c Indian Academy of Sciences ONLINE RESOURCES Assessment of demographic bottleneck in Indian horse and endangered pony breeds A. K. GUPTA 1, MAMTA CHAUHAN 1, ANURADHA BHARDWAJ 1 and R. K. VIJH 2 1 National Research Centre on Equines, Sirsa Road, Hisar 125 001, India 2 National Bureau of Animal Genetic Resource, Karnal 132 001, India [Gupta A. K., Chauhan M., Bhardwaj A. and Vijh R. K. 2015 Assessment of demographic bottleneck in Indian horse and endangered pony breeds. J. Genet. 94, e56 e62. Online only: http://www.ias.ac.in/jgenet/onlineresources/94/e56.pdf] Introduction Bottleneck study of any continuously decreasing population is important and crucial issue in its conservation strategies including the analysis of simulated and real populations (Williamson-Natesan 2005; Busch et al. 2007). A bottleneck in a population can increase the rate of inbreeding, loss of genetic variation, fixation of deleterious alleles, thereby reducing evolutionary potential of animals to adapt to new selective pressures, such as climatic change or shift in available resources and increasing the probability of population extinction (Frankham 1995). The genetic changes caused by a bottleneck in a population s effective size can lower the possibility of population s persistence (Vrijenhoek 1994; Newman and Pilson 1997). Various endangered or threatened populations have been reported to have low levels of genetic variations (Vrijenhoek 1994; Gibbs et al. 1998). However, all the populations that have been reduced in size did not show quantifiable lower levels of genetic diversity (Waldman et al. 1998) which also necessitates the assessment of bottlenecks with molecular marker for their conservation and evolutionary genetics. India is bestowed with a rich biodiversity of equids in the form of two horses (Marwari and Kathiawari) and four endangered pony breeds (Bhutia, Spiti, Manipuri and Zanskari) besides indigenous donkeys and wild asses (Gupta et al. 2012a,b; 2014). Overall population of these breeds, specially endangered pony breeds has declined in most of the pockets in their home tracts (less than 1000) which is due to their decreased utility and increased modernization of transport system even in hilly and difficult terrains (Gupta et al. 2012a, b). It is expected that bottleneck might have taken For correspondence. E-mail: akguptanrce@hotmail.com. place in some of the endangered pony breeds. Therefore it is important to identify bottlenecked populations for conservation of breed(s) as conservation of any breed is very important because the loss of animal species or subspecies may represent a social or economic loss to human population, especially in developing countries. Further, India being a signatory to the State of the World Animal Genetic Resources (SoWAnGR) needs to characterize, document and conserve these indigenous breeds. DNA-based molecular genetics methods, which provide a powerful tool for inferring the demographic history of a population namely multilocus genotypes from microsatellite were used along with three single-sample methods, namely heterozygosity excess, mode shift and M-ratio tests for assessing the presence of bottlenecks in the Indian breeds. Breeds and blood samples Material and methods Genetically unrelated, adult, healthy animals (n = 284) of all the six indigenous registered horse (Marwari and Kathiawari) and pony breeds (Spiti, Zanskari, Manipuri and Bhutia) selected in different geographic locations in India were chosen for evaluating bottleneck in these populations. Fifty horses/ponies of each breed except Bhutia (34) and Spiti (16) breeds were selected based on their phenotypic characteristics. All these animals were selected from different and somewhat isolated pockets in their home tracts. Thirty-four Indian Thoroughbred horses were also included in the study as an out-group. Blood samples (5 8 ml) were collected from jugular vein using EDTA (0.5 mm, ph 8.0) coated tubes. Genomic DNA was isolated from blood using standard procedure of digestion with proteinase K, separation Keywords. bottleneck; Indian equine breeds; mutation-drift equilibrium; mode shift indicator. e56
