Assessment of Demographic Bottleneck in Indian Horse and Endangered Pony Breeds

c Indian Academy of Sciences

ONLINE RESOURCES

Assessment of demographic bottleneck in Indian horse and endangered pony breeds

A. K. GUPTA1∗, MAMTA CHAUHAN1, ANURADHA BHARDWAJ1 and R. K. VIJH2

1National Research Centre on Equines, Sirsa Road, Hisar 125 001, India 2National 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 place in some of the endangered pony breeds. Therefore it is important to identify bottlenecked populations for con- Bottleneck study of any continuously decreasing popula- servation of breed(s) as conservation of any breed is very tion is important and crucial issue in its conservation strate- important because the loss of animal species or subspecies gies including the analysis of simulated and real populations may represent a social or economic loss to human pop- (Williamson-Natesan 2005;Buschet al. 2007). A bottle- ulation, especially in developing countries. Further, India neck in a population can increase the rate of inbreeding, loss being a signatory to the State of the World Animal Genetic of genetic variation, fixation of deleterious alleles, thereby Resources (SoWAnGR) needs to characterize, document and reducing evolutionary potential of animals to adapt to new conserve these indigenous breeds. DNA-based molecular selective pressures, such as climatic change or shift in avail- genetics methods, which provide a powerful tool for infer- able resources and increasing the probability of population ring the demographic history of a population namely multi- extinction (Frankham 1995). The genetic changes caused locus genotypes from microsatellite were used along with by a bottleneck in a population’s effective size can lower three single-sample methods, namely heterozygosity excess, the possibility of population’s persistence (Vrijenhoek 1994; mode shift and M-ratio tests for assessing the presence of Newman and Pilson 1997). Various endangered or threatened bottlenecks in the Indian breeds. 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 Material and methods show quantifiable lower levels of genetic diversity (Waldman et al. 1998) which also necessitates the assessment of bot- Breeds and blood samples tlenecks with molecular marker for their conservation and Genetically unrelated, adult, healthy animals (n = 284) evolutionary genetics. of all the six indigenous registered horse (Marwari and India is bestowed with a rich biodiversity of equids in Kathiawari) and pony breeds (Spiti, Zanskari, Manipuri and the form of two horses (Marwari and Kathiawari) and Bhutia) selected in different geographic locations in India four endangered pony breeds (Bhutia, Spiti, Manipuri and were chosen for evaluating bottleneck in these populations. Zanskari) besides indigenous donkeys and wild asses (Gupta Fifty horses/ponies of each breed except Bhutia (34) and et al. 2012a,b; 2014). Overall population of these breeds, Spiti (16) breeds were selected based on their phenotypic specially endangered pony breeds has declined in most of the characteristics. All these animals were selected from dif- pockets in their home tracts (less than 1000) which is due to ferent and somewhat isolated pockets in their home tracts. their decreased utility and increased modernization of trans- Thirty-four Indian Thoroughbred horses were also included port system even in hilly and difficult terrains (Gupta et al. in the study as an out-group. Blood samples (5–8 mL) were 2012a, b). It is expected that bottleneck might have taken collected from jugular vein using EDTA (0.5 mM, pH 8.0) coated tubes. Genomic DNA was isolated from blood using ∗ For correspondence. E-mail: [email protected]. standard procedure of digestion with proteinase K, separation Keywords. bottleneck; Indian equine breeds; mutation-drift equilibrium; mode shift indicator.

Journal of Genetics Vol. 94, Online Resources e56 A. K. Gupta et al. with phenol : chloroform : isoamyl alcohol and precipitation the third method to further conﬁrm the problem of bot- with ethanol (Sambrook et al. 1989). tlenecks by applying the m_p_val.exe program (Garza and Williamson 2001).

