Molecular genetic characterization of Madras Red sheep in Tamil Nadu, India using microsatellite markers

R Selvam, P S Rahumathulla, S N Sivaselvam, S M K Karthickeyan and R Rajendran

Department of Animal Genetics and Breeding, Madras Veterinary College
Tamilnadu Veterinary and Animal Sciences University, Chennai - 600 007, India
drselvam@gmail.com

Abstract

Genetic variation in Madras Red sheep breed in South India was studied using 10 FAO recommended ovine-specific microsatellite markers. The number of observed alleles ranged from 7 to 19. The size of alleles ranged from 92 to 168 bp. All the 10 loci were found to be highly polymorphic. The polymorphism information content values ranged from 0.737 to 0.902 with a mean of 0.809. The observed heterozygosity ranged from 0.923 to 1.000 with a mean value of 0.972 whereas the expected heterozygosity ranged from 0.688 to 0.883 with a mean value of 0.785 indicating the heterogeneous nature of the population distributed in the breeding tract. The markers used in the study were highly informative and high heterozygosity value is indicative of the higher amount of genetic variability.

Key words: alleles, DNA, genetic variation, Hardy-Weinberg equilibrium, heterozygosity, PCR, polymorphism information content, polyacrylamide gel, silver staining

Introduction

India has vast genetic diversity with respect to domestic animal species. Particularly, the State of Tamil Nadu is endowed with several breeds of sheep. They are well known for their heat tolerance, mutton production and adaptability to the local agro-ecological conditions.  Preliminary surveys in the native tract of these breeds revealed that the population of indigenous breeds of sheep has declined considerably due to indiscriminate crossing with non-descripts.  At present, there is genetic dilution of these breeds and a few of them are threatened with extinction (Tantia and Vij 2000).  Hence, for effective and meaningful conservation, genetic characterisation of native breeds is the first step to safeguard our valuable germplasm. . Madras Red breed is distributed in Chennai, Kancheepuram, Thiruvallur, Villupuram districts of Tamil Nadu, India. The animals are medium-sized, well-built, hair sheep. The color generally varies from light to dark tan.

Due to the tremendous progress in the field of molecular biology, new class of markers called molecular markers has emerged during the last two decades (Mac-Hugh et al 1998).    Of these, microsatellites are the markers of choice as they are abundantly distributed throughout the mammalian genome and because of easy genotyping.  They have a large number of alleles, a high level of heterozygosity and are inherited in a Mendelian fashion.  These characteristics make them valuable for genome mapping, linkage analysis and phylogenetic studies (Koreth et al 1996).

The application of microsatellites for characterising breeds of domestic animals is still a relatively new issue and only recently, research has been focused on this point.  Hence, the present work was undertaken with the objective to characterize Madras Red sheep breed of Tamil Nadu based on microsatellite polymorphism. This study will be of immense use in identifying genetic uniqueness of the breed, tracing the evolutionary origin and formulating effective conservation strategy.

Materials and methods 

Blood sample collection and DNA extraction

Blood samples were collected at random from 50 unrelated sheep belonging to different villages in the breeding tract. DNA was extracted from the blood by a rapid, non-enzymatic method (Lahiri and Nurnberger 1991). The quantity and quality of the isolated DNA samples were tested by a standard spectrophotometer analysis.

Genotyping

A total of 10 microsatellite markers were genotyped on 50 DNA samples of Madras Red breed. The microsatellite markers were selected as per the guidelines of Food and Agriculture Organisation of United Nations (FAO 2004).

The markers analysed were BM 8125, CSSM 31, Oar AE 129, Oar CP 34, Oar FCB 128, Oar HH 35, Oar JMP 29, Oar JMP 8, RM 4 and TGLA 377. The PCR amplications were carried out in a 20µl final reaction volume containing at least 20-50 ng of genomic DNA, 20 picomoles of each primer, 1.5mM MgCl₂, 100µM dNTPs, 0.25U Taq polymerase and 10x buffer. The PCR protocol was as follows: 3 min at 94°C followed by 30 cycles of 1 min at 94°C, 1 min at annealing temperature (varies for primers)  and 1 min at 72°C. The last elongation step was for 10 min.

After confirmation of amplification on 2% agarose gel, products were electrophoresed on 6% denatured polyacrylamide gel and visualized by silver staining (Comincini et al 1995) along with a single stranded 10 bp DNA ladder (Invitrogen, USA) as marker.  Estimation of allele size was done using Diversity Database software (Bio-Rad, USA). The polymorphism information content (PIC) was analysed using Nei's formula (Nei 1973). Genetic variation was quantified by calculating observed and effective number of alleles, observed heterozygosity and expected heterozygosity. Hardy-Weinberg equilibrium (HWE) test was performed and co-efficient of gene differentiation was calculated.

