Genetic and demographic bottleneck analysis of Indian camel breeds by microsatellite markers

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Genetic and demographic bottleneck analysis of Indian camelbreeds by microsatellite markers

Sharat Chandra Mehta

Received: 19 May 2014 /Accepted: 23 July 2014# Springer Science+Business Media Dordrecht 2014

Abstract The genetic and demographic bottleneck analysisof Indian camel breeds was carried out utilizing 40 microsat-ellite markers. Allelic polymorphism was observed at 20 lociin the Indian dromedary breeds. A total of 66 alleles werescored. The average number of alleles, expected heterozygos-ity and polymorphic information content were, respectively,3.25±0.27, 0.56±0.04 and 0.49±0.04 in Bikaneri; 3.25±0.25,0.53±0.03 and 0.46±0.03 in Jaisalmeri; 3.0±0.21, 0.53±0.04and 0.45±0.03 in Kachchhi and 3.1±0.19, 0.51±0.03 and0.44±0.03 in Mewari breed. Higher genetic variation wasobserved in most numerous Bikaneri breed. Genetic distanceswere least between the breed pair Bikaneri and Jaisalmeriwhich was closely placed with the Kachchhi breed. TheMewari camels had relatively higher genetic distance fromthe other three Indian dromedary breeds. The bottleneck anal-ysis revealed the presence of genetic bottleneck in all fourbreeds of Indian dromedary. However, the qualitative graph-ical method resulted in normal L-shaped distribution of allelefrequencies in Jaisalmeri breeds and shifted mode in Bikaneri,Kachchhi and Mewari breeds. The demographic bottleneckanalysis revealed minimum reduction (−9.65 %) in the popu-lation of camels in Jaisalmeri breeding tract as compared tothat of Bikaneri (−14.18 %), Kachchhi (−27.78 %) andMewari (−32 %) breeding tracts. Conclusively, the geneticbottleneck analysis could explain the demographic bottleneckin the Indian dromedary populations. Therefore, appropriateconservation and improvement efforts are needed in all fourdromedary breeds with immediate attention on Mewari andKachchhi breeds. The present study is the first report indemonstrating the genetic basis of demographic bottleneckin the Indian dromedary populations.

Keywords Camel . Conservation .Microsatellite . Geneticdistance . Bottleneck

Introduction

The dwindling camel (Camelus dromedarius) population inseveral camel rearing countries is a matter of major concernfor the conservation biologist. The world has 26.68 millioncamels spread across 47 countries. About 85 % of the camelpopulation inhabits mainly eastern and northern Africa(Somalia, Ethiopia, Sudan, Djibouti and Kenya) and the restin Indian subcontinent and Middle East counties (FAOSTAT2012). The population of camel in India is 0.516 million and ithas gone down by 18.2 % during the years 2003 to 2007(BAHS 2010). Under such circumstances, the structure andcomposition of the populations get distorted due to the dis-proportionate reduction in the animals of the two sexes, loss ofelite animals, irrational breeding and associated factors. It iswidely believed that population bottlenecks should be avoidedin threatened species because they can increase the rate ofinbreeding, loss of genetic variation and fixation of mildlydeleterious alleles, and thereby reduces adaptive potential andincreases the probability of extension (Cornuet and Luikart1996).

India has four major breeds viz. Bikaneri, Jaisalmeri,Kachchhi and Mewari (Fig. 1). The Bikaneri breed is predom-inantly bred in the Bikaner, Sri Ganganagar, Hanumangarh,Churu, Jhunjhunu, Sikar and Nagaur districts of Rajasthan.The home tract of this breed is arid and sandy with extremehot and cold climates. It is a multipurpose breed but the camelsof this breed have been classified as baggage type due to theirhigher potential to carry loads. The Jaisalmeri breed is pre-dominantly bred in the Jaisalmer, Barmer, Jalore and Jodhpurdistricts of Rajasthan. The breeding tract has very poor vege-tation. Sand dunes are the typical features of the tract.