Molecular techniques Results A panel of 48 microsatellite markers that were used previ- ously for assessing genetic diversity among different indige- Data on overall range and mean values for observed num- nous horse breeds were followed (Gupta et al. 2014). The ber of alleles (Na), expected alleles (Ne), heterozygosity, electropherograms drawn through Gene Scan were used to both observed and expected (Ho and He) along with poly- detect DNA fragment sizing details using Gene Mapper soft- morphic information content (PIC) in each breed indicated ware, ver. 3.0 (Applied Biosystems, Foster City, USA). Numbers high genetic diversity among these breeds (table 1)andall of alleles at each locus were recorded for all the microsatel- the microsatellite used were polymorphic in nature. Further, lites which amplified correctly in different multiplexes. some of microsatellites, namely ASB002, UM011, TKY333, HMS004, TKY321, AHT004, TKY337, LEX033, TKY312, COR007, HTG010, AHT016, TKY287 and LEX073 had Statistical analysis allele number more than 10 along with high heterozygosity Software PopGene (Kimura and Crow 1964) was used to cal- in most of the breeds. To characterize bottleneck in different culate allele number, allele frequency, expected and observed equine populations along with Thoroughbred horse, the heterozygosity (data on these parameters can be provided power of three statistical tests: sign, standard and Wilcoxon by author upon request). The bottleneck in the populations tests for mutation–drift equilibrium studies were used along was studied by estimating the heterozygosity excess using with mode-shift indicator test and M-ratio measurements. software BOTTLENECK accessible at http://www.ensam. inra.fr/URLB. Three tests: sign, standardized differences and Mutation–drift equilibrium Wilcoxon sign-rank tests under three models of microsatellite evolution: IAM, SMM and TPM were used to compute In IAM model of microsatellite evolution, observed numbers the distribution of gene diversity expected from the observed of loci with heterozygotic excess were significantly higher number of alleles, given sample size under the assumption than expected number of loci in all the breeds (table 2). of mutation–drift equilibrium (Cornuet and Luikart 1996). The probability values revealed that all the seven popula- IAM and SMM represent the extremes of how new alle- tions are not in mutation–drift equilibrium (P<0.05). The les were introduced in the population. TPM has been pro- TPM model revealed that excess heterozygotes to be sig- posed as an intermediate model that provides a more realistic nificant only in sign test of Manipuri, Zanskari and Bhutia picture of how some DNA sequences evolve (Di Rienzo et al. breeds depicting deviation from mutation–drift equilibrium. 1994). A qualitative descriptor of allele frequency distribu- However, probability values under sign test revealed that all tion, mode shift indicator which discriminates bottlenecked populations except Zanskari breed were in mutation–drift populations from stable populations was also used (Luikart equilibrium as these values were not significant (P > 0.05) et al. 1998). M-ratio measurements were also carried out as 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 Na Ne Ho He 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

Na, observed number of alleles; Ne, expected number of alleles; Ho, observed heterozygosity; He, expected heterozygosity; PIC, polymorphic information content.

