Sushma Prasad et al.

INTRODUCTION

Indian sub-continent is divided on the basis of different geographical climatic conditions as arid, semi-arid, and high land region. Camelus dromedarius is widely distrib- uted in North- West India. The success of the camel in hotter and drier climates than other domestic animals is due to its particular physiology. Its ability to endure torrid heat and extreme desiccation in its environment are of paramount importance in determining its distribution. In India camels are dweller of hot arid land as in Rajasthan and Gujarat while few population are present in semi-arid region of the country. There has been very few genetic studies on Indin dromedary ie. Jaisalmeri (Gautam et al., 2004), Bikaneri, Kutchchi and Mewari (Vijh et al., 2007, Mehta et al. 2007). There are three recognized breeds of C. dromedarius but few non-descript population also exist in the interior parts of the country as Malvi (Prasad et al 2011, Mewati Sindhi, Vijh et al., 2000).

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 -3.75%. Main threat to C. dromedarius has been its lack of utility because of development of roads and other means of transportation. Camel population may also show genetic decline in response to process such as inbreeding depression and genetic drift in the years to come. Microsatellites are the markers of choice for the detection of genetic diversity analysis of camel (Mburu et al., 2003, Nolte et al., 2005, Vijh et al., 2007).There have been several other genetic studies on the family Camelidea in Dubai, Germany, Australia, Kenya and Ethopia hat have primarily reported the development of new microsatellite loci in Alpacas (Obraque et al., 1998), Llama (Lang et al., 1996) and camels (Han et al., 2000).

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 (8-10 ml) were collected using ethyl- ene diamine tetra acetic acid (EDTA)-coated vacutainer tubes (Becton Dickinson, USA) from different breeds from different geographical areas (Table 1) wiz Rajas- than, Gujarat, Madhya Pradesh (Figure 1). Total genomic DNA was extracted using DNA extraction kit (Roche DNA extraction kit). The set of primers for polymer- ase chain reaction (PCR) ampli!cation of microsatellite loci were synthesized from Applied Biosystems and the forward primes were labelled with different #urescent dyes. The 25 microsatellite primers used along with their labels, Accession No=Reference, sequence, repeat motif, No of alleles and size range are given in Table 1.

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).

GENETIC STRUCTURE

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 Hardy-Weinberg equilibrium (Nei, 1978). Genetic structure was accessed by Wright’s (1951) statistics: f, and F, obtained from an analysis of vari- ance of allele frequencies (Cockerham, 1969). As most mutations in microsatellites involve the addition or sub- traction of a small number of repeat units, according to a stepwise mutation model (Ota and Kimura, 1973; Valdes et al., 1993; Slatkin, 1995), population genetic differen-

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

2.9.3.2software (Goudet, 2002). Statistical signi!cance tests were based on 10,000 randomizations followed by sequential Bonferroni’s correction (Goudet et al., 1996).

BOTTLENECK ANALYSIS

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 mutation-drift equilibrium than loss of heterozy-

gosity under Hardy-Weinberg equilibrium. Hence, popu- lations that have experienced recent reduction in effec- tive population size may present higher allele diversity (HE under Hardy-Weinberg equilibrium) than HE under mutation-drift equilibrium HE for a given number of alleles in the population (Cornuet and Luikart, 1996; Luikart et al., 1998a, b). The distribution of HE under mutation-driff equilibrium for each locus and popula- tion was obtained by a simulation of a coalescent proc- ess. Because microsatellite loci may evolve following the stepwise mutation model (SMM; Ota and Kimura, 1973) or the two-phase mutation model (TPM; Di Rienzo et al., 1994), we performed the bottleneck test using 100% SMM and combining the two models of mutation, with 70% SMM and 30% TPM (Luikart et al., 1998b).

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

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

FIXATION INDICES

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 -0.060, -0.028, 0.086, 0.223, 0.342 and 0.299 for Malvi, Mewari, Mewati, Bikaneri, Jaisalmeri and Kutchhi camel respectively. The negative value of FIS pointing towards out breeding i.e. mating of individuals who are less related than the average relationship of the population amounting to gene #ow. Nineteen of the 25 loci showed signi!cant deviation from Hardy Weinberg equilibrium. On the other hand, the FST and RST which is the relative measure of gene differentiation among populations was 0.265 and 0.292 which were highly sig- ni!cant. This result implies a high differentiation among the six camel populations as a result of structuring of population as in Bikaneri and Jaisalmeri, where the observed heterozygosity and number of allele are low.

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 non-signi!cant popula-

tion structure.

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 non-parametric test gave the estimated value more than

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.

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