S. C. Mehta (*)National Research Centre on Camel, Post Box 07, Bikaner 334001,Indiae-mail: [email protected]

Trop Anim Health ProdDOI 10.1007/s11250-014-0653-y

Jaisalmeri camels are lightly built, slightly lean and thin inappearance and well known for riding and race potential. TheKachchhi breed inhabits the Rann of Kachchh in the Gujaratstate. The major breeding tract encompasses the Kachchh andBanaskantha districts of Gujarat. Sizable population of thebreed is also found in Patan, Mehsana, Sabarkantha,Gandhinagar, Ahmedabad, Anand, Jamnagar and Porbandardistricts of Gujarat. The land is marshy with abundant saltbushes. The Kachchhi breed is a multipurpose breed and thefemales are good milk producers. The Mewari breed is pre-dominantly bred in the Udaipur, Chittorgarh, Rajsamand,Pratapgarh, Banswara and Dungarpur districts of Rajasthanand adjoining districts of Madhya Pradesh. The tract consistsof hills of the Aravali in the Mewar area. Mewari camels arestouter and well adapted to travel and carry loads across hills.They are exclusively used for milk production (Mehta et al.2007). The population of Bikaneri, Jaisalmeri, Kachchhi andMewari camels in the respective breeding tract has beenestimated to be 306,135; 106,867; 29,505 and 8,834 heads(Mehta et al. 2011).

The study of genetic diversity had always been an area ofinterest for the geneticists and conservation biologist. Themicrosatellite markers are now considered as the most pow-erful genetic markers for characterisation of plant and animalgenetic resources (Goldstein and Pollock 1997). Also, themicrosatellite loci are probably the best markers currentlyavailable for detecting recent bottlenecks because of theirgenerally high level of variability (Cornuet and Luikart

1996). Therefore, this study was planned to investigate thegenetic variation using microsatellite markers and to carry outgenetic and demographic bottleneck analysis due to severereduction in the population of Indian dromedary.

Materials and methods

Sampling

The blood samples were collected from unrelated camels ofthe Bikaneri, Jaisalmeri, Kachchhi and Mewari breed main-tained at the National Research Centre on Camel, Bikaner.The centre maintains around 100 camels of Bikaneri breed, 80camels of Jaisalmeri breed and 70 camels each of theKachchhi and Mewari breeds. The outbred males of eachbreed are regularly procured and introduced in the centre’sherd to avoid inbreeding. Samples were also collected fromthe breeding tract of each breed to ensure a collection of 100samples of each breed (Fig. 1). Out these 100 samples, 50samples of unrelated camels were identified based on thepedigree records of the centre and herd information gatheredfrom the camel owners for present investigation.

Genotyping of microsatellite loci

DNA was isolated using phenol–chloroform method(Sambrook et al. 1989) with minor modifications and 40

Fig. 1 Habitat of Bikaneri, Jaisalmeri, Kachchhi and Mewari breeds of camel in India

Trop Anim Health Prod

microsatellite primer pairs (Obreque et al. 1998, 1999; Langet al. 1996; Penedo et al. 1998, 1999a, b; Mariasegaram et al.2002) were selected for the present study. PCR amplificationswere carried out in 25-μl reactions containing 50 ng DNA,5 pmol each primer (Geneset, Singapore and Genetix, India),1 U Taq DNA polymerase, 0.2 mM each dNTP, 2.5 μl 10XTaqDNA polymerase buffer containing 10mMTris–HCl (pH9.0), 1.5 mM MgCl2, 50 mM KCl and 0.01 % gelatin. ThePCR amplification programme, performed on an EppendorfMastercycler gradient, consisted of an initial denaturationtemperature of 94 °C for 5 min, then 30 cycles at 94 °C for45 s, 50–64 °C for 1 min and 72 °C for 1 min. Final extensionwas carried out at 72 °C for 15 min. The microsatellite bandswere resolved by 6 % Urea Polyacrylamide GelElectrophoresis (Bio-Rad: Sequi-Gen® GT), stained with sil-ver nitrate (Bassam et al. 1991) and sized using GeneTools(Syngene) software.

The allele frequency was calculated by using the equationpi=Ki/N, where pi = frequency of ith allele, Ki = number ofobservation of ith allele andN = total number of alleles scored.Observed heterozygosity (Ho) was calculated by taking theratio of the number of heterozygous animals to the totalnumber of animals scored. The expected heterozygosity (He)

was calculated by using the equation He ¼ 1−∑i¼1

n

pi2 , where

He = expected heterozygosity and Pi = frequency of ith allele.The polymorphism information content (PIC) is anoth-er important measure of DNA polymorphism. Besidesbeing a measure of genetic variation, it is also used inthe context of gene mapping. The values of PIC arelower than heterozygosity for the corresponding markerbecause in PIC, a quantity is subtracted from hetero-zygosity that corresponds to the probability of theoffspring being uninformative. Thus, PIC also indicatesthe potential of a marker to differentiate the closelyrelated individuals or populations. The polymorphic in-formation content was calculated (Botstein et al. 1980)