A. K. Gupta et al. with phenol : chloroform : isoamyl alcohol and precipitation with ethanol (Sambrook et al. 1989). Molecular techniques A panel of 48 microsatellite markers that were used previously for assessing genetic diversity among different indigenous horse breeds were followed (Gupta et al. 2014). The electropherograms drawn through Gene Scan were used to detect DNA fragment sizing details using Gene Mapper software, ver. 3.0 (Applied Biosystems, Foster City, USA). Numbers of alleles at each locus were recorded for all the microsatellites which amplified correctly in different multiplexes. Statistical analysis Software PopGene (Kimura and Crow 1964) was used to calculate allele number, allele frequency, expected and observed heterozygosity (data on these parameters can be provided by author upon request). The bottleneck in the populations was studied by estimating the heterozygosity excess using software BOTTLENECK accessible at http://www.ensam. inra.fr/urlb. Three tests: sign, standardized differences and Wilcoxon sign-rank tests under three models of microsatellite evolution: IAM, SMM and TPM were used to compute the distribution of gene diversity expected from the observed number of alleles, given sample size under the assumption of mutation drift equilibrium (Cornuet and Luikart 1996). IAM and SMM represent the extremes of how new alleles were introduced in the population. TPM has been proposed as an intermediate model that provides a more realistic picture of how some DNA sequences evolve (Di Rienzo et al. 1994). A qualitative descriptor of allele frequency distribution, mode shift indicator which discriminates bottlenecked populations from stable populations was also used (Luikart et al. 1998). M-ratio measurements were also carried out as the third method to further confirm the problem of bottlenecks by applying the m_p_val.exe program (Garza and Williamson 2001). Results Data on overall range and mean values for observed number of alleles (N a ), expected alleles (N e ), heterozygosity, both observed and expected (H o and H e ) along with polymorphic information content (PIC) in each breed indicated high genetic diversity among these breeds (table 1) and all the microsatellite used were polymorphic in nature. Further, some of microsatellites, namely ASB002, UM011, TKY333, HMS004, TKY321, AHT004, TKY337, LEX033, TKY312, COR007, HTG010, AHT016, TKY287 and LEX073 had allele number more than 10 along with high heterozygosity in most of the breeds. To characterize bottleneck in different equine populations along with Thoroughbred horse, the power of three statistical tests: sign, standard and Wilcoxon tests for mutation drift equilibrium studies were used along with mode-shift indicator test and M-ratio measurements. Mutation drift equilibrium In IAM model of microsatellite evolution, observed numbers of loci with heterozygotic excess were significantly higher than expected number of loci in all the breeds (table 2). The probability values revealed that all the seven populations are not in mutation drift equilibrium (P<0.05). The TPM model revealed that excess heterozygotes to be significant only in sign test of Manipuri, Zanskari and Bhutia