Results 

The results of the microsatellite analysis of Madras Red sheep size, polymorphisms are furnished in Table 1.

The number of alleles was in the range of 7 (BM8125 and TGLA 377) to 19 (CSSM 31) with the mean number of 9.8 per microsatellite locus. The allele sizes ranged from 92 bp (TGLA 377) to 168bp (Oar AE 129). The frequency of the alleles ranged from 0.0108 (Oar JMP 29) to 0.8333 (Oar JMP 8). The polymorphism information content (PIC) values ranged from 0.737 (Oar FCB 128) to 0.902 (CSSM 31) indicating that all these loci were highly polymorphic in nature. The sheep population under study was not in Hardy-Weinberg equilibrium for all microsatellite loci, except Oar JMP 8.The observed heterozygosity ranged from 0.923 to 1.000 while the expected heterozygosity ranged from 0.688 to 0.883 across the microsatellite markers studied.

Discussion 

Allele numbers and sizes

It should be noted that the microsatellite loci analyzed in this study is as per the recommended list of loci by FAO (2004). 

The mean number of alleles observed in the present study was generally higher than those reported for Nilagiri and Coimbatore sheep, the other breeds of sheep from the same State of Tamilnadu. In Nilagiri sheep, the number of alleles across all the loci studied ranged from 3 to 8 with a mean of 5 and the size of alleles ranged from 72 to 228 bp. The frequency of alleles ranged from 0.0104 to 0.5781(Haris et al 2007). Kumarasamy et al (2008) analysed genetic structure of Coimbatore breed with 27 sheep specific markers and observed that number of alleles ranged from 3 to 8 with a mean of 6 and the size of alleles ranged from 72 to 220 bp. The frequency of the alleles ranged from 0.0166 to 0.7245.In another study with 25 markers on Ganjam sheep,distributed in Orissa state of India, the mean number of allele was found to be 5.48 (Arora et al 2008). 

The number of alleles identified at Oar AE 129, Oar CP 34, Oar FCB 128, and Oar JMP 29 loci were 7, 8, 8, and 20 in Swiss breeds of sheep respectively (Saitbekova-Stahlberger et al 2001).  Using the same set of microsatellite primers, the numbers of alleles identified in the present study were found to be almost similar in Madras Red sheep. 

 The number of alleles at Oar CP 34 locus was 8 similar reports were also made for Oar CP 34 and Oar FCB 128 (Diez-Tascon et al 2000) and for Oar CP 34 (Arranz et al 2001).  In Nilagiri sheep the size of alleles ranged from 72 to 228 bp. The frequency of alleles ranged from 0.0104 to 0.5781. In total, 125 alleles were observed at the 25 loci studied (Haris et al 2007). The product sizes obtained for the loci are within the ranges of previous workers.  The differences observed in the present study might be due the breeds and population differences.

Polymorphism information content

In general, PIC values are suggestive of polymorphic nature of the microsatellite loci analyzed. The mean polymorphism information content value for all ten microsatellite value is 0.809.  The Oar loci such as HH35, JMP 29, JMP 8, CP 34 and CSSM 31 were found to be highly polymorphic with the PIC values being 0.871, 0.856, 0.835, 0.807 and 0.902 respectively.  Other loci, RM4 and TGLA 377, had the PIC estimates of 0.777 and 0.739 respectively. In some of the Spanish sheep breeds the PIC values ranged from a minimum of 0.30 for BM 1258 locus to a maximum of 0.89 for MAF 70 locus (Arranz et al 2001).  The PIC values observed are comparable with those observed in Garole sheep (Sodhi et al 2003) and Nilagiri sheep (Haris et al 2007).

Hardy-Weinberg equilibrium

The results of the χ² test for goodness of fit revealed that the population of Madras Red sheep was not in Hardy-Weinberg equilibrium proportions (P<0.01) for all the microsatellite loci except Oar JMP 8.  The disequilibrium proportions observed in most of the loci in the study might be due to both the systematic and dispersive processes operating in the population. Similar population disequilibria were noticed for Nilagiri (Haris et al 2007), Muzzafarnagri (Arora 2004), Garole (Sodhi et al 2003) and Bellary sheep (Kumar et al 2007) and for all the five loci studied in Madras Red, Mecheri, and Nilagiri sheep (Satyanarayana 2001). 