by using PIC ¼ 1− ∑i¼1

n

p2i

� �− ∑n−1

i¼1∑

j¼iþ1

n

2p2i p2j , where pi is

the frequency of the ith allele and pj is the frequency ofthe jth allele. PHYLIP 3.6 (Felsenstein 1985) was uti-lized for calculating the genetic distance among Indiancamel breeds. The Nei’s distance (Ds), Cavalli-SforzaChord distance (Dc) and Reynold’s distance (Fst) werecalculated. The Nei’s (1972) standard distance (Ds) esti-mates value linearly related to the time since divergence,assuming that all loci have the same rate of neutral muta-tion, and that the genetic variation is maintained by theequilibrium between infinite allele mutation and geneticdrift, with the effective population size of each populationremaining constant. In populations X and Y, if ij p and ij qare the frequencies of ith allele at the jth locus, while aj is

the number of alleles at the jth locus, and m is the number ofloci examined, then it is defined as:

Ds ¼ −ln J xy=ffiffiffiffiffiffiffiffiffiffiJ x J y

p� �

Where

J x ¼Xj¼1

m Xi¼1

a j

p2ij=m; J y ¼Xj¼1

m Xi¼1

a j

q2ij=m; J xy ¼Xj¼1

m Xi¼1

a j

pijqijm

The other two genetic distances assume that there is nomutation, and that all gene frequency changes are by geneticdrift alone. The Cavalli-Sforza chord distance (Dc) (Cavali-Sforza and Edwards 1967) gives the chord distance betweenthe two populations, if we represent the two populations onthe surface of a multidimentional hypersphere using allelefrequencies at the jth locus, the distance is the geometricdistance between the points in multi-dimensional space:

Dc ¼ 2

πm

Xj¼1

mffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi2 1−

Xi¼1

a j ffiffiffiffiffiffiffiffiffipijqij

p !vuut

Reynolds et al. (1983) genetic distance is based on thedegree of relationship by descent between two individuals:

θ2 ¼Xj¼1

m Xi¼1

a j

pij−qij� �2

2Xj¼1

m

1−Xi¼1

a j

pijqij� � !

The phylogenetic tree was constructed from all the threemeasures of the genetic distance using the unweighted pair-group method with arithmetic mean (UPGMA) method ofclustering by the DrawGram programme of the PHYLIPpackage.

Bottleneck analysis

The bottleneck hypothesis was investigated usingBOTTLENECK 1.2.02 (Cornuet and Luikart 1996). The bot-tleneck tests for the departure from mutation drift equilibriumbased on heterozygosity (not heterozygote) excess or deficien-cy. This does not require information on historical populationsizes or level of genetic variations. It requires only a measure-ment of allele frequencies from 5 to 20 polymorphic loci in asample of approximately 20–30 individuals. The bottleneckcompares heterozygosity expected (He) from the observednumber of alleles at Hardy-Weinberg equilibrium to the het-erozygosity expected (Heq) at mutation drift equilibrium insame sample, that has the same size and the same number ofalleles. This is different from the difference between observedand expected heterozygosities in the sense of Nei’s (1978)


gene diversity. The bottleneck tests rely on the assumption thateach sample is representative of a well-defined population andthe independence of the results among pairs of loci, which isachieved if all loci scored on a given sample are geneticallyindependent. Microsatellite loci are considered to be selective-ly neutral. Various models have been put forward to explainthe mutation processes that affect microsatellite evolution.The infinite allele model (IAM) (Kimura and Crow 1964)assumes that microsatellite mutations may create an infinitenumber of repeated units/allelic states and the probability ofbackmutation to the original allele would be low enough to benegligible. Under the stepwise mutation model (SMM) (Ohtaand Kimura 1973), microsatellite mutations have the sameprobability of gaining or contracting one repeat unit. Thus,this model also takes into account back mutations. The two-phase model (TPM) (Di Rienzo et al. 1994) addresses this byallowing mutations of one repeat unit (one phase) with prob-ability p and mutations of ≥1 unit(s) (two phase) with proba-bility 1−p, while the distribution of the lengths of multiunitmutations is geometric.

Bottleneck events in the population were tested by twomethods. The first method consisted of three excess heterozy-gosity tests developed by Cornuet and Luikart (1996) like signtest, standardized differences test and a Wilcoxon signed ranktest. The probability distribution was established using 1,000simulations based on allele frequency and heterozygosity un-der three models such as infinite allele model (IAM), stepwisemutation model (SMM) and two-phase model of mutation(TPM). The second method was the graphical representationof the mode-shift indicator (Luikart and Cornuet 1998) origi-nally proposed by Luikart et al. (1998). Loss of rare alleles inbottlenecked populations was detected when one or more ofthe common allele classes have a higher number of alleles thanthe rare allele class (Luikart et al. 1998). Livestock censusfigures (BAHS 2010) were utilized for the carrying out demo-graphic bottleneck analyses in dromedary breeds.

