ISSN: 2278-4136 P-ISSN: 2349-8234 JPP 2018; 7(2): 1940-1942 Genetic diversity analysis of Deoni cattle using Received: 01-01-2018 Accepted: 02-02-2018 microsatellite markers

K Swathi Department of Animal Genetics K Swathi, S Sai Reddy and B Ramesh Gupta and Breeding, College of Veterinary Science, PV Abstract Narsimha Rao Telangana The present study was undertaken for population genetic analysis of microsatellite variation in Deoni Veterinary University, cattle using 15 microsatellite markers selected from the list suggested by FAO. All the 15 microsatellite Hyderabad, Telangana, India markers were amplified and observed number of alleles ranged from five (in INRA005, INRA063, S Sai Reddy ETH225 and ILSTS006) to fourteen (in HAUT024) with total 109 alleles. The overall mean observed Department of Animal Genetics heterozygosity and expected heterozygosity values were 0.272 (±0.068) and 0.793 (±0.014), and ranged and Breeding, College of from 0.000 to 0.926 and 0.695 to 0.897, respectively. The overall mean PIC was 0.763. All the 15 Veterinary Science, PV primers used in the present study were found to be polymorphic and highly informative with the PIC Narsimha Rao Telangana values ranging from 0.643 to 0.888. The overall mean inbreeding coefficient (FIS) obtained in the present Veterinary University, study was 0.661, suggested excess heterozygosity in the population. The χ2 test for Hardy – Weinberg Hyderabad, Telangana, India equilibrium revealed that there was significant deviation of observed allele frequencies from expected for all the loci studied. The high information of polymorphic loci throw light on the scope for maintaining B Ramesh Gupta variation in the population. Department of Animal Genetics and Breeding, College of Keywords: deoni cattle, microsatellite, genetic variability, inbreeding coefficient, hardy - weinberg Veterinary Science, PV Narsimha Rao Telangana equilibrium Veterinary University, Hyderabad, Telangana, India Introduction India, the top milk producer in the world, is progressing at a faster rate in animal husbandry sector. India has diversified and unique cattle genetic resources having 30 well recognized breeds that constitute 7.75% (FAO1995) of the total cattle breeds of the world. Indian cattle

breeds also known as Zebu cattle (Bos indicus) are broadly categorized based upon their utility into dairy, draft and dual purpose breeds. Zebu breeds of cattle from southern part of India are well known for their draught efficiency and disease resistance. Deoni is one of the famous dual purpose cattle breeds of India. The dual purpose breeds have specific characters like disease resistance, ability to survive and reproduce under adverse climatic conditions with low inputs

and heat tolerance. Microsatellites are short DNA sequence stretches, in which a motif of one to six bases is tandemly repeated and these sequences differ in repeat number among the individuals (Schlotter and Tautz, 2000). Microsatellites are highly polymorphic, dispersed throughout genome at a frequency of one at every 6 kb sequence (Bruford and Wayne, 1993) [1] and

amenable to PCR amplification to make them potentially useful DNA markers in gene mapping studies. The present study was carried out with an objective of assessing the molecular genetic variability of Deoni cattle using microsatellite markers.

Materials and Methods

DNA samples The microsatellite analysis was carried out on a sample of 28 unrelated Deoni cattle collected from Institutional Livestock Farm Complex, College of Veterinary Science, Rajendranagar, Hyderabad. Genomic DNA was isolated from peripheral blood using standard Phenol- Chloroform method (Sambrook et al., 2001) with minor modifications. The quality and

quantity of DNA was evaluated on spectrophotometer and through 0.8% agarose gel electrophoresis, respectively.

Correspondence Microsatellite analysis K Swathi A set of 15 dinucleotide microsatellite marker primers were chosen from the list recommended Department of Animal Genetics and Breeding, College of by FAO. Polymerase chain reaction (PCR) amplification of microsatellite loci was carried out Veterinary Science, PV in a 12.5µl reaction volume containing 1.5 mM MgCl2, 2.5 mM dNTPs, 60 pM of forward ⁄ Narsimha Rao Telangana reverse primer, approximately 100 ng of genomic DNA and 0.5 U of Taq DNA polymerase. Veterinary University, PCR was carried out using cycling conditions as: 5 min at 95 oC, followed by 34 cycles Hyderabad, Telangana, India ~ 1940 ~ Journal of Pharmacognosy and Phytochemistry

of 1 min at 94 oC, 30 s at annealing temperature (depending unrelated individuals and also due to the presence of more upon locus), 30 s at 72 oC and final extension at 72 oC for 5 homozygous individuals (less variation in the breed) in the min. Amplified PCR products were checked on 2% agarose samples analyzed. gel containing ethidium bromide and visualized through UV The coefficient of inbreeding (FIS) measures the reduction in illumination. Allele sizes were estimated by running a 20 bp the heterozygosity of individuals within population and higher DNA ladder (GeneRuler, Fermentas) along with the PCR values of FIS indicate closer relationship with the individual. products and genotypes scored manually. The FIS value calculated in present study ranged from -0.158 (BM1824) to 1.000 (MM12), with a mean of 0.512 across the Results and Discussion loci in this study indicated the presence of excess The ratio of optical density in this study at 260 and 280 nm heterozygosity in the investigated cattle population. were consistently between 1.69 to 1.80 indicating the The χ2 test for Hardy – Weinberg equilibrium revealed that deproteinisation and good quality of DNA (Sambrook and there was significant deviation of observed allele frequencies Russell, 2001). Similar observations were recorded by many from expected for all the loci studied. This indicated that the authors in various breeds of catlle Muralidhar et al. (2003); population was not in equilibrium status which was due to Srinivas et al. (2006) in Ongole and Deoni; Chennakesavulu long term selection being practiced in the population. The et al. (2003) in Punganur cattle. Various within breed disequilibrium exhibited in most of loci revealed that there diversity estimates, Viz. Mean number of alleles (Na), might be unobserved null alleles. Effective number of alleles (Ne), Shannon’s information index (I), Observed heterozygosity (Ho), Expected heterozygosity Table 1: Mean number of alleles (Na), Effective number of alleles (He), Polymorphism information content (PIC), Fixation (Ne), Shannon’s Information Index (I), Observed heterozygosity (Ho), Expected heterozygosity (He) Polymorphism Information indices (FIS) at each microsatellite marker analysed in Murrah buffaloes are presented in Table 1. content (PIC) and Fixation Indices (FIS) at various microsatellite loci studied. A total of 109 alleles were amplified across 15 microsatellite loci. Allele size ranged from 74 to 300 bp. The number of Locus N Na Ne I Ho He F PIC alleles per locus varied from 5 to 14, with an overall mean of INRA005 24 5.000 4.235 1.517 0.583 0.764 0.236 0.726 7.27±0.714. The observed number of alleles demonstrated TGLA126 15 6.000 5.556 1.751 0.333 0.820 0.593 0.794 that almost all the microsatellite loci utilized in the present INRA035 24 6.000 4.129 1.567 0.208 0.758 0.725 0.721 study were sufficiently polymorphic. The mean number of ILSTS005 23 6.000 4.133 1.556 0.435 0.758 0.426 0.722 alleles observed is more than the number reported in Deoni INRA063 24 5.000 3.282 1.333 0.208 0.695 0.700 0.643 (5.9) and Sahiwal (5.2) cattle (Mukesh et al., 2004) [8], in TGLA122 23 13.000 7.723 2.278 0.435 0.871 0.501 0.859 Hariana (4.2), Sahiwal (4.1) and Tharparker (3.6) cattle HEL001 24 6.000 4.590 1.606 0.083 0.782 0.893 0.748 ETH225 25 5.000 3.743 1.432 0.000 0.733 1.000 0.691 (MacHugh et al., 1997) and Deoni (5.9) (Srinivas et al., [12] ILSTS006 27 5.000 3.701 1.416 0.000 0.730 1.000 0.684 2006). Sodhi et al. (2005) reported the mean number of CSRM60 25 8.000 6.757 1.982 0.360 0.852 0.577 0.834 alleles as 5.82 in and 5.86 in Deoni cattle. BM1824 27 7.000 4.993 1.733 0.926 0.800 -0.158 0.772 The effective number of alleles (Ne) is an estimate of the TGLA227 23 7.000 5.845 1.824 0.043 0.829 0.948 0.806 number of alleles with equal frequencies corresponding to a MM12 27 8.000 5.321 1.827 0.000 0.812 1.000 0.787 particular PIC value. The mean effective number of alleles HAUT024 23 14.000 9.706 2.427 0.391 0.897 0.564 0.888 was 5.25±044, values were in range of 9.71 (HAUT024) to HAUT027 26 8.000 5.007 1.804 0.077 0.800 0.904 0.776 3.28 (INRA 063). The mean Ne was higher compared to 3.42 in Hariana and 2.95 in reported by Mukesh et References al. (2004) [8]. 1. Bruford MW, Wayne RK. Microsatellites and Their The PIC values which is the statistical assessment of Application to Population Genetic Studies. Current informativeness of markers, ranged from 0.64 (INRA063) to Option in Genetics and Development. 1993; 3:939-943. 0.89 (HAUT024), with a mean PIC of 0.763. All the loci 2. 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