ABSTRACT: Random amplification of polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) analysis was carried out using DNA samples of 30 animals of Rathi cattle and 42 animals of Tharparkar cattle. Genomic DNA was isolated as per standard protocol and evaluated for its quality, purity and concentration. Twenty-three random primers were screened out of which 15 primers yielded satisfactory amplifications and were used for further analysis. Average numbers of polymorphic fragments per primer were 7.07±0.86 in Rathi and 6.80±0.61 in Tharparkar cattle. The percentage of polymorphic bands in these two cattle breeds were 86 and 87%, respectively. Within breed genetic similarities for pooled over primers in the animals of Rathi and Tharparkar breeds were 0.577±0.30 and 0.531±0.02, respectively on the basis of band frequency (BF) and 0.645±0.04 and 0.534±0.04, respectively on the basis of band sharing (BS). Averages of between-breed genetic similarities for pooled over primers were 0.97 and 0.92 according to BF and BS, respectively, which reflect the higher degree of genetic similarity between Rathi and Tharparkar cattle breeds. Index of genetic distance based on BF and BS for pooled over primers was 0.030±0.011 and 0.088±0.031, respectively. Percentage of polymorphic bands and within-breed genetic similarities on the basis of band frequency (BF) and band sharing (BS) for pooled over primers revealed higher genetic similarity in Rathi than Tharparkar cattle population. High estimates of between-breed genetic similarities for pooled over primers indicated that either Rathi is having decent from Tharparkar or both the cattle breeds are having common descent. The low value of Index of genetic distances between these two cattle breeds may be due to the fact that Rathi and Tharparkar cattle breeds are the native of the Thar Desert in Northwest India. The results of between-breed genetic distances also confirm the existence of the high degree of genetic similarity between these two breeds of cattle. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 9: 1204-1209)
INTRODUCTION Characterization of Indian zebu cattle has attempted by biometrical methods, which are based exclusively on quantitative traits. As these traits are controlled by multiple loci and are affected by polygene and environmental factors, genetic improvement in these traits relatively slow. Efforts were also made to explore polymorphisms on the basis of biochemical, immunological and cytogenetic markers. However, since these polymorphisms could not exploit the genetic variation at DNA level, hence they were of little importance and could not be used in the precise identification of an individual. Molecular genetic techniques are efficient for evolutionary, ecological and population genetics studies. Among these a technique termed as Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPDPCR) has been successfully applied in genetic studies of various animals and plant species (Michelmore et al., 1991; Welsh and McClelland, 1991; Baird et al., 1992; Chapaco et al., 1992) as well as for molecular characterization of bovine populations (Bardin et al., 1992; Kemp and Teale, 1992). RAPD-PCR is based on amplification of DNA in the polymerase chain reaction (PCR) by short random primers. This technique has several advantages over others as it is non-radioactive, easy to perform, involves low cost, readable directly on the gel, does not require knowledge of prior sequence and also requires very little amount of DNA. India has 26 recognized cattle breeds among these Rathi is a dual-purpose cattle breed having the home track in Alwar and Rajputana region of Rajasthan and spread up to Bikaner district. Milk yield of Rathi cow is about 4.5 kg per day. Bullocks of this breed are used for the draft purpose. Selective breeding of Rathi cattle in closed herd system at Livestock Research Station (LRS), Nohar of Rajasthan Agricultural University, Bikaner resulted in the production of germ plasma having dam’s yield 2,233 kg per lactation in the elite herd. Tharparkar is also dual-purpose cattle breed and the preferred synonyms for this breed are Thari, Grey Sindhi and White Sindhi. Cows of this breed are considered to be temperamental and having average milk yield of 1,000 to 7,500 lb. (Wahid, 1971). Bullocks of this breed are of medium size and steers and having a reputation of willing workers. This breed is known for their high disease resistance. Very few reports are available on molecular characterization of indigenous breeds; therefore present investigation was conducted to analyze the genetic variability and similarity between and within Rathi and Tharparkar cattle populations by RAPD-PCR.
MATERIALS AND METHODS
Experimental animals
Randomly selected 30 Rathi and 40 Tharparkar animals maintained at Livestock Research Station (LRS) of Rajasthan Agricultural University, Nohar, DistrictHanumangarh, Rajasthan and Central Cattle Breeding Farm (CCBF), Suratgarh, District-Hanumangarh, Rajasthan, respectively were used in the present investigation.