Heterozygosity

Genetic diversity can be measured as the amount of actual or potential heterozygosity and diversity within the breed is measured as the estimate of heterozygosity.  The observed heterozygosities were high approaching unity. In an earlier study, similar higher heterozygosity values were noticed for Oar AE 129, Oar LP 34, Oar FCB 128, and Oar JMP 29 in Swiss sheep breeds (Saitbekova-Stahlberger et al 2001).  The results are comparable with the previous findings in Nilagiri sheep in which the observed heterozygosity ranged from 0.4222 to 1.000 with a mean value of 0.7610 and the (Haris et al 2007).

The panel of microsatellites evaluated in Madras Red sheep breed in the present study showed a very high polymorphism and heterozygosity. Microsatellite based analysis of sheep breed structure is an initial step towards estimation of genetic distances and relationships among Indian sheep breeds and to help in conservation plans. We propose that this set of microsatellites may be used for identifying individuals and for genetic diversity studies for selection and conservation of native sheep breeds.

References 

Arora S B 2004 Genetic structure of Muzzafarnagri sheep based on microsatellite analysis. Small Ruminant Research 54: 227-230

Arranz J J, Bayon Y and San Primitivo F 2001 Genetic variation at microsatellite loci in Spanish sheep.  Small Ruminant Research 39: 3-10

Comincini S, Leone P, Redaelli L, De Giuli L, Zhang Y and Ferretti L 1995 Characterization of bovine microsatellites by silver staining. Journal of Animal Breeding and Genetics 112: 415-420

Diez-Tascon C, Little John R P, Almedia P A R, and Crawford A M 2000 Genetic variation within the Merino sheep breed: analysis of closely related populations using microsatellites.  Animal Genetics 31: 243-251

FAO 2004 Secondary guidelines for development of national farm animal genetic resources management plans for global management of cattle genetic resources using reference microsatellites global projects for the maintenance of domestic animal genetic diversity (MoDAD). [http://www.fao.org/dad-is/]

Haris G, Sivaselvam S N, Karthickeyan S M K and Saravanan R 2007 Molecular characterisation of Nilagiri sheep (Ovis aries) of south India based on microsatellites. Asian-Australasian Journal of Animal Sciences 20: 633-637

Koreth J, Jhon L and James M 1996 Microsatellites and PCR genomic analysis.  Journal of Pathology 178: 239-248

Kumar D, Sharma1 R, Pandey A K, Gour1 D S, Malik G, Ahlawat S P S  and Jain A 2007 Genetic diversity and bottleneck analysis of Indian Bellary sheep by microsatellite markers. Russian Journal of Genetics 43: 996-1005

Kumarasamy P, Prema S, Ganapathi P, Karthickeyan S M K and Kanakaraj P 2008 Genome analysis in Coimbatore sheep using microsatellite markers. In: National Symposium on Redefining  Role of Indigenous Animal Genetic Resources in Rural Development, Bangalore,India, February 15 and 16,2008

Lahiri D K and Nurnberger J I 1991 A rapid non enzymatic method for the preparation HMW DNA from blood for RFLP studies. Nucleic Acids Research 19:544

Mac-Hugh D E, Loftus R T, Cunningham P and Bradley D G 1998 Genetic structure of seven European cattle breeds assessed using 20 microsatellite markers. Animal Genetics 29: 333-340

Nei M 1973 The theory and estimation of genetic distance. In: Morton N E (ed).Genetic Structure of Populations pp.45-54 University of Hawaii Press Honolulu

Saitbekova-Stahlberger N, Schlapfer J, Dolf G and Gaillord C 2001 Genetic relationships in Swiss sheep breeds based on microsatellite analysis. Journal of Animal Breeding and Genetics 118:379-387

Satyanarayana R 2001 Estimation of polymorphic information content in sheep using cattle microsatellites.  M.V.Sc.  Thesis submitted to the Tamil Nadu Veterinary and Animal Sciences University, Chennai – 51

Sodhi M, Mukesh M, Arora R, Tantia M S and Bhatia S 2003 Genetic structure of Garole- a unique Indian micro sheep assessed using microsatellite markers. Indian Journal of Dairy Science 56:167-173

Tantia M S and Vij P K 2000 Population estimates of sheep and goat breeds of India.  Indian Journal of Animal Research 34: 60-63

Received 20 May 2008; Accepted 16 January 2009; Published 18 April 2009

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