breeds depicting deviation from mutation drift equilibrium. However, probability values under sign test revealed that all populations except Zanskari breed were in mutation drift equilibrium as these values were not significant (P > 0.05) and hence null hypothesis was accepted in favour of Table 1. Various measure of genetic variability among different individual horse and pony breeds. Breed Parameter N a N e H o H e PIC Kathiawari Range 3.0 14.0 1.76 6.99 0.12 0.98 0.44 0.87 0.37 0.833 Mean 7.90±0.41 3.88±0.20 0.67±0.03 0.70±0.02 0.662±0.0179 Marwari Range 5.0 20.0 1.54 10.73 0.26 0.94 0.35 0.92 0.34 0.87 Mean 10.06±0.36 5.04±0.23 0.67±0.02 0.76±0.02 0.727±0.0166 Manipuri Range 3.0 14.0 2.04 9.58 0.29 1.00 0.52 0.91 0.41 0.85 Mean 8.42±0.42 4.63±0.22 0.72±0.02 0.76±0.01 0.708±0.0131 Spiti Range 3.0 10.0 1.68 7.42 0.21 1.00 0.42 0.89 0.37 0.85 Mean 5.52±0.42 3.46±0.31 0.67±0.06 0.67±0.03 0.626±0.0189 Thoroughbred Range 3.0 12.0 1.14 7.32 0.21 1.00 0.30 0.88 0.27 0.84 Mean 6.27±0.35 3.62±0.25 0.66±0.04 0.68±0.02 0.632±0.0212 Zanskari Range 3.0 15.0 2.14 9.22 0.32 0.98 0.54 0.90 0.43 0.85 Mean 8.52±0.35 4.68±0.20 0.68±0.02 0.76±0.01 0.772±0.0136 Bhutia Range 3.0 16.0 2.11 10.47 0.29 1.00 0.54 0.92 0.51 0.85 Mean 8.70±0.46 4.89±0.31 0.71±0.04 0.77±0.02 0.704±0.0134 N a, observed number of alleles; N e, expected number of alleles; H o, observed heterozygosity; H e, expected heterozygosity; PIC, polymorphic information content. e57
Bottlenecks assessment in Indian horse and pony breeds Table 2. Test for null hypothesis in six Indian horse and pony breeds along with English Thoroughbred horses. IAM TPM SMM Breed/model Test/model OHE EHE OHE EHE OHE EHE Manipuri Sign test: number of loci with heterozygosity excess (probability) 44 28.47 (0.00000) 34 28.52 (0.06977) NS 22 28.38 (0.4320) NS Standard difference test: Ti values (probability) 5.786 (0.00000)*** 1.994 (0.02305)* 5.377 (0.00000)*** Wilcoxon rank test: (probability of heterozygosity excess) 0.00000*** 0.00450* 0.06376NS Zanskari Sign test: number of loci with heterozygosity excess 46 28.46 (0.00000)* 40 28.50 (0.00033)* 16 28.21 (0.00033)* Standard difference test: Ti values (probability) 6.178 (0.00000)* 2.745 (0.00302)* 4.167 (0.00002)* Wilcoxon rank test: (probability of heterozygosity excess) 0.00000*** 0.00006*** 0.00073*** Spiti Sign test: number of loci with heterozygosity excess (probability) 39 28.02 (0.00067)*** 32 28.46 (0.18644) NS 22 28.70 (0.03506)* Standard difference test: Ti values (probability) 4.032 (0.00003)*** 1.401 (0.08063) NS 2.242 (0.01248)* Wilcoxon rank test: (probability of heterozygosity excess) 0.00000* 0.02034* 0.14785 NS Bhutia Sign test: number of loci with heterozygosity excess 40 28.14 (0.00017)* 33 28.07 (0.09212) 18 27.75 (0.00327)** Standard difference test: Ti values (probability) 4.772 (0.00000)*** 1.195 (0.11603) NS 5.637 (0.00000)*** Wilcoxon rank test: (probability of heterozygosity excess) 0.00000*** 0.03876* 0.00319** Marwari Sign test: number of loci with heterozygosity excess (probability) 37 28.89 (0.01054)* 25 28.38 (0.19822) NS 12 28.16 (0.01200) NS Standard difference test: Ti values (probability) 3.785 (0.00008)*** 1.22 (0.13086) NS 10.687 (0.00000)*** Wilcoxon rank test: (probability of heterozygosity excess) 0.000000*** 0.87485 NS 0.00000*** Kathiawari Sign test: number of loci with heterozygosity excess 40 28.27 (0.00026)*** 32 28.45 (0.18570) NS 13 28.25 (0.06001) NS Standard difference test: Ti values (probability) 3.665 (0.00012)*** 1.216 (0.11203) NS 10.453 (0.00000)*** Wilcoxon rank test: (probability of heterozygosity excess) 0.00009*** 0.35122 NS 0.00001*** Thoroughbred Sign test: number of loci with heterozygosity excess 41 28.22 (0.00007)*** 32 28.33 (0.17646) NS 24 28.58 (0.11574) NS Standard difference test: Ti values (probability) 4.589 (0.00000)*** 1.446 (0.07402) NS 4.532 (0.00000)*** Wilcoxon rank test: (probability of heterozygosity excess) 0.00000*** 0.02034* 0.07849 NS OHE, observed heterozygosity excess; EHE, expected heterozygosity excess; NS nonsignificant; * P<0.05%; ** P<0.01%. e58