Results

Allelic distribution and gene diversity

Successful amplification of 40 Camelidae microsatellite lociwas carried out in Indian dromedary breeds. Twenty loci werepolymorphic and rests viz. VOLP32, VOLP77, YWLL29,YWLL36, YWLL40, YWLL43, YWLL46, LCA8, LCA19,LCA24, LCA30, LCA36, LCA65, LCA68, LCA5, LCA37,LCA77 were monomorphic. The observed number of allelesranged from two to six in Bikaneri and Jaisalmeri breeds andtwo to five in Kachchhi and Mewari breeds. The averagenumbers of alleles across the microsatellite loci were 3.25±0.27 in Bikaneri, 3.25±0.25 in Jaisalmeri, 3.0±0.21 inKachchhi and 3.1±0.19 Mewari (Table 1).

The observed heterozygosity ranged from 0.12 to 0.72 inBikaneri, 0.15 to 0.73 in Jaisalmeri, 0.13 to 0.88 in Kachchhiand 0.14 to 0.83 inMewari breed. In the present investigation,the average expected heterozygosity was 0.56±0.04 inBikaneri, 0.53±0.03 in Jaisalmeri, 0.53±0.04 in Kachchhiand 0.51±0.03 in Mewari breed. Pooled over loci, the poly-morphism information content (PIC) values was 0.49±0.04 inBikaneri, 0.46±0.03 in Jaisalmeri, 0.45±003 in Kachchhi and0.44±0.03 in Mewari breed (Table 1).

Genetic relationship

The PHYLIP 3.6 was utilized to estimate the genetic distancesamong the camel breeds. Nei’s genetic distance, Cavalli-Sforza Chord Measure and Reynold’s genetic distance wereestimated (Table 2). The phylogenetic trees were constructedfrom all the three measures of the genetic distance using theUPGMA method of clustering by DRAWGRAM programmeof PHYLIP package (Fig. 2). One thousand boot-strappingswere also done and the consensus trees were constructed. Thesame tree topology was derived with the three measures.

Genetic bottleneck analysis

The sign test, standardized differences test and Wilcoxon testcarried out for bottleneck analysis in all four dromedarybreeds utilizing allele frequency as well as heterozygosityhave been depicted in Table 3. The analyses indicated thatthere were few loci with heterozygosity deficiency (Hd) and alarge number of loci were with heterozygosity excess (He) ascompared to the expected number of loci with heterozygosityexcess (Hee) in 1,000 simulations based on allele frequencyand heterozygosity in all four dromedary breeds. In sign test,all the three models (IAM, TPM and SMM) in both themethods had significantly (P<0.01) higher He than Hee in allfour breeds except Mewari in TPM (He=16 and Hee=11; He=16 and 10.98) and SMM (He=16 and Hee=11.44; He=16 and11.39) where the significance level was 5 % (P<0.05), and inJaisalmeri in SMM where the He was non-significantly(P>0.05) higher than Hee (He=15 and Hee=11.30; He=15and 11.38). The standardized differences test revealed thatthe standardized differences between He and Hee were signif-icantly (P<0.01) different from zero at most loci in all fourbreeds in all three mutation models except for Jaisalmeri [T2=2.097; P=0.018 and T2=1.557; P=0.060] and Mewari [T2=1.631; P=0.051 and T2=1.301; P=0.097] in SMM. InWilcoxon test, the probability values for heterozygosity ex-cess (P—one tail for He) were significant (P<0.001) in all thebreeds, in all three mutation models based on both themethods except Mewari in TPM (P<0.01) and SMM(P>0.05), and Jaisalmeri in SMM (P<0.05). So, the nullhypothesis that the dromedary populations are at mutation–drift equilibrium was rejected. In addition, qualitative


graphical method of Luikart and Cornuet (1998) was used tovisualize the allele frequency spectra. The microsatellite al-leles were classified into 10 frequency classes. The normal L-shaped form was observed in Jaisalmeri whereas in the rest,i.e. Bikaneri, Kachchhi and Mewari breeds, shifted mode wasobserved (Fig. 3).