Journal of Genetics Vol. 94, Online Resources e57 Bottlenecks assessment in Indian horse and pony breeds NS NS NS NS NS NS 5.637 (0.00000)*** 4.532 (0.00000)*** 5.377 (0.00000)*** 2.242 (0.01248)* 4.167 (0.00002)* 10.687 (0.00000)*** 10.453 (0.00000)*** 0.00000*** 0.00001*** − − − − − − − 22 28.38 (0.4320) 22 28.70 (0.03506)* 12 28.16 (0.01200) 13 28.25 (0.06001) 24 28.58 (0.11574) NS NS NS NS NS NS NS NS NS NS NS NS 1.22 (0.13086) 1.216 (0.11203) − − 34 28.52 (0.06977) 0.01%. < ∗∗∗ 0.05%; ** P < IAM TPM SMM nonsigniﬁcant; * P NS Standard difference test: Ti values (probability) 5.786 (0.00000)***Standard difference test: Ti values (probability)Standard difference test: Ti values 1.994 (probability) (0.02305)* Standard difference test: Ti values (probability) 3.785 (0.00008)*** 3.665 (0.00012)*** 4.589 (0.00000)*** 1.446 (0.07402) Wilcoxon rank test: (probability of heterozygosity excess)Standard difference test: Ti valuesWilcoxon (probability) rank test: (probability of heterozygosity excess) 0.00000*** 0.00000*** 6.178 (0.00000)* 0.00450* 0.00006*** 2.745 (0.00302)* 0.06376NS 0.00073*** Standard difference test: Ti values (probability) 4.772 (0.00000)*** 1.195 (0.11603) Standard difference test: Ti values (probability) 4.032 (0.00003)*** 1.401 (0.08063) Wilcoxon rank test: (probability of heterozygosity excess) 0.00000***Wilcoxon rank test: (probability of heterozygosity excess) 0.03876* 0.00000*** 0.00319** 0.02034* 0.07849 Wilcoxon rank test: (probability of heterozygosity excess) 0.00000* 0.02034* 0.14785 Wilcoxon rank test: (probability of heterozygosity excess)Wilcoxon rank test: (probability of heterozygosity excess) 0.000000*** 0.00009*** 0.87485 0.35122 Test for null hypothesis in six Indian horse and pony breeds along with English Thoroughbred horses. Table 2. Breed/model Test/modelManipuri Sign test: number of loci with heterozygosity excess (probability) 44 28.47 (0.00000) Bhutia Sign test: number of loci with heterozygosity excess OHE 40 28.14 (0.00017)* EHE 33 28.07 (0.09212) OHE 18 EHE 27.75 (0.00327)** OHE EHE ZanskariSpiti Sign test: number of loci with heterozygosity excess Sign test: number of loci with heterozygosity excess (probability) 46 39 28.46 28.02 (0.00000)* (0.00067)*** 32 40 28.46 (0.18644) 28.50 (0.00033)* 16 28.21 (0.00033)* Kathiawari Sign test: number of lociThoroughbred with heterozygosity excess Sign test: number of loci with heterozygosity excess 40 28.27 41 (0.00026)*** 32 28.22 (0.00007)*** 28.45 32 (0.18570) 28.33 (0.17646) Marwari Sign test: number of loci with heterozygosity excess (probability) 37 28.89 (0.01054)* 25 28.38 (0.19822) OHE, observed heterozygosity excess; EHE, expected heterozygosity excess;