DNA samples
Blood samples were collected in sterile polypropylene centrifuge tubes containing Experimental animals Randomly selected 30 Rathi and 40 Tharparkar animals maintained at Livestock Research Station (LRS) of Rajasthan Agricultural University, Nohar, DistrictHanumangarh, Rajasthan and Central Cattle Breeding Farm (CCBF), Suratgarh, District-Hanumangarh, Rajasthan, respectively were used in the present investigation. DNA samples Blood samples were collected in sterile polypropylene centrifuge tubes containing anticoagulant. The blood was gently mixed with anticoagulant and kept on ice to maintain the low temperature in order to prevent cell lysis. Subsequently, the blood samples were transported to the laboratory and stored at 4°C until the isolation of genomic DNA. The genomic DNA was isolated by phenol extraction method (Anderson et al., 1986) with some modifications. Amplification of genomic DNA by PCR The amplifications were carried out in 0.2 ml of PCR reaction tubes using a programmable thermal cycler (M. J. Research, USA). The reaction mix was prepared in 25 µl reaction volume having 50 ng of genomic DNA, 100 µl each of dNTPs, 1 µM of Tetra Methyl Ammonium Chloride (TMAC), 40 ng of primer, 0.5-1.0 U of Taq DNA Polymerase and 2.5 µl of 10 X Taq DNA Polymerase buffer (500 mM KCl, 100 mM Tris HCl, 1.5 mM MgCl2, 1% Triton X-100). Various PCR programmes were tested for obtaining reproducible results and ultimately amplification of DNA was carried out by initial denaturation at 95°C for 5 min, 45 cycles of denaturation, annealing and primer extension at 94, 36 and 72°C for 1 min each, respectively followed by final extension at 72°C for 5 min. The amplified products were electrophoresed by running in a 1.4% w/v agarose gel at 1-2 V/cm for approximately 4-6 h in
Amplification of genomic DNA by PCR
The amplifications were carried out in 0.2 ml of PCR reaction tubes using a programmable thermal cycler (M. J. Research, USA). The reaction mix was prepared in 25 µl reaction volume having 50 ng of genomic DNA, 100 µl each of dNTPs, 1 µM of Tetra Methyl Ammonium Chloride (TMAC), 40 ng of primer, 0.5-1.0 U of Taq DNA Polymerase and 2.5 µl of 10 X Taq DNA Polymerase buffer (500 mM KCl, 100 mM Tris HCl, 1.5 mM MgCl2, 1% Triton X-100). Various PCR programmes were tested for obtaining reproducible results and ultimately amplification of DNA was carried out by initial denaturation at 95°C for 5 min, 45 cycles of denaturation, annealing and primer extension at 94, 36 and 72°C for 1 min each, respectively followed by final extension at 72°C for 5 min. The amplified products were electrophoresed by running in a 1.4% w/v agarose gel at 1-2 V/cm for approximately 4-6 h in 1×TAE buffer. The gels were stained with Ethidium Bromide, viewed under UV light and documented for further analysis.
Recording of data and statistical analysis Only distinct and prominent bands were scored and the presence and absence of a band was recorded as ‘1’ and ‘0’, respectively. RAPD patterns of Rathi and Tharparkar animals were compared within as well as between breeds. Genetic similarity within and between the populations was calculated using two measures of genetic similarity i.e. band sharing and band frequency.
Genetic similarity based on band sharing
The band sharing (BS) frequency between two animals was calculated as an expression of similarity of RAPD Recording of data and statistical analysis Only distinct and prominent bands were scored and the presence and absence of a band were recorded as ‘1’ and ‘0’, respectively. RAPD patterns of Rathi and Tharparkar animals were compared within as well as between breeds. Genetic similarity within and between the populations was calculated using two measures of genetic similarity i.e. band sharing and band frequency. Genetic similarity based on band sharing The band sharing (BS) frequency between two animals was calculated as an expression of similarity of RAPD
fingerprints of animals from either the same or different breeds (Jeffrey and Morton, 1987; Wetton et al., 1987; Dunnington et al., 1990) using the formula.
BS=2 Nab/(Na+Nb)
Where, Nab is a number of common fragments observed in a and b animals and Na and Nb are the total numbers of fragments scored in animals a and b, respectively. Within breed similarity (WSi) was calculated as the average of Bab across all the possible comparisons between animals within i th breed. Between breed similarity (BSij), corrected for within-breed similarity were estimated (Lynch, 1990) as.
BS ij=1+BS’ij-(WSi+ WSj)/2
Where, WSi and WSj are the values of within breed similarity in ith and jth breeds, respectively and BS’ij is the average similarity between all possible comparisons between animals of the ith and jth breeds. The value of BS’ij may exceed 1. The genetic distance (Dij) was estimated as.
D’ij=-In [BS’ij/√ (WSi⋅WSj)]
Genetic similarity based on band frequency
The genetic similarity within the breed was estimated as. Where, Vi is the frequency of occurrence on the ith band and N is the total number of bands scored. The genetic similarity between two breeds was obtained as follows.
U=1/N∑Vi
Where, Vi is the frequency of occurrence on the ith band and N is the total number of bands scored. The genetic similarity between two breeds was obtained as follows.
I=1/N∑(2.Vi (1)⋅Vi (2))/[{Vi (1)}2 +{Vi (2)}2 ]
Where Vi (1) and Vi (2) are the frequency of occurrence of i th band in breeds 1 and 2 respectively and N is the total number of bands scored. The index of genetic distance was estimated as -In
For more reference: https://www.ajas.info/upload/pdf/17_195.pdf
Thank you: Amit Kumar Sharma, Bharat Bhushan*, Sanjeev Kumar1, Pushpendra Kumar, Arjava Sharma and Satish Kumar Animal Genetics Division, Indian Veterinary Research Institute, Izatnagar, Bareilly-243 122 (Uttar Pradesh), India
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