Discussion

Allelic distribution and gene diversity

The number of alleles at different marker loci and theirfrequencies are simple indicators of the population

structure and its breeding history. It is evident from theabove observation that Kachchhi and Mewari breeds haverelatively less genetic variation as compared to theBikaneri and Jaisalmeri, it is further substantiated by thefact that the Mewari is the least numerous and Kachchhiis the second least numerous breed of camel in India(Mehta et al. 2011). The number of alleles and allele sizerange observed in the present investigation were broadlyin agreement with the literature (Obreque et al. 1998;Sasse et al. 2000; Jianlin et al. 2000; Vijh et al. 2007;Spencer and Woolnough 2010; Banerjee et al. 2012).Slight differences in the number of alleles and hencefrequencies were expected due to the existence of actualgenetic differences among the stocks used by differentresearchers. Further, the observation of monomorphic pat-tern at 20 microsatellite loci, which are known to bepolymorphic in the camelids, may be attributed to theexistence of higher degree of genetic homozygosityamong and within the individuals of the Indian dromedarybreeds at these loci.

The observed heterozygosity depends on the number ofheterozygous individuals in the population and the expectedheterozygosity depends on the number of alleles and theirfrequency in a population at a particular locus. In the presentinvestigation little higher allelic variation was observed in

Table 2 Genetic distances among Indian dromedary breeds

Breed Bikaneri Jaisalmeri Kachchhi Mewari

Bikaneri 0.000a,b 0.046/0.039b 0.072/0.057b 0.117/0.118b

Jaisalmeri 0.051a 0.000a,b 0.062/0.051b 0.109/0.110b

Kachchhi 0.075a 0.063a 0.000a,b 0.136/0.129b

Mewari 0.169a 0.148a 0.176a 0.000a,b

a Nei’s genetic distanceb Cavalli-Sforza chord measure/Reynold’s genetic distance

Table 1 Genetic variation at microsatellite loci in Indian dromedary breeds

Locus Reference Number Size (bp) Bikaneri Jaisalmeri Kachchhi Mewari

n Ho He PIC n Ho He PIC n Ho He PIC n Ho He PIC

VOLP03 Obreque et al. (1998) 50 144–168 5 0.59 0.72 0.68 4 0.28 0.54 0.46 3 0.48 0.43 0.37 4 0.36 0.42 0.37




YWLL09 Lang et al. (1996) 50 160–162 2 0.59 0.45 0.35 2 0.29 0.31 0.26 2 0.38 0.30 0.26 2 0.41 0.48 0.37

YWLL38 Lang et al. (1996) 50 180–186 3 0.64 0.53 0.42 3 0.67 0.54 0.44 3 0.55 0.57 0.48 3 0.57 0.50 0.42

YWLL44 Lang et al. (1996) 50 104–106 2 0.31 0.37 0.30 3 0.45 0.50 0.41 2 0.58 0.45 0.39 3 0.31 0.35 0.30

YWLL58 Lang et al. (1996) 50 173–177 2 0.17 0.38 0.30 2 0.15 0.44 0.34 2 0.13 0.47 0.36 2 0.58 0.50 0.38

YWLL59 Lang et al. (1996) 50 115–117 2 0.64 0.43 0.34 2 0.73 0.46 0.36 2 0.46 0.35 0.29 2 0.60 0.46 0.35

CVRL01 Mariasegaram et al. (2002) 50 208–240 3 0.46 0.66 0.59 3 0.48 0.59 0.51 3 0.62 0.65 0.57 3 0.14 0.26 0.24





LCA18 Penedo et al. (1998) 50 224–230 3 0.72 0.66 0.58 3 0.26 0.57 0.48 3 0.51 0.66 0.59 3 0.56 0.49 0.43

LCA22 Penedo et al. (1998) 50 170–180 4 0.45 0.64 0.58 4 0.73 0.62 0.54 4 0.66 0.62 0.54 4 0.60 0.67 0.62

LCA33 Penedo et al. (1998) 50 122–130 3 0.12 0.18 0.17 3 0.27 0.31 0.29 3 0.16 0.15 0.14 3 0.55 0.50 0.44

LCA56 Penedo et al. (1999a) 50 134–138 2 0.50 0.38 0.30 2 0.42 0.41 0.32 2 0.38 0.30 0.26 2 0.56 0.36 0.28

LCA63 Penedo et al. (1999b) 50 210–222 5 0.53 0.64 0.58 5 0.38 0.63 0.59 5 0.67 0.68 0.64 5 0.23 0.66 0.61

LCA66 Penedo et al. (1999a) 50 234–238 3 0.25 0.64 0.57 3 0.49 0.59 0.51 3 0.57 0.53 0.47 3 0.83 0.65 0.57

n number of alleles; Ho observed heterozygosity; He expected heterozygosity; PIC polymorphic information content


Bikaneri followed by Jaisalmeri and Kachchhi. Least variationwas observed in the Mewari breed. The measured expectedheterozygosity commensurate with the population base of theIndian dromedary breeds in the breeding tract (Mehta et al.2011). Pooled over loci, the PIC ranged from 0.14 to 0.78. Itwas evident that the markers VOLP08 and LCA56 were theleast informative and VOLP67 and CVRL03 were the mostpowerful markers. Still, the markers used in the study wereable to explain the genetic variation existing among the

individuals of the four Indian dromedary breeds as per theirpopulation base in the breeding tract (Mehta et al. 2011).Slightly wider range of PIC has been reported in Old WorldCamelids (Sasse et al. 2000; Jianlin et al. 2000; Mariasegaramet al. 2002; Vijh et al. 2007; Banerjee et al. 2012). This wasexpected due to the existence of more number of alleles inother breeds of dromedary, selective breeding with in popula-tions, and status of population of the breeds in the breedingtract. Nevertheless, the studied microsatellite markers can

Bikaneri

0.0255

0.0345

Jaisalmeri

0.0823 Kachchhi

Mewari

Fig. 2 Phylogenetic relationshipamong Indian dromedary breeds

Table 3 Mutation–drift equilibrium, heterozygosity excess/deficiency under different mutation models in Indian dromedary breeds

Model Breed Method based onfrequency orheterozygosity

Sign test Standardized differencestest

Wilcoxon test

Hee Hd He p T2 p p (one tail for Hd) p (one tail for He) p (two tails for He

or Hd)

1. IAM Bikaneri Frequency 10.26 1 19 0.000 5.284 0.000 1.000 0.000 0.000

Heterozygosity 10.27 1 19 0.000 4.887 0.000 0.999 0.000 0.000

Jaisalmeri Frequency 10.31 2 18 0.000 4.408 0.000 1.000 0.000 0.000

Heterozygosity 10.42 2 18 0.000 4.121 0.000 1.000 0.000 0.000

Kachchhi Frequency 10.02 1 19 0.000 5.062 0.000 1.000 0.000 0.000

Heterozygosity 09.93 1 19 0.000 4.878 0.000 1.000 0.000 0.000

Mewari Frequency 10.25 3 17 0.002 4.223 0.000 0.999 0.000 0.000

Heterozygosity 10.27 4 16 0.008 3.909 0.000 0.999 0.000 0.000

2. TPM Bikaneri Frequency 10.95 1 19 0.000 4.266 0.000 0.999 0.000 0.000

Heterozygosity 10.91 1 19 0.000 3.899 0.000 0.999 0.000 0.001


Heterozygosity 11.01 3 17 0.005 2.978 0.001 0.999 0.000 0.000


Heterozygosity 10.81 1 19 0.000 3.881 0.000 1.000 0.000 0.000


Heterozygosity 10.98 4 16 0.018 2.741 0.004 0.997 0.004 0.007

3. SMM Bikaneri Frequency 11.22 2 18 0.001 3.199 0.001 0.999 0.001 0.001

Heterozygosity 11.26 2 18 0.001 2.752 0.003 0.999 0.001 0.002


Heterozygosity 11.38 5 15 0.076 1.557 0.060 0.978 0.024 0.048


Heterozygosity 11.07 3 17 0.005 2.926 0.002 0.999 0.000 0.000


Heterozygosity 11.39 4 16 0.028 1.301 0.097 0.923 0.082 0.165

Parameters for TPM: Variance=30.00; Proportion of SMM in TPM=70 %; Estimation based on 1,000 replications

IAM infinite allele model; TPM two-phase model; SMM stepwise mutation model; Hee heterozygosity excess expected; He heterozygosity excess; Hd

heterozygosity deficiency; p probability, T2 standardized difference test statistic


potentially be used for diverse genetic applications includinglinkage mapping, individual identification and parentagetesting.