A. K. Gupta et al. mutation drift equilibrium. SM model revealed heterozygotic deficiency in all the populations as values of observed heterozygosity excess values were quite less than values of expected heterozygosity excess (table 2). Probability values under sign test were significantly higher than 0.05 in Manipuri, Marwari, Kathiawari and Thoroughbred populations, indicating the acceptance of null hypothesis. In standardized difference test, Ti values under IAM model were significantly higher than 1.645 at 5% level indicating the rejection of null hypothesis of mutation drift equilibrium. However, Ti values under TPM model indicated the acceptance of null hypothesis in all the breeds except Manipuri and Zanskari, while negative probability values indicated heterozygote deficient in Marwari, Kathiawari, Spiti, Bhutia and Thoroughbred only. Under SMM model, Ti was highly negative for all the breeds indicating heterozygosity deficiency. The Wilcoxon rank tests revealed significantly low values of probabilities (P<0.05) indicating the rejection of null hypothesis under IAM in all the breeds. Under TPM model, probability values were significantly low (<0.05) in Manipuri, Zanskari, Spiti, Thoroughbred and Bhutia populations, while in Marwari and Kathiawari, the values were nonsignificant. Under SMM, mutation drift equilibrium was accepted as all the values were nonsignificant. Allele frequency distribution: mode-shift indicator test Recent bottleneck in the populations (i.e. within past few dozen generations) was examined by the graphical method analysing distortion of allele frequency distribution. All the seven breeds showed normal L shaped curve (figure 1) reflecting no bottleneck occurred in the recent past. M-ratio test The M-ratio values at individual locus in different populations ranged significantly in all the breeds, namely Marwari (0.360 to 1.00), Kathiawari (0.381 to 1.00), Manipuri (0.333 to 1.00), Spiti (0.333 to 1.00), Thoroughbred (0.280 to 1.00), Zanskari (0.321 to 1.00) and Bhutia (0.323 to 1.00). Although range seem to be quite wide but the lowest values as well as values less than 0.500 were only in one or two loci in each population. Average values of M-ratio were 0.751, 0.735, 0.775, 0.652, 0.718, 0.794, 0.691 in Marwari, Kathiawari, Manipuri, Spiti, Thoroughbred, Zanskari and Bhutia populations, respectively (table 3). These values were more than 0.7 or very close to it and were not significant at 0.05 level, indicating thereby that all the populations have not undergone severe reduction in population size or critical levels. Discussion Among Indian horse and pony breeds, all the four pony breeds, namely Manipuri, Zanskari, Spiti and Bhutia are endangered and are declining (Gupta et al. 2012a, b). In demographic bottlenecked population, it is expected that the population has decreased along with low genetic diversity. However, high genetic diversity as observed in all the breeds is in agreement with previous findings (Chauhan et al. 2011; Gupta et al. 2005, 2013) and all the microsatellites can be used effectively for genetic diversity studies. In the present study, 14 microsatellites which showed very high number of alleles along with maximum heterozygosity can be used effectively for similar study with any of the horse breed. Mutation drift equilibrium Populations showing a significant heterozygosity excess would be considered as having experienced a recent bottleneck. However, heterozygosity excess should not be confused with excess of heterozygotes (Cornuet and Luikart 1996). In sign test under IAM, null hypothesis (mutation drift equilibrium) was rejected in favour of overall heterozygosity Figure 1. Graphic distribution of proportion of alleles and their distribution in different breeds. e59
Bottlenecks assessment in Indian horse and pony breeds Table 3. Observed number of alleles (Na) and average M-ratio values at each locus in Indian breeds and English Thoroughbred horses. Kathiawari Marwari Manipuri Spiti Thoroughbred Zanskari Bhutia Locus Na M-ratio Na M-ratio Na M-ratio Na M-ratio Na M-ratio Na M-ratio Na M-ratio ASB002 6.0 0.54545 12.0 0.66667 9.0 0.90000 7.0 0.70000 9.0 0.50000 13.0 0.72222 13.0 0.76471 EB2B8 7.0 0.53846 9.0 0.69231 9.0 0.60000 8.0 0.61538 9.0 0.64286 10.0 0.71429 9.0 0.75000 HTG006 8.0 0.61538 10.0 0.66667 4.0 0.33333 8.0 0.66667 6.0 0.37500 8.0 0.57143 6.0 0.60000 UM011 14.0 0.87500 14.0 0.87500 10.0 0.90909 4.0 0.80000 8.0 0.80000 9.0 0.90000 12.0 0.75000 TKY301 8.0 0.88889 11.0 1.00000 8.0 0.72727 5.0 0.83333 6.0 1.00000 8.0 0.80000 8.0 0.80000 TKY333 14.0 0.38889 20.0 0.55556 14.0 0.40000 9.0 0.30000 12.0 0.33333 15.0 0.42857 16.0 0.45714 TKY374 10.0 0.47619 16.0 0.53333 10.0 0.66667 6.0 0.40000 8.0 0.66667 6.0 0.85714 13.0 0.86667 HMS001 6.0 0.50000 10.0 0.71429 8.0 0.66667 6.0 0.50000 5.0 0.45455 8.0 0.53333 6.0 0.54545 HMS004 9.0 0.81818 11.0 1.00000 10.0 0.90909 6.0 0.60000 7.0 0.77778 9.0 0.90000 11.0 1.00000 HTG003 8.0 0.72727 9.0 0.81818 8.0 0.72727 4.0 0.44444 5.0 0.45455 9.0 0.81818 8.0 0.72727 HTG007 13.0 0.92857 14.0 0.93333 14.0 0.93333 10.0 0.66667 9.0 0.60000 12.0 0.85714 8.0 0.72727 TKY321 12.0 0.70588 13.0 0.76471 10.0 0.58824 7.0 0.63636 10.0 0.83333 9.0 0.81818 10.0 0.58824 TKY394 10.0 0.71429 14.0 0.87500 11.0 0.73333 8.0 0.66667 7.0 1.00000 9.0 0.81818 9.0 0.64286 UM032 7.0 1.00000 8.0 0.88889 6.0 1.00000 5.0 0.83333 5.0 0.71429 6.0 1.00000 5.0 0.71429 AHT004 11.0 1.00000 12.0 1.00000 11.0 1.00000 7.0 0.70000 8.0 0.88889 9.0 0.90000 10.0 1.00000 HMS003 11.0 1.00000 10.0 0.71429 10.0 0.90909 7.0 0.41176 9.0 0.90000 8.0 0.47059 8.0 0.80000 TKY294 8.0 1.00000 7.0 1.00000 6.0 0.85714 5.0 1.00000 6.0 0.85714 13.0 1.00000 7.0 1.00000 TKY337 7.0 0.87500 11.0 0.91667 7.0 0.87500 5.0 0.71429 5.0 1.00000 7.0 0.87500 10.0 0.71429 ASB023 7.0 0.50000 11.0 0.73333 7.0 0.58333 5.0 0.45455 5.0 0.50000 9.0 0.69231 9.0 0.75000 HMS006 5.0 0.83333 5.0 0.83333 6.0 1.00000 3.0 0.50000 4.0 0.66667 6.0 1.00000 6.0 1.00000 LEX033 9.0 0.36000 8.0 0.38095 10.0 0.43478 4.0 0.36364 7.0 0.28000 8.0 0.72727 12.0 0.41379 TKY312 6.0 0.54545 11.0 0.68750 10.0 0.83333 5.0 0.55556 5.0 0.55556 10.0 0.90909 11.0 0.68750 TKY297 7.0 0.77778 8.0 0.57143 8.0 0.72727 4.0 0.36364 5.0 1.00000 8.0 0.72727 8.0 0.66667 AHT005 14.0 0.87500 10.0 0.62500 12.0 0.80000 6.0 0.50000 9.0 0.56250 9.0 0.56250 9.0 0.60000 ASB017 4.0 0.33333 9.0 0.52941 3.0 0.75000 3.0 0.75000 5.0 0.29412 3.0 0.75000 3.0 0.75000 HTG004 4.0 1.00000 5.0 0.50000 7.0 1.00000 4.0 1.00000 4.0 1.00000 6.0 1.00000 0.0 1.00000 VHL020 6.0 0.91667 5.0 0.91667 5.0 0.75000 4.0 0.66667 4.0 0.54545 7.0 1.00000 4.0 0.81818 ASB043 11.0 0.62500 11.0 0.50000 9.0 0.62500 6.0 0.50000 6.0 0.80000 11.0 0.72727 9.0 0.60000 COR069 5.0 0.66667 8.0 0.77778 5.0 0.63636 4.0 0.77778 4.0 0.75000 8.0 0.66667 9.0 0.53846 HMS002 6.0 0.72727 7.0 0.83333 7.0 0.72727 7.0 0.63636 3.0 0.83333 6.0 0.81818 7.0 0.63636 I18 8.0 0.41667 10.0 0.69231 8.0 0.85714 7.0 0.60000 5.0 0.71429 9.0 0.69231 7.0 0.53846 LEX078 5.0 1.00000 9.0 0.60000 6.0 0.80000 3.0 1.00000 5.0 1.00000 9.0 1.00000 7.0 0.77778 AHT031 3.0 1.00000 6.0 0.85714 4.0 0.77778 3.0 0.75000 3.0 0.50000 3.0 1.00000 7.0 0.66667 COR022 9.0 1.00000 6.0 0.54545 7.0 0.85714 3.0 0.75000 5.0 1.00000 6.0 1.00000 4.0 0.42857 COR007 4.0 0.58333 6.0 0.55556 6.0 0.50000 3.0 0.33333 3.0 0.50000 5.0 0.75000 6.0 0.45833 HTG010 7.0 0.92308 10.0 0.85714 8.0 0.90000 4.0 0.58333 5.0 0.61538 9.0 0.83333 11.0 0.68421 SGCV28 12.0 0.55556 18.0 0.66667 9.0 0.54545 7.0 1.00000 8.0 0.83333 10.0 0.75000 13.0 0.75000 AHT016 5.0 0.75000 8.0 0.63158 6.0 0.75000 5.0 0.58333 5.0 0.50000 6.0 0.69231 6.0 0.61111 LEX054 9.0 0.66667 12.0 0.66667 9.0 1.00000 7.0 0.55556 6.0 1.00000 9.0 0.87500 11.0 0.57143 UCDEQ425 6.0 0.90000 8.0 0.90909 7.0 1.00000 5.0 1.00000 6.0 0.77778 7.0 0.90000 8.0 0.90000 LEX003 9.0 0.75000 10.0 0.76923 10.0 0.83333 6.0 0.66667 7.0 0.90909 9.0 0.83333 9.0 0.58333 TKY287 9.0 0.64286 10.0 0.62500 10.0 0.92857 6.0 0.66667 10.0 0.60000 10.0 0.78571 7.0 0.80000 TKY341 9.0 0.80000 10.0 1.00000 13.0 0.87500 6.0 0.62500 6.0 0.77778 11.0 0.88889 12.0 0.52941 LEX073 4.0 0.85714 7.0 0.62500 7.0 0.66667 5.0 0.41176 7.0 1.00000 8.0 0.82353 9.0 0.61111 TKY325 6.0 1.00000 10.0 0.81250 12.0 1.00000 7.0 0.87500 7.0 0.88889 14.0 1.00000 11.0 0.43750 TKY344 11.0 0.71429 13.0 0.80000 8.0 1.00000 7.0 0.66667 8.0 0.71429 11.0 0.62500 7.0 0.87500 UM002 5.0 1.00000 8.0 0.40625 7.0 0.52000 4.0 1.00000 5.0 0.83333 5.0 0.32143 7.0 0.32353 Mean 5.0 0.75144 13.0 0.73454 13.0 0.77477 5.0 0.65158 5.0 0.71809 9.0 0.79438 11.0 0.69054 Range 7.895 0.333 1.0 10.0625 0.381 1.00 8.4167 0.333 1.00 5.5208 0.300 1.00 6.2708 0.280 1.00 8.5208 0.321 1.00 8.7021 0.323 1.00 SD 2.889 0.20083 3.1784 0.16579 2.5751 0.17593 1.7009 0.19223 2.0289 0.21289 2.5010 0.16256 2.6858 0.16811 e60
A. K. Gupta et al. excess and a genetic bottleneck in all the horse and pony populations. IAM model of mutation in microsatellites assumes that each mutational process generates new allele therefore, gene diversity excess (H e > H eq ) is demonstrated only for loci evolving under this model. Overall populations of all the pony breeds has decreased and as such demographic bottleneck seems to hold good under IAM, but it needs to be confirmed through other tests also. Under TPM model, all the breeds except Zanskari were observed to be in mutation drift equilibrium with the acceptance of null hypothesis. Situation was totally different under SMM model as heterozygosity deficiency was observed in all the population which indicated that none of the population have recently undergone bottleneck. SMM model is the most conserved model of microsatellite evolution, any heterozygosity excess is likely to confirm a recent reduction in effective population. If the locus evolves under the strict SMM, there can be situation where this gene diversity excess is not observed (Cornuet and Luikart 1996). This model under sign test indicated the existence of mutation drift equilibrium in all the breeds. Among all the models, TPM model is intermediate to IAM and SMM which assumes that most mutational changes result in increase or decrease of one repeat unit but mutations of larger magnitude also occur. Hence, most of microsatellite loci fit the TPM rather than IAM or SMM model (Ellegren 2004). Although populations of all the endangered pony breeds have declined but there is mutation drift equilibrium in all the seven populations which could be possibly due to presence of fragmented population in the home tracts also (Redeker et al. 2006). Earlier studies with limited set of microsatellites also revealed presence of mutation drift equilibrium