Journal of Genetics Vol. 94, Online Resources e58 A. K. Gupta et al. mutation–drift equilibrium. SM model revealed heterozy- (0.360 to 1.00), Kathiawari (0.381 to 1.00), Manipuri (0.333 gotic deficiency in all the populations as values of observed to 1.00), Spiti (0.333 to 1.00), Thoroughbred (0.280 to heterozygosity excess values were quite less than values of 1.00), Zanskari (0.321 to 1.00) and Bhutia (0.323 to 1.00). expected heterozygosity excess (table 2). Probability values Although range seem to be quite wide but the lowest values under sign test were significantly higher than 0.05 in as well as values less than 0.500 were only in one or two loci Manipuri, Marwari, Kathiawari and Thoroughbred popula- in each population. Average values of M-ratio were 0.751, tions, indicating the acceptance of null hypothesis. In stan- 0.735, 0.775, 0.652, 0.718, 0.794, 0.691 in Marwari, Kathi- dardized difference test, Ti values under IAM model were awari, Manipuri, Spiti, Thoroughbred, Zanskari and Bhutia significantly higher than 1.645 at 5% level indicating the populations, respectively (table 3). These values were more rejection of null hypothesis of mutation–drift equilibrium. than 0.7 or very close to it and were not significant at 0.05 However, Ti values under TPM model indicated the accep- level, indicating thereby that all the populations have not tance of null hypothesis in all the breeds except Manipuri undergone severe reduction in population size or critical and Zanskari, while negative probability values indicated levels. heterozygote deficient in Marwari, Kathiawari, Spiti, Bhutia and Thoroughbred only. Under SMM model, Ti was highly Discussion negative for all the breeds indicating heterozygosity deficiency. The Wilcoxon rank tests revealed significantly low Among Indian horse and pony breeds, all the four pony values of probabilities (P<0.05) indicating the rejection of breeds, namely Manipuri, Zanskari, Spiti and Bhutia are null hypothesis under IAM in all the breeds. Under TPM endangered and are declining (Gupta et al. 2012a, b). In model, probability values were significantly low (<0.05) in demographic bottlenecked population, it is expected that the Manipuri, Zanskari, Spiti, Thoroughbred and Bhutia popu- population has decreased along with low genetic diversity. lations, while in Marwari and Kathiawari, the values were However, high genetic diversity as observed in all the breeds nonsignificant. Under SMM, mutation–drift equilibrium was is in agreement with previous findings (Chauhan et al. 2011; accepted as all the values were nonsignificant. 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 Allele frequency distribution: mode-shift indicator test of alleles along with maximum heterozygosity can be used Recent bottleneck in the populations (i.e. within past few effectively for similar study with any of the horse breed. dozen generations) was examined by the graphical method analysing distortion of allele frequency distribution. All the Mutation–drift equilibrium seven breeds showed normal ‘L’ shaped curve (figure 1) reflecting no bottleneck occurred in the recent past. Populations showing a significant heterozygosity excess would be considered as having experienced a recent bottleneck. However, heterozygosity excess should not be confused M-ratio test with excess of heterozygotes (Cornuet and Luikart 1996). In The M-ratio values at individual locus in different popula- sign test under IAM, null hypothesis (mutation–drift equi- tions ranged significantly in all the breeds, namely Marwari librium) was rejected in favour of overall heterozygosity

Figure 1. Graphic distribution of proportion of alleles and their distribution in different breeds.