Genetic relationship

The least genetic distance was observed between the breedpair Bikaneri and Jaisalmeri which was closely associatedwith the Kachchhi. The Mewari camels had relatively highergenetic distance from the other three Indian dromedary breeds.Though the genetic relationship between the four dromedarybreeds remained same in the three different measures of thegenetic distance but the closeness between the measures basedon different assumptions viz. genetic variation is maintainedby genetic drift only (Cavalli-Sforza chord distance andReynold’s genetic distance) versus the genetic variation ismaintained by the equilibrium between infinite allele mutationand genetic drift (Nei’s genetic distance), was quite evident.Comparable genetic relationship among the Indian dromedarybreeds has been reported by Mehta et al. (2006) and Banerjee

et al. (2012). Since long, the Bikaneri, Jaisalmeri andKachchhi breeds were utilized for their draught potential butthe Mewari breed of camel is the only breed which hasexclusively been reared for milk production. The documenta-ry evidences for its’ utilization as milch animals are availablefor the last four decades and hence the choice to retain or cullan animal must have been based on their utility to mankind.Further, the Mewari is the least numerous of the Indian camelbreeds and the impact of small population size (Mehta et al.2011) in terms of restricted gene flow and increased homozy-gosity may be among the major reasons for its’ differentiationfrom other Indian dromedary breeds.

Genetic bottleneck analysis

The sign test as well as the Wilcoxon test rejects the nullhypothesis of existence of the populations at mutation–driftequilibrium on the basis of excess heterozygosity. However,the sign test is more conservative and rejects the null hypoth-esis based on excess heterozygosity at a significant number of

0

0.05

0.1

0.15

0.2

0.25

1 2 3 4 5 6 7 8 9 10

Alle

le F

requ

encie

sAllele Frequency Class

(a) Bikaneri

0

0.05

0.1

0.15

0.2

0.25

0.3

1 2 3 4 5 6 7 8 9 10

Alle

le F

requ

encie

s

Allele Frequency Class

(b) Jaisalmeri

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

1 2 3 4 5 6 7 8 9 10

Alle

le F

requ

encie

s


(c) Kachchhi

0

0.05

0.1

0.15

0.2

0.25

1 2 3 4 5 6 7 8 9 10

Alle

le F

requ

encie

s


(d) Mewari

Fig. 3 Mode-shift graph showingpresence of bottleneck in Bikaneri(a), Kachchhi (c), Mewari (d) andabsence in Jaisalmeri (b) camelpopulations


loci, whereas Wilcoxon test is more sensitive and comparesthe magnitude of observed heterozygosity excess with expect-ed levels (Cornuet and Luikart 1996). On the other hand, thestandardized differences test establishes if the average ofstandardized differences between observed and expected het-erozygosities is significantly different from zero. Positivevalues of the bottleneck statistic T2 are indicative of genediversity excess caused by a recent reduction in effectivepopulation size, while negative value are consistent with arecent population expansion without immigration.

The higher magnitude of loci with heterozygosity excess(He) with their respective expected number of loci with het-erozygosity excess (Hee) in majority of cases reflected thepresence of genetic bottleneck in the recent past populationdynamics of all four breeds of Indian dromedary. When abottleneck event occurs in a population, the population expe-riences a reduction of its effective size, the allele number isreduced faster than the heterozygosity, i.e. the observed het-erozygosity is larger than the heterozygosity expected fromthe observed allele number when the locus is at mutation–driftequilibrium. In addition, the qualitative graphical method ofLuikart and Cornuet (1998) was used to visualize the allele

frequency spectra. The normal L-shaped form was observedin Jaisalmeri whereas for the rest, i.e. Bikaneri, Kachchhi andMewari breeds, shifted mode was observed. According toLuikart and Cornuet (1998), population bottlenecks cause acharacteristic mode shift distortion in the distribution of allelefrequencies at selectively neutral loci. Bottlenecks cause al-leles at low frequency (<0.1) to become less abundant thanalleles in one or more intermediate allele frequency class (e.g.,0.1–0.2). This distortion is transient and likely to be detectablefor only a few dozen generations. This reflects that theBikaneri, Kachchhi and Mewari breeds had the bottleneckevent in recent past. Further, the Jaisalmeri breed had alsoundergone the genetic bottleneck event but still the effectivepopulation size has not reduced significantly and the geneticvariation is being maintained within the threshold limits.

