Biosci. Biotech. Res. Comm. 8(2):
Population genetic structure of the camel, Camelus dromedarius based on microsatellite loci:
Sushma Prasad,*,** Sharique A. Ali,* Priyanka Banerjee,** Jyoti Joshi,**
Upasana Sharma,** and R.K. Vijh**
*Department of Biotechnology, Sai•a Science College, Bhopal, M.P., India **National Bureau of Animal Genetic Resources,
Camelus dromedarius is a widely distributed animal of
KEY WORDS: MICROSATELLITE, GENETIC STRUCTURE, CAMELUS DROMEDARIUS, BOTTLENECK.
*Corresponding Author: email@example.com Received 10th July, 2015
Accepted after revision 15th October, 2015 BBRC Print ISSN:
© A Society of Science and Nature Publication, 2015. All rights153 reserved.
Online Contents Available at: http//www.bbrc.in/
Sushma Prasad et al.
There has been a dramatic reduction in the camel population of India. According to 18th livestock census India has only 0.52 million camels remaining while the population was 0.63 million in 2003. Estimated growth rate is
In recent years some systematic studies have been reported on characterization of breeds of camel of other countries as South Africa (Nolte et al., 2005) Saudi Arabia (Abdulaziz et al., 2009), Australia (Spencer et al., 2010), Tunisia (Ahmed et al., 2010) and Canaria (Urusula et al., 2010).
Throughout the last three decades, the identi!cation of genetically distinct populations has been a key issue in conservation biology (Wayne, 1992; Paetkau, 1999; Frankham et al., 2002). Ryder (1986) and Moritz (1994) distinguished two types of conservation units, namely evolutionary signi!cant unit and management unit. An evolutionary signi!cant unit is de!ned as a population that is substantially reproductively isolated from other groups and that represents an important component in the evolutionary process of a species, whereas a management unit is a population with signi!cant allelic divergence at nuclear or mitochondrial loci between populations. The relevance of these concepts and the genetic information
in species conservation, including data on population structure, has been intensively recognized to develop and apply adequate conservation and management strategies (Paetkau et al., 1999). In the present study, we report on the population genetic structure of C. dromedarius based on twenty !ve microsatellite loci and bottleneck analy- sis to test the hypothesis of mutation drift equilibrium or recent reduction in effective population size.
MATERIAL AND METHODS
SAMPLES AND GENETIC ANALYSIS
689 blood Samples
The PCR ampli!cation was carried out in 25 ml reac- tion volume consisting of 50 ng genomic DNA, 1.5mM MgCl2, 200 mM dNTPs, 5 p mol of each primer, and 0.5 u of Taq (New England Biolabs, UK). The PCR reaction was carried out in GeneAmp 9700 (Applied Biosystems, USA). The thermocycling conditions utilized were initial denaturation at 94oC for 2 min, followed by 30 cycles of 60 s at 94oC, 45 s at annealing temperature, and 60 s at 72oC. The !nal extension at 72oC was prolonged for 10 min. The samples were analyzed using Avant 3130 Automated DNA Sequencer (Applied Biosystems) with Rox 400 as internal lane standard. The data was col- lected and analyzed using GeneScanTM (Ver 3.7.1) and GenotyperTM (Ver 3.0) software (Applied Biosystems).
Microsatellite loci were characterized for the number of alleles per locus and population, the allelic richness for population, and for observed (Ho) and expected hetero- zygosity (HE) under
Sushma Prasad et al.
FIGURE 1: Geographical distribution areas of different camel breeds.
tiation was also estimated by RST (Slatkin, 1995). RST is obtained by analysis of variance of allele size and may be interpreted as the correlation between allele sizes of different individuals in the same population (Goodman, 1997). This is analogous to , with the exception that for
the correlation between allele frequencies of different individuals in the same population considers an in!nite allele model (Cockerham, 1969; Weir and Cockerham, 1984). Analyses were performed using the FSTATS
22.214.171.124software (Goudet, 2002). Statistical signi!cance tests were based on 10,000 randomizations followed by sequential Bonferroni’s correction (Goudet et al., 1996).
To verify if populations were affected by size reduction leading to a bottleneck, we determined the heterozygos- ity excess by the Wilcoxon sign rank test using the Bot- tleneck software (Cornuet and Luikart, 1996). The bot- tleneck process may cause a faster loss of heterozygosity under
RESULTS AND DISCUSSION
The present study included 689 individuals of Indian dromedary belonging to six breeds/population (Malvi, Mewari, mewati, Bikaneri, Kutchi and Jaisalmeri). These
Sushma Prasad et al.