in some of the individual Indian breeds (Gupta et al. 2005, 2012a, b; Chauhan et al. 2011). Another test, standardized difference test rejects the null hypothesis of population at mutation drift equilibrium if probability of heterozygosity excess at the 5% confidence level is <0.05. Under IAM model, this hypothesis was rejected in all the Indian horse and pony populations along with Thoroughbred horses. IAM model is of little importance as it is based on presumption of existence of infinite number of alleles. Under TPM model, null hypothesis was not accepted in Manipuri and Zanskari breeds only while Ti were highly negative for all the breeds indicating heterozygosity deficiency under SMM model. Under TPM and SMM models, heterozygote deficiency was observed in most of the populations indicating no bottleneck in all the populations. Taking all the results together, it is clear that serious bottleneck has not taken place in any of the pony or horse breed. Mode-shift indicator test The mode-shift indicator test, a qualitative test for detection of bottleneck was also used as a second method to detect potential bottlenecks, as the nonbottlenecked populations that are near mutation drift equilibrium are expected to have a large number of alleles at low frequencies resulting in L-shaped graph (Luikart 1997; Luikart et al. 1998). In all the seven breeds, L-shaped curves indicated the absence of any recent bottleneck in them which could be due to existence of high diversity among them as reported earlier (Gupta et al. 2014). In some mammalian species, which are characterized by highly fluctuating or cyclical population dynamics, high levels of genetic diversity has been maintained in them in face of frequent bottlenecks (Redeker et al. 2006; Busch et al. 2007). In Spiti, Zanskari and Marwari breeds, similar observations have been earlier reported with different set of microsatellites which supports the present findings (Gupta et al. 2005, 2013; Chauhan et al. 2011). M-ratio test In the present study, M-ratio analysis clearly revealed that all the horse and pony populations are genetically static and have not undergone severe genetic reduction in their population sizes (Garza and Williamson 2001). Actual decrease in overall populations of different breeds without any bottleneck may be due to maintenance of genetic diversity in fragmented subpopulations in home tract of respective breeds (Redeker et al. 2006; Gupta et al. 2014). This test revealed that in spite of decrease in populations of various Indian breeds, genetically bottleneck has not taken place which is quite encouraging for adopting conservation and preservation strategies. Conclusion This study indicated that although the populations of all the horse and pony breeds have decreased rapidly during the last couple of decades but no recent bottlenecks had taken place in any of the Indian horse or pony breed. Small fragmented subpopulations of each pony/horse breed in their home tract might have a very good genetic diversity among them. Since populations of all the pony breeds is continuously declining, it may sooner or later lead to bottlenecks in these populations and breeds may become extinct if suitable measure are not taken in time to conserve them. References Busch J. D., Waser P. M. and Dewoody A. 2007 Recent demographic bottlenecks are not accompanied by a genetic signature in banner tailed kangaroo rats (Dipodomys spectabilis). Mol. Ecol. 16, 2450 2462. Chauhan M., Gupta A. K. and Dhillon S. 2011 Genetic diversity and population structure of three Indian horse breeds. Mol. Biol. Rep. 38, 3505 3511. Cornuet J. 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