Indian J. Anim. Res., 49 (1) 2015: 36-39 AGRICULTURAL RESEARCH COMMUNICATION CENTRE Print ISSN:0367-6722 / Online ISSN:0976-0555 www.arccjournals.com/www.ijaronline.in

Molecular characterization of using microsatellite markers

A. Barani*, P.S. Rahumathulla1, R. Rajendran2, P. Kumarasamy3, P.Ganapathi4 and P. Radha5 Department of Animal Genetics and Breeding, Madras Veterinary College, Chennai- 600 007, India. Received: 07-09-2012 Accepted: 28-05-2013 doi: 10.5958/0976-0555.2015.00007.2 ABSTRACT The present study estimates genetic variability in Pulikulam cattle (n=50), a draught breed of India, by use of 18 microsatellite markers recommended by the Food and Agriculture Organization. Microsatellite genotypes were derived and allelic and genotypic frequencies, heterozygosities and gene diversity were estimated. A total of 142 alleles were distinguished by the 18 microsatellite markers used. All the microsatellites were highly polymorphic, with mean (± s.e.) allelic number of 7.89 ± 0.72, ranging 3-12 per locus. The observed heterozygosity in the population ranged between 0.1316 and 0.8958, with mean (± s.e.) of 0.5758 ± 0.0535, indicating considerable genetic variation in this population. Genetic bottleneck hypotheses were also explored. Our data suggest that the Pulikulam breed has not experienced a genetic bottleneck in the recent past.

Key words: Genetic bottleneck, Genetic diversity, Microsatellite markers, Pulikulam cattle.

INTRODUCTION mainly used for penning and for manure in the agricultural India is one of the mega bio-diversity centres of the fields and bulls are used for “bull baiting” (Rajendran et al., world. As per the 17th Livestock Census, the cattle population 2008). in India is 185.18 million, of which 160.50 million are DNA-based molecular markers have a very high indigenous (Anonymous, 2004). Mechanization, unplanned level of polymorphism and have been successfully used to and indiscriminate breeding among native stocks as well as evaluate genetic variation of populations in breeding human bias in favour of certain breeds have directly or programs (Martin-Burriel et al., 1999; Almeida et al., 2000). indirectly lead to the dilution of indigenous germplasm (FAO, Due to the ubiquity, PCR typability, Mendelian co-dominant 2000). Hence, there is an urgent need to prevent the rapid inheritance, extreme polymorphism and high heterozygosity, erosion of animal genetic resources. microsatellites have assumed an increasingly important role A large proportion of indigenous livestock as markers in the genome (Koreth et al., 1996). populations in the developing world have yet to be MATERIALS AND METHODS characterised or evaluated at phenotypic and genetic levels Sample collection and genomic DNA extraction: Blood (Hanotte and Jianlin, 2005). The Pulikulam breed of cattle is samples were collected from 50 unrelated Pulikulam cattle primarily a draught breed, small in size and capable of much from its breeding tract. Genomic DNA was isolated by endurance (Littlewood, 1936). Cows are generally smaller Phenol-Chloroform method (Sambrook et al., 1989). The in size. Calves are generally grey in colour at birth. Heifers purity and concentration of DNA samples were estimated by and cows are generally grey in colour. The presence of reddish UV spectrophotometer. The quality of DNA samples were or brownish tinges in any one or more of the points viz., also checked by agarose gel (1%) electrophoresis. muzzle, eyes, perineum, switch and back is observed (Pattabhiraman, 1958). Pulikulam cattle is a quick trotting A total of 18 microsatellite primer sets, specific for breed and got the name from the village Pulikulam in cattle, were used in the study as recommended by FAO and Sivaganga district of Tamilnadu from where it originated and International Society for Animal Genetics (BM1824, CSSM8, the approximate population size was 45,000. The herds are CSSM66, ETH3, ETH10, ETH225, HAUT24, HEL1, HEL5, *Corresponding author’s e-mail: [email protected] and Dairy Production Division, SRS of NDRI, Adugodi, Bangalore- 560 030., 1 Veterinary College and Research Institute,Tirunelveli., 2TANUVAS, Madavaram Milk Colony, Chennai., 3Department of Bioinformatics and ARIS Cell, MVC, Chennai.4 & 5 - IVPM, Ranipet, Chennai, India. Vol. 49 Issue 1, (February 2015) 37 HEL9, ILSTS6, ILSTS11, ILSTS34, MM8, MM12, observed as twelve in Pulikulam cattle falls within the range TGLA53, TGLA122 and TGLA227). Only forward primer of 8-13 recorded for maximum number of alleles among the of each pair was labeled (FAM, PET, VIC, NED, TAMRA, various breeds of cattle (Manjunatha Prabhu, 2004; Mukesh HEX and TET) whereas the reverse primers were kept et al.,2004). The allele frequencies ranged from 0.0100 (107 unlabeled. bp, 109 bp at HAUT24 and 105 bp at HEL1) to 0.8816 (261 Microsatellite markers and PCR amplification: PCR was bp at ILSTS11) in the present study. However, the allele frequencies ranged from 0.0306(ETH10) to 0.8673(ETH152) carried out in 251 reaction volume containing 1.5 mM Mgcl2, 200M dNTPs, 50ng of each primer, 100ng of template DNA in (Kumar et al., 2006) and from 0.0104 to 0.7614 and 0.5U of Taq DNA polymerase. PCR cycling conditions in Krishna Valley (Kumar et al., 2009). were: 5 min at 95oC, followed by 35 cycles of 45 sec at 95oC, Polymorphism information content: PIC values for 45 sec at annealing temperature (52-64oC) of each primer, Pulikulam cattle in the present study ranged from 0.5920 45 sec at 72oC, and final extension of 10 min at 72oC. The (HEL5) to 0.9100 (TGLA122) with a mean value of 0.8251 PCR products with different fluorescent labels were mixed ± 0.0221. The mean PIC value recorded in the present study with Hi Di formamide and Liz™internal size standard, in Pulikulam cattle was higher than those reported in denatured for 5 minutes at 95ºC and snap chilled on ice for 5 Tamilnadu breeds (Ganapathi, 2007; Karthickeyan et al., minutes before run on ABI PRISM 3730XL DNA sequencer. 2007) as well as in other Indian breeds of cattle. Sizing of alleles and extraction of genotypic data were Heterozygosity: The observed heterozygosity values (Ho) performed by GENEMAPPER version 3.0. varied between 0.1316 (ILSTS11) and 0.8958 (HEL9), while

Statistical analyses: Microsatellite allele frequencies, the expected heterozygosity (He) ranged from 0.2168 effective number of alleles, observed and expected (ILSTS11) to 0.8838 (HEL9). The mean values were 0.5758 heterozygosity, F-statistics were calculated and test of ± 0.0535 and 0.6699 ± 0.0425 for Ho and He respectively. Hardy-Weingberg equilibrium was carried out using the The mean observed heterozygosity recorded in the present Popgene version 1.31 (Yeh et al., 1999). The study on Pulikulam cattle was lesser than those reported for polymorphism information content (PIC) was calculated (Karthickeyan et al., 2007) and Bargur according to Nei (1978) using the individual frequencies (Ganapathi, 2007) breeds of cattle. The mean expected in which the allele occurred at each locus (http:// heterozygosity in the present study was higher than that www.genomics.liv.ac.uk/animal/pic.html.). Bottleneck reported for Umblachery (Karthickeyan et al., 2007) and events in the population were tested by two methods. The lesser than that reported for Bargur (Ganapathi, 2007). first method consisted of three excess heterozygosity tests Within population inbreeding estimate: The inbreeding developed by Cornuet and Luikart (1996). The second estimates values ranged from -0.1460 (ETH10) to method was the graphical representation of the mode- 0.5631(HEL5).The overall inbreeding estimate or shift indicator originally proposed by Luikart et al. heterozygote deficiency within population calculated in the (1998). present study was 0.1500 ± 0.0455. This reflected a moderate RESULTS AND DISCUSSION level of inbreeding or heterozygote deficiency in the

Number, size and frequency of microsatellite alleles: A population. The mean FIS value recorded in Pulikulam cattle total of 142 alleles were observed in Pulikulam cattle is higher than that reported for Bargur breed (Ganapathi, population, over all 18 markers under investigation, with a 2007). Umblachery cattle had a negative mean FIS value mean of 7.89 ± 0.72 alleles per locus. This value reflected indicating absence of inbreeding (Karthickeyan et al., 2007). the high level of allelic variability in Pulikulam population. Hardy-Weinberg Equilibrium: Majority of the loci under

The effective number of alleles (ne) ranged from investigation (12 out of 18 loci) returned highly significant 1.2722 (ILSTS11) to 7.9723 (HEL9). The mean number of Chi-square values suggesting departure from Hardy-Weinberg effective alleles considering all loci was 3.73 ± 0.42. The ‘ne’ Equilibrium (HWE). The loci HEL1 revealed statistically values provide information about the number of predominant significant departure from HWE. Thus, 12 loci in this study alleles at a particular locus. departed from HWE. Minimum number of alleles at a particular locus Bottleneck analysis: Bottleneck occurs when populations observed as three in this study was in agreement with the experience severe temporary reduction in size. It influences findings in Krishna Valley (Karthickeyan et al., 2006; Kumar the distribution of genetic variation within and among et al., 2009). Maximum number of alleles at a few loci populations. Cornuet and Luikart (1996) introduced 38 INDIAN JOURNAL OF ANIMAL RESEARCH heterozygosity excess as a method for detection of alleles were organized into 10 frequency classes, which bottlenecks. This method is based on the premise that permit checking whether the scattering followed the normal populations experiencing recent reduction in size develop an L-shaped form, where alleles with low frequencies (0.01- excess of heterozygosity at selectively neutral loci relative 0.1) are the most numerous. This approach detected no mode to the heterozygosity expected at mutation-drift equilibrium. shift in the frequency distribution of alleles and a normal The sign test for I.A.M revealed that 9 loci suffered L-shaped curve was observed which ruled out genetic heterozygote deficiency while the remaining 9 loci had bottleneck in Pulikulam cattle. heterozygotes in excess. The sign test for T.P.M revealed that CONCLUSION 11 loci suffered heterozygote deficiency and 7 loci were with Despite unplanned breeding, this breed still has heterozygotes in excess. Under S.M.M, 15 loci recorded sufficient genetic variability. The significant level of heterozygote deficiency and 3 loci were with heterozygotes variability in Pulikulam cattle, notwithstanding its small in excess. The number of loci with observed heterozygosity population size, is indicative of a valuable reservoir of genetic excess was lower than the expected heterozygosity excess in diversity in this breed that may be used in future. This fact, all the three models of microsatellite evolution. coupled with its evident environmental adaptation, Standardized differences test revealed highly emphasizes the importance of genetic regulation and significant T2 values (P<0.01) under TPM and SMM indicating conservation of this indigenously evolved draught breed and the possible deviation of population from mutation drift its sustainable utilization. It is now critical to initiate planned equilibrium. However, the negative T2 values revealed and organized breeding, as our measured inbreeding significant deficit in observed gene diversity of Pulikulam cattle coefficient indicates moderate level of inbreeding in the (compared to that of equilibrium gene diversity estimated under population. the assumption of constant population size) thus ruling out the High-priority action is also necessary considering possibility of occurrence of recent genetic bottleneck. the husbandry practices adopted by the farmers, which may Similarly, the Wilcoxon sign rank test also ruled out weaken the diversity levels through breeding of relatives. 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