Demographic bottleneck analysis

The livestock census figures reveal that the camel populationin the country was 0.61 million in the year 1951 and itcontinued growing till the year 1972. After a short staticperiod from 1972 to 1977, the camel population in the countryhad a declining trend and it was 0.516 million in the year 2007(Fig. 4). With a generation interval of 5.5 years (Mehta andSahani 2009), the camels in the country have declined overlast five to six generations. The observation of shifted-mode inBikaneri, Kachchhi and Mewari breeds and normal L-shapedform in Jaisalmeri breed was a matter of curiosity, especiallybecause the Bikaneri is the most numerous and Mewari is theleast numerous breed of camel in India (Mehta et al. 2011).The demographic distribution of different breeds was there-fore studied. Instead of considering breed-wise population, thecensus figures were preferred for analysis because in thebreeding tract, there was neither any restriction nor any pref-erence to breed the animals as per their breed. Hence, the geneflow was across all the camels of the tract. The breeding tractof Bikaneri had 315,881 camels in the year 2003, which were

0.6

0.8

0.9

1

1.1 1.1 1.081 1.03

0.91

0.64

0.52

0

0.2

0.4

0.6

0.8

1

1.2

1951

1956

1961

1966

1972

1977

1982

1987

1992

1997

2003

2007

Cam

el P

opul

a�on

(m)

Year

Fig. 4 Camel population in India as per livestock census 1951–2007

-50000

0

50000

100000

150000

200000

250000

300000

350000

Bikaneri Jaisalmeri Kachchhi Mewari

Num

ber o

f Cam

els

Breeds

Census 2003

Census 2007

Reduc�on

Fig. 5 Status of dromedarypopulation in India as perlivestock census 2003 and 2007


reduced to 271,074 in the year 2007 with a negative growth of14.18 % in 5 years (Fig. 5). It was observed that the femaleswere not being bred regularly and even they were being usedfor their draught power, which used to be the task of males.The reduced utility of males has led to excessive and indis-criminate culling of male sex. Therefore, the individualsbreeding for the next generation have reduced considerably.The breeding tract of Mewari had 24,919 camels in the year2003, which were reduced to 16,944 in the year 2007 with thehighest negative growth of 32 % in 5 years mainly due to thedevelopmental factors. Also, the availability of free pastureland is the least in this tract. Further, the Mewari camels arebeing reared exclusively for milk production, because of thisthe rearing of camels in the tract is much similar to that of anyother dairy animal, i.e. rearing of one breeding male in a herdof about 25–30 camels. The breeding tract of Kachchhi had53,246 camels in the year 2003, which were reduced to 38,454in the year 2007 with a negative growth of 27.78 % in 5 yearsmainly due to the tremendous development in the Kachchhdistrict of Gujarat in the last decade. However, least reductionin camel population was observed in the habitat of Jaisalmericamel. The tract had 146,208 camels in the year 2003, whichwere reduced to 132,102 in the year 2007 with a negativegrowth of 9.65 % in 5 years (Fig. 5). It was observed thatJaisalmer was the only district in the country which had apositive growth of 5.49 % (2,028 heads) in population ofcamels during this period. This is because the tract is sparselypopulated and huge area is still available for grazing. Further,the major area of the breeding tract lies in the Jaisalmer andBarmer districts of Rajasthan which have been recognized asone among the most backward districts of the country by theGovernment of India. It is therefore evident that the changedutility and developmental factors have resulted in the reduc-tion of effective population in the recent past. TheMewari andKachchhi were the most affected breeds followed by theBikaneri and Jaisalmeri. The distortion of the L-shaped formalso presents the similar results. Conclusively, the recent de-mographic bottlenecks were accompanied by genetic bottle-necks in the Indian dromedary populations.

It is evident from the above study that Mewari andKachchhi breeds have relatively less genetic variation ascompared to the Bikaneri and Jaisalmeri. The Mewari camelshad also relatively higher genetic distance from the rest of theIndian dromedary breeds due to selection. The bottleneckanalysis revealed the presence of genetic bottleneck in all fourbreeds of Indian dromedary. However, the qualitative graph-ical method resulted in normal L-shaped distribution of allelefrequencies in Jaisalmeri breed and shifted mode in Bikaneri,Kachchhi and Mewari breeds. The study warrants that appro-priate measures to increase the effective breeding populationmust be taken up in the habitat of all four breeds of camel.Preventing indiscriminate culling of males and regular breed-ing of all available breedable females may change the genetic

constitution of the generations to come and help us in main-taining variability and overcoming bottlenecks in thepopulations.

Acknowledgments This work was carried out under an InstitutionalProject in the Animal Genetics Laboratory of National Research Centreon Camel, Bikaner, India. The entire funding for the work was receivedfrom our parent organization, the Indian Council of Agricultural Re-search, New Delhi, India. The laboratory assistance provided by Sh. R.L. Vyas, Senior Technical Assistant in carrying out this task is highlyacknowledged.

Conflict of interest There is no conflict of interest. The manuscriptdoes not contain clinical studies or patient data.

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Genetic and demographic bottleneck analysis of Indian camel breeds by microsatellite markers