Table 1: Sampling localities and sample size (N = 689) of C. dromedarius.
were genotyped using 25 loci (Table 2). All the loci were polymorphic in nature and were successfully ampli!ed. The various genetic parameters estimated for the six populations have been given in Table 3. The number of alleles ranged from 9 to 25 in all six populations. They displayed high levels of polymorphism, CMS104 having 6 allele and LCA 70 loci with 23 alleles, respectively. A total of 390 alleles were detected in the six popula- tions in the 25 loci studied. The effective numbers of alleles are signi!cantly less than the number of alleles observed (Table 2) revealing large number of alleles at low frequency. The mean observed heterozygosity was
0.626±0.065, 0.577±0.063, 0.557±0.064, 0.298±0.057, 0.324±0.061, 0.432±0.043 for Malvi, Mewari, Mewati, Bikaneri, Jaisalmeri and Kutchi camel population, respectively. The observed heterozygosity values were less than the expected heterozygosity values in Mewati, Bikaneri, Jaisalmeri and Kachchhi while observed het- erozygosity were high in Malvi and Mewari camel strain pointing toward that Mewati, Bikaneri, Jaisalmeri and Kachchhi breed have de!ned population structure rather than Malvi and Mewari. High observed heterozygosity in Malvi and Mewari camel may be due to gene #ow from adjoining areas leading higher heterozygosity value.
OMMUNICATIONSC ESEARCHR IOTECHNOLOGYB IOSCIENCEB
157 DROMEDARIUS CAMELUS ,CAMEL THE OF STRUCTURE GENETIC POPULATION
Table 2: Microsatellite alleles and heterozygosities in six camel breeds wiz Malvi, Mewari, Mewati, Bikaneri, Jaisalmeri and Kutchi
No Observed number of alleles; Ne Effective number of alleles;
Ho Observed heterozygosity; He Expected heterozygosity
.al et Prasad Sushma
Sushma Prasad et al.
Table 3: Estimates of F statistics at each locus in six camel breeds.
The mean estimates of F statistics were FIT=0.161, FIS=0.320 and FST=0.213. All these estimates were signif- icantly different from zero (P>0.05). The population wise FIS index were
158 POPULATION GENETIC STRUCTURE OF THE CAMEL, CAMELUS DROMEDARIUS
The high value of observed heterozygosity in Malvi and Mewari reveal exchange of germplasm (in#ow) leading to higher heterozygosity. This is also evident from nega-
tive FIS value in Malvi and Mewari. The FIS value close to zero for Mewati camel showing
The assignment test based on likelihood method uti- lizing allelic frequencies (30) and with the ‘‘leave one out’’ procedure (31) as implemented in Genealex soft- ware assigned 94% of the individuals correctly to their respective populations. However all the 39 Mewari, 152 Kutchhi individuals were correctly assigned exhibiting its distinctiveness among the six breeds. Malvi (137) Mewati (20) and Bikaneri (143) were also signi!cantly assigned. 172 out of 192 Jaisalmeri were correctly assigned while 20 individual assigned to Bikaneri breed. The 6 camel populations formed three clusters clearly. Malvi camel came out as a separate population as it formed a separate cluster, Mewari and Mewati were in
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
FIGURE 2: Graphical representation of population assignment.
another cluster, while Bikaneri, Jaisalmeri and Kutchhi came near, it may due to geographical closeness between them and exchange of germ plasm amongst them.
The data were also subjected to AMOVA for estima- tion to !nd overall signi!cant difference among the six camel populations. The AMOVA values indicated 38% variation among breeds and 62% among individuals. It means to specify that the individuals within the breed/ populations contribute more to the variability than the individuals between the populations. Among the camel population degree of freedom was 5.
We utilized three test viz., sign test, standardised differences test and Wilcoxon test to !nd out whether these six camel populations have undergone recent bot- tleneck. All the three models of Microsatellite evolution IAM, SMM and TPM were utilised for the purpose. Esti- mated values are given in table..In sign test expected heterozygosity excess and observed heterozygosity excess was not signi!cant in IAM, whereas this was sig- ni!cant for heterozygote de!ciency in TPM and SMM showing lack of severe reduction in effective population size. In standardized difference test (statistic T2) all the calculated values for T2 are less than 1.645 in IAM, TPM and SMM model in all camel breeds (value from normal distribution) and thus null hypothesis of mutation drift equilibrium was accepted. Wilcoxon Rank test which is a
0.05for IAM thus accept the null hypothesis of mutation drift equilibrium while this hypothesis was rejected for TPM and SMM favouring heterozygotic de!ciency.
Mode shift a qualitative method was also applied. The population that are near mutation drift equilib- rium are expected to have a large proportion of allele at low frequency. The graphical method (Luikart et al., 1998) utilised allelic classes and proportion of allele and exhibited the L shaped curve, with most alleles with low frequency and few alleles with high frequency.
Sushma Prasad et al.
The levels of observed genetic diversity of camels can be used to decide which populations should be pri- oritized for conservation purposes especially when the head counts are showing steep decline. The information shall also provide scienti!c basis for future management of camel population in India.
IMPLICATIONS FOR CONSERVATION
The rapid and intense destruction and fragmentation of natural habitats and infrastructural development have reduced the utility of C. dromedarius leading to the reduction in populations of camel (Rollefson 1993). These threats may interfere directly in the spatial dis- tribution and in population size of the single humped camel in the medium and long term. Nevertheless, the results obtained in this study suggest that these threats still have not had a strong impact on the genetic struc- ture and diversity of the single humped camel popu- lations because of their spread over large geographical area except for decrease in number of animals due to economic conditions and utility. We suggest that conser- vation strategies for C. dromedarius should consider the evidence of distribution of allele frequency and morpho- logical differentiation for conservation. It is important to maintain the high level of genetic diversity by design- ing a planned breeding strategy and utilizing camel for alternative usage of milk production, tourism and as meat animal.