Journal of Genetics Vol. 94, Online Resources e59 Bottlenecks assessment in Indian horse and pony breeds 1111 1111 M-ratio a N M-ratio a N M-ratio a N M-ratio a N M-ratio a N M-ratio and average M-ratio values at each locus in Indian breeds and English Thoroughbred horses. ) a a N N ( M-ratio Kathiawari Marwari Manipuri Spiti Thoroughbred Zanskari Bhutia a N Observed number of alleles Table 3. Locus ASB002EB2B8HTG006 6.0UM011TKY301 7.0 8.0 0.54545TKY333 14.0TKY374 0.53846 8.0 0.61538 14.0 12.0HMS001 0.87500 10.0HMS004 0.88889 10.0 9.0HTG003 0.38889 6.0 0.66667 14.0HTG007 0.47619 9.0 11.0TKY321 20.0 8.0 0.50000 0.66667 0.69231 13.0TKY394 9.0 16.0 0.81818 0.87500 12.0UM032 1.00000 0.72727 10.0 10.0AHT004 4.0 0.55556 0.92857 9.0 10.0 11.0 0.90000HMS003 0.53333 0.70588 7.0 11.0TKY294 14.0 8.0 0.71429 14.0 9.0 0.71429 0.33333 0.60000 11.0TKY337 10.0 0.90909 13.0 1.00000 7.0ASB023 1.00000 1.00000 14.0 8.0 0.72727 0.40000 8.0HMS006 1.00000 0.81818 0.93333 7.0 10.0 8.0 8.0 0.66667 4.0LEX033 0.76471 0.70000 12.0 7.0 1.00000 8.0TKY312 14.0 0.87500 10.0 5.0 5.0 9.0 0.66667 8.0 0.87500TKY297 10.0 0.90909 0.66667 0.61538 6.0 9.0 0.80000 0.50000AHT005 9.0 11.0 1.00000 7.0 6.0 0.83333 0.88889 0.93333 11.0 0.71429ASB017 0.72727 0.83333 0.30000 6.0 7.0 0.58824 0.36000 6.0 14.0 6.0 11.0HTG004 9.0 11.0 0.40000 8.0 0.54545 0.73333 10.0 10.0VHL020 6.0 0.50000 1.00000 5.0 12.0 4.0 0.91667 0.77778 6.0 4.0ASB043 0.50000 0.87500 4.0 7.0 8.0 0.60000 1.00000 0.73333 11.0 8.0 0.37500COR069 0.64286 0.80000 6.0 8.0 0.90909 13.0 6.0 1.00000 0.66667 0.33333 0.83333HMS002 11.0 7.0 10.0 8.0 0.33333 1.00000 0.44444 5.0 1.00000I18 0.63636 7.0 0.38095 7.0 5.0 7.0 10.0 0.68750 0.91667 0.66667LEX078 8.0 0.85714 0.66667 0.62500 6.0 7.0 5.0 9.0 9.0 6.0 0.72222 9.0 0.87500 15.0AHT031 0.57143 0.62500 10.0 5.0 10.0 5.0 10.0 0.66667 0.58333 0.45455COR022 8.0 0.70000 0.77778 5.0 0.71429 11.0 0.72727 5.0 13.0 0.57143 7.0 5.0COR007 6.0 12.0 1.00000 0.41176 0.83333 0.52941 0.90000 3.0 0.60000 5.0 8.0 8.0 0.43478 0.42857HTG010 0.50000 0.83333 8.0 9.0 0.45455 0.83333 5.0 1.00000 0.80000 8.0 8.0SGCV28 0.91667 0.50000 7.0 9.0 9.0 4.0 12.0 0.76471 1.00000 0.80000 6.0 1.00000 3.0 9.0 0.85714 3.0 0.41667 5.0 1.00000 12.0AHT016 16.0 0.72727 0.71429 7.0 7.0 4.0 1.00000 5.0 12.0 0.77778 0.45455 9.0 9.0 9.0 5.0 0.58333 8.0 9.0 0.53333 0.88889 0.83333 10.0 0.75000 13.0 0.90000 0.75000 6.0 0.75000 5.0 6.0 6.0 0.60000 0.50000 0.92308 0.90000 0.85714 0.71429 5.0 4.0 0.45714 9.0 0.55556 1.00000 0.36364 6.0 5.0 0.55556 5.0 0.75000 0.60000 0.62500 0.81818 0.81818 6.0 7.0 0.80000 11.0 6.0 0.75000 0.69231 10.0 0.86667 9.0 3.0 0.85714 0.50000 18.0 4.0 0.85714 8.0 0.36364 0.81818 8.0 6.0 1.00000 4.0 7.0 0.54545 0.63636 5.0 10.0 6.0 4.0 0.72727 0.50000 6.0 0.55556 8.0 8.0 0.54545 1.00000 8.0 4.0 0.85714 0.75000 0.90000 9.0 13.0 0.66667 0.66667 5.0 0.72727 7.0 9.0 1.00000 0.47059 1.00000 4.0 7.0 0.28000 0.80000 0.55556 0.58824 6.0 0.66667 0.50000 7.0 0.85714 0.72727 8.0 9.0 0.63158 10.0 0.77778 9.0 5.0 1.00000 0.56250 0.85714 6.0 4.0 1.00000 0.64286 8.0 5.0 0.77778 0.87500 10.0 3.0 8.0 0.50000 4.0 6.0 0.63636 6.0 0.90000 7.0 0.69231 3.0 1.00000 0.54545 0.29412 7.0 3.0 9.0 10.0 1.00000 0.80000 0.71429 4.0 1.00000 8.0 1.00000 0.90909 0.72727 3.0 0.75000 3.0 0.60000 0.54545 4.0 0.80000 0.75000 9.0 7.0 1.00000 3.0 0.75000 0.56250 0.71429 6.0 11.0 12.0 0.72727 0.75000 6.0 5.0 0.33333 5.0 11.0 5.0 0.58333 0.83333 3.0 7.0 1.00000 0.75000 5.0 0.75000 9.0 1.00000 0.68750 0.41379 8.0 1.00000 3.0 8.0 1.00000 0.58333 5.0 0.72727 0.71429 8.0 1.00000 6.0 0.50000 3.0 1.00000 0.60000 0.66667 0.0 0.66667 5.0 0.50000 9.0 9.0 0.61538 4.0 9.0 0.81818 0.83333 3.0 0.75000 6.0 1.00000 9.0 0.50000 1.00000 5.0 0.60000 10.0 0.81818 9.0 7.0 0.69231 1.00000 1.00000 0.53846 7.0 0.75000 6.0 0.75000 0.83333 0.63636 7.0 7.0 4.0 13.0 0.77778 11.0 6.0 0.69231 0.53846 0.66667 0.42857 0.75000 0.45833 0.68421 6.0 0.6 LEX054UCDEQ425LEX003 6.0 9.0TKY287TKY341 0.90000 9.0 0.66667LEX073 9.0TKY325 9.0 0.75000 12.0 8.0TKY344 4.0 0.64286UM002 6.0 0.80000 11.0 10.0Mean 0.66667 0.90909 0.85714 10.0Range 1.00000 10.0 5.0SD 0.71429 0.76923 9.0 7.0 7.0 0.62500 5.0 10.0 1.00000 13.0 7.895 1.00000 10.0 10.0 0.333–1.0 1.00000 1.00000 0.75144 0.62500 13.0 0.81250 2.889 8.0 0.80000 10.0625 0.83333 0.20083 13.0 0.92857 0.381–1.00 12.0 7.0 5.0 7.0 0.87500 8.4167 0.40625 8.0 6.0 3.1784 0.333–1.00 0.73454 6.0 1.00000 0.66667 0.55556 1.00000 0.16579 6.0 7.0 1.00000 5.5208 0.66667 13.0 0.300–1.00 0.66667 7.0 6.0 6.0 5.0 2.5751 0.52000 0.62500 6.2708 7.0 0.17593 0.77477 7.0 10.0 0.280–1.00 0.87500 0.41176 1.00000 0.77778 4.0 6.0 0.66667 5.0 1.7009 8.5208 0.90909 0.60000 7.0 0.19223 7.0 0.321–1.00 9.0 7.0 1.00000 8.0 0.77778 0.65158 8.7021 10.0 9.0 2.0289 0.88889 0.323–1.00 1.00000 5.0 0.87500 0.90000 11.0 0.71429 5.0 0.21289 0.78571 0.83333 14.0 11.0 8.0 8.0 0.88889 0.83333 11.0 0.71809 2.5010 7.0 9.0 1.00000 0.57143 0.16256 12.0 0.82353 0.90000 0.62500 5.0 9.0 0.80000 0.58333 11.0 2.6858 0.52941 9.0 7.0 0.32143 0.16811 0.79438 0.43750 0.6 0.87500 7.0 11.0 0.32353 0.69054

Journal of Genetics Vol. 94, Online Resources e60 A. K. Gupta et al. excess and a genetic bottleneck in all the horse and pony in L-shaped graph (Luikart 1997; Luikart et al. 1998). In all populations. IAM model of mutation in microsatellites the seven breeds, L-shaped curves indicated the absence of assumes that each mutational process generates new allele any recent bottleneck in them which could be due to exis- therefore, gene diversity excess (He > Heq) is demonstrated tence of high diversity among them as reported earlier (Gupta only for loci evolving under this model. Overall populations et al. 2014). In some mammalian species, which are charac- of all the pony breeds has decreased and as such demographic terized by highly fluctuating or cyclical population dynamics, bottleneck seems to hold good under IAM, but it needs to high levels of genetic diversity has been maintained in them be confirmed through other tests also. Under TPM model, all in face of frequent bottlenecks (Redeker et al. 2006;Busch the breeds except Zanskari were observed to be in mutation– et al. 2007). In Spiti, Zanskari and Marwari breeds, simi- drift equilibrium with the acceptance of null hypothesis. lar observations have been earlier reported with different set Situation was totally different under SMM model as het- of microsatellites which supports the present findings (Gupta erozygosity deficiency was observed in all the population et al. 2005, 2013; Chauhan et al. 2011). which indicated that none of the population have recently undergone bottleneck. SMM model is the most conserved model of microsatellite evolution, any heterozygosity excess M-ratio test is likely to confirm a recent reduction in effective popula- In the present study, M-ratio analysis clearly revealed that tion. If the locus evolves under the strict SMM, there can all the horse and pony populations are genetically static be situation where this gene diversity excess is not observed and have not undergone severe genetic reduction in their (Cornuet and Luikart 1996). This model under sign test indi- population sizes (Garza and Williamson 2001). Actual cated the existence of mutation–drift equilibrium in all the decrease in overall populations of different breeds without breeds. Among all the models, TPM model is intermediate to any bottleneck may be due to maintenance of genetic diver- IAM and SMM which assumes that most mutational changes sity in fragmented subpopulations in home tract of respec- result in increase or decrease of one repeat unit but mutations tive breeds (Redeker et al. 2006; Gupta et al. 2014). This test of larger magnitude also occur. Hence, most of microsatellite revealed that in spite of decrease in populations of various loci fit the TPM rather than IAM or SMM model (Ellegren Indian breeds, genetically bottleneck has not taken place 2004). Although populations of all the endangered pony which is quite encouraging for adopting conservation and breeds have declined but there is mutation–drift equilibrium preservation strategies. 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 Conclusion microsatellites also revealed presence of mutation–drift equilibrium in some of the individual Indian breeds (Gupta et al. This study indicated that although the populations of all the 2005, 2012a, b; Chauhan et al. 2011). horse and pony breeds have decreased rapidly during the last Another test, standardized difference test rejects the null couple of decades but no recent bottlenecks had taken place hypothesis of population at mutation–drift equilibrium if in any of the Indian horse or pony breed. Small fragmented probability of heterozygosity excess at the 5% confidence subpopulations of each pony/horse breed in their home tract level is <0.05. Under IAM model, this hypothesis was might have a very good genetic diversity among them. Since rejected in all the Indian horse and pony populations along populations of all the pony breeds is continuously declining, with Thoroughbred horses. IAM model is of little impor- it may sooner or later lead to bottlenecks in these populations tance as it is based on presumption of existence of infinite and breeds may become extinct if suitable measure are not number of alleles. Under TPM model, null hypothesis was taken in time to conserve them. not accepted in Manipuri and Zanskari breeds only while Ti were highly negative for all the breeds indicating heterozy- References gosity deficiency under SMM model. Under TPM and SMM models, heterozygote deficiency was observed in most of the Busch J. D., Waser P. M. and Dewoody A. 2007 Recent demo- populations indicating no bottleneck in all the populations. graphic bottlenecks are not accompanied by a genetic signature in Taking all the results together, it is clear that serious bottle- banner–tailed kangaroo rats (Dipodomys spectabilis). Mol. Ecol. neck has not taken place in any of the pony or horse breed. 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. Mode-shift indicator test 38, 3505–3511. Cornuet J. M. and Luikart G. 1996 Description and power analy- The mode-shift indicator test, a qualitative test for detec- sis for two tests for detecting recent population bottlenecks from tion of bottleneck was also used as a second method to allele frequency data. Genetics 144, 2001–2014. Di Rienzo A., Peterson A. C., Garza J. C., Valdes A. M., Slatkin M. detect potential bottlenecks, as the nonbottlenecked popula- and Freimer N. B. 1994 Mutational processes of simple sequence tions that are near mutation–drift equilibrium are expected to repeat loci in human populations. Proc. Natl. Acad. Sci. USA 91, have a large number of alleles at low frequencies resulting 3166–3170.

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Received 22 December 2014, in ﬁnal revised form 17 April 2015; accepted 27 April 2015 Unedited version published online: 1 May 2015 Final version published online: 6 November 2015

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