Ahmed, M.O., SalemF.B., BedhiafS., RekikB. and DjemaliM., (2010). Genetic diversity in Tunisian dromedary (Camelus dromedarius) populations using microsatellite markers. Live- stock Science; 132:
Cockerham C (1969). Variance of gene frequencies. Evolution 23:
Cornuet J.M. and Luikart G. (1996). Description and power analysis of two tests for detecting recent population bottle- necks from allele frequency data. Genetics 144:
Di Rienzo A., Peterson A. C., Garza J. C., Valdes A. M. (1994). Mutational processes of
Frankham R., Ballou J. D. and Briscoe D. A. (2002). Introduc- tion to Conservation Genetics. Cambridge University Press, New York.
Gautam L., Mehta S. C., Gahlot R. S., Gautam K. (2004); Genetic characterization of Jaisalmeri camel using microsatellite mark- ers. Indian J Biotech;
Sushma Prasad et al.
Goodman S. J. (1997). RstCalc: a collection of computer pro- grams for calculating estimates of genetic differentiation from microsatellite data and determining their signi!cance. Mol. Ecol. 6:
Goudet J. (2002). FSTAT: a program to estimate and test gene diversities and !xation indices. Available at http://www.unil. ch/izea/softwares/fstat.html.
Goudet J., Raymond M., de Meeus T. and Rousset F. (1996). Testing differentiation in diploid populations. Genetics 144:
Han Y., Jedoschenko D., Hue G., Reiner G., Geldermann H. (2000). Screening and Analysis of New Microsatellite Loci in Camels; University of Hohenhein, Stuttgart, Germany.
Rollefson Ilse Köhler and Rathore H.S., (1996). The Malvi camel: a newly discovered breed from India, Animal Genetic Resources
Lang KDM, Vang Y, Plante Y. (1996). Fifteen polymorphic dinucleotide microsatellites in llamas and alpacas. Anim Genet; 27:293.
Luikart G., Allendorf F. W., Cornuet J. M. and Sherwin W. B. (1998b). Distortion of allele frequency distributions provides a test for recent population bottlenecks. J. Hered. 89: 238- 247.
Luikart G., Sherwin W. B., Steele B. M. and Allendorf F. W. (1998a). Usefulness of molecular markers for detecting popula- tion bottlenecks via monitoring genetic change. Mol. Ecol. 7:
Mburu D.N, Ochieng J. W., Kuria S. G., Jianlin H., Kaufmann B., Rege J. E., Hanotte O. (2003); Genetic diversity and rela- tionships of indigenous Kenyan camel (Camelus dromedar- ius) populations: implications for their classi!cation. Animal Genetics;
Mehta S.C., Goyal A., Sahani M.S. (2007). Microsatellite mark- ers for genetic characterization of Kachchhi camel. Indian Journal of Biotechnology, 6:
Moritz C. (1994). Applications of mitochondrial DNA in con- servation: a critical review. Molecular Ecology;3:
Nei M. (1978). Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89:
Nolte M., Kotze A., van der Bank F. H., Grobler J. P. (2005). Microsatellite markers reveal low genetic differentiation
among South American Camelus dromedrius populations. South African J Anim Sci;
Obraque V., Coogle L., Henney P. J., Bailey E., Mancilla R.,
Ota H. and Kimura M. (1973). A model of mutation appropriate to estimate the number of electrophoretically detectable alleles in a !nite population. Genet. Res. 22:
Paetkau D. (1999). Using genetic to identify intraspeci!c con- servation units: a critique of current proposals. Conserv. Biol. 13:
Ryder, O. A. (1986). Species conservation and systematics: the dilemma of subspecies. Trends in Ecology and Evolution
Slatkin M. (1995). A measure of population subdivision based on microsatellite allele frequencies. Genetics 139:
Slatkin, M., (1993). Isolation by distance in equilibrium and
Spencer, P.B.S and WoolnoughA.P., (2010). Assessment and genetic characterisation of Australian camels using microsat- ellite polymorphisms. Livest. Sci., 129:
Ursula Schulz, Isabel
Valdes A. M., Slatkin M and Freimer N. B. (1993). Allele fre- quencies at microsatellite loci: the stepwise mutation model revisited. Genetics 133:
Vijh R.K., Tantia M.S., Mishra B.,Bharani Kumar S.T. (2007); Genetic diversity and differentiation of Dromedarian camel. Animal biotechnology;
Vijh RK, Sahai R. Camel breeds of India and their conservation. In Domestic Animal Diversity; Eds; (2000); S I publications, Karnal,
Wayne R. K. (1992). On the use of molecular genetic characters to investigate species status. Conservation Biol. 6:
Weir B. S. and Cockerham C. C. (1984). Estimating
Wright S. (1951). The genetical structure of populations. Ann. Eugenics 15: