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Molecular characterization of camel breeds of Gujarat using microsatellite markers
Livestock Science 181 (2015) 85–88
Contents lists available at ScienceDirect
Livestock Science journal homepage: www.elsevier.com/locate/livsci
Short communication
Molecular characterization of camel breeds of Gujarat using microsatellite markers A.C. Patel a, T.K. Jisha a, Disha Upadhyay a, Rakesh Parikh a, Maulik Upadhyay a, Riddhi Thaker a, S. Das b, J.V. Solanki a, D.N. Rank a,b,n a b
Department of Animal Genetics and Breeding, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand 388001, Gujarat, India Sahjeevan Trust, Vijay Nagar, Bhuj 370001, Gujarat, India
art ic l e i nf o
a b s t r a c t
Article history: Received 2 May 2014 Received in revised form 3 October 2015 Accepted 6 October 2015
Camels are the economic backbone of many nomadic tribes of the world including India. Camel population in India is restricted to western part of the country and is represented by eight breeds. The western most state of the country, Gujarat, possesses two camel breeds, Kachchhi and Kharai inhabiting in the same area. Populations of both are facing severe decline which calls for their immediate conservation. In the present study we examined genetic variability and structure in Kachchhi and two populations of Kharai (n ¼193) using 27 microsatellite markers. A total of 138, 112 and 95 alleles were observed in Kachchhi, Kharai (K) and Kharai (A) respectively with the mean effective number of alleles per locus 2.818 70.303; 2.373 7 0.245 and 2.313 7 0.224. The mean observed heterozygosity was 0.4467 0.039 for Kachchhi, 0.272 7 0.040 for Kharai (K) and 0.423 7 0.044 for Kharai (A), which was lower than expected heterozygosity 0.53570.045, 0.461 7 0.051 and 0.474 7 0.043 respectively. The average inbreeding coefficient (FIS) 0.2337 0.037 was substantially high in both the breeds. The test for Hardy–Weinberg equilibrium showed significant deviations at most of loci. The mean multilocus FST value (0.237) suggested significant population differentiation. This was also supported by AMOVA (Analysis of Molecular Variance), Principal component analysis and Bayesian cluster analysis. The genetic distinctness of these camel breeds as revealed by microsatellite analysis may have significant impact on issues concerning conservation and biodiversity. & 2015 Elsevier B.V. All rights reserved.
Keywords: Camel Genetic variability Microsatellite markers Population differentiation
1. Introduction Camels belong to the family Camiladae. Genus Camelus consists of Camelus dromedarius, dromedary (one hump) camel and Camelus bactrianus, Bactrian (two humped) camel. India inhabits mainly dromedary camels and its distribution is restricted to the western part of the country particularly in Rajasthan and Gujarat states with eight recognized camel breeds (www.nbagr.res.in/regcamel.html). Camels in India are mainly reared by landless nomadic or semi-nomadic tribes as an ancestral business. India has experienced recently a sharp decline in camel population. According to recent Livestock Census, the population of Kachchhi camel has declined from 10,477 in 2003 to 8575 in 2007 and that of Kharai just 2173 in 2007 thus, registering approximately 20 percent decline in Kachchhi camel population in last four years. Hence their conservation assumes national priority. n Corresponding author at: Department of Animal Genetics and Breeding, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand388001, Gujarat, India. Fax: þ 91 2692 261486. E-mail address: [email protected] (D.N. Rank).
http://dx.doi.org/10.1016/j.livsci.2015.10.007 1871-1413/& 2015 Elsevier B.V. All rights reserved.
Characterization at morphological and genetic level is the first step towards formulating breeding policies and prioritizing the breeds for conservation in an effective and meaningful way. Recent studies have established the usefulness of microsatellite loci as genetic tools for the study of dromedary and Bactrian camelids (Mburu et al., 2003). Gujarat possesses two breeds, Kachchhi and Kharai (a recently recognized breed). Kachchhi breed inhabit Kachchh and Banaskantha, dry and semi-arid districts of north Gujarat. Kharai (meaning saline adapted) mainly thrive on mangroves and marine vegetation. They are restricted to coastal areas of Kachchh district (Kharai K). A small population of Kharai camel was translocated in the past (300–400 years before) at Aliabet (an erstwhile Narmada river delta) in Bharuch district (Kharai A). Though both, Kachchhi and Kharai camels share many characters they are not only morphologically distinct but differ with respect to milk and wool quality (Anonymous, 2011). The present study was undertaken to evaluate the genetic diversity and to estimate its relationship among camel population using 27 microsatellite markers.
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A.C. Patel et al. / Livestock Science 181 (2015) 85–88
2. Materials and methods Blood samples were collected from the Jugular vein in 9 ml capacity vacutainer (EDTA, K3) from non-related animals of Kachchhi breed (n ¼75), Kharai (K) (n ¼64) and Kharai (A) populations (n ¼ 54). Not more than five samples were collected per herd (22 herds per breed were sampled). The collected blood samples were brought to the laboratory on ice. Genomic DNA was extracted manually (John et al., 1991) or by using Kit (HiPurATM Mammalian Genomic DNA Purification Spin Kit, HiMedia). PCR amplification of DNA samples, using 27 microsatellite markers was carried out in seven multiplex panels. Samples were genotyped by capillary electrophoresis on automated DNA sequencer (ABI PRISMs 310 Genetic Analyzer) using GSLiz500 as size standard. Further analysis of the samples was done using Gene mapper 4.0 version. 2.1. Statistical analysis Allele frequency, observed and effective number of alleles, observed and expected heterozygosity estimates and Pair wise Nei's genetic distance, mean number of migrants (Nm) were computed using GenAlEx6.41 software. F-statistics (FIS, FIT, and FST) and Allelic Richness (AR) were computed using FSTAT v 2.9.3.2 program (Goudet, 2002) with Jackknifing procedure applied over loci in deriving their significance levels. AR was calculated using a rarefied sample size of 40 diploid individuals per breed. Polymorphism Information Content (PIC) was measured using SAS software. A Fisher's exact test was performed to determine possible deviations from the HWE and genotype linkage disequilibrium for all pairs of loci using GENEPOP v 4.1.4 (Rousset, 2008). The same software was also used for finding the frequency of private alleles. Pair wise distance matrix based on the
proportion of shared alleles with individuals as taxonomic unit was utilized to construct neighbor-joining tree using PHYLIP version 3.5 (Felsenstein, 1993). Pair-wise chord distances between individual animals were utilized to perform principal component analysis using SPSS version 13.0.
3. Results 3.1. Microsatellite polymorphism and alleles Our observation on 193 samples showed that except two loci (YWLL40 and YWL08) all other loci were polymorphic in nature. The number of alleles per polymorphic locus ranged from 2 to 15 in Kachchhi breed, 2 to 13 in Kharai population (K) and 2 to 10 in Kharai population (A). Out of 27 markers, 14 markers in Kachchhi, 11 markers in Kharai (K) and 5 markers in Kharai (A) showed high polymorphism (having more than 4 observed alleles). In the pooled population (all population together), the number of alleles in polymorphic markers ranged from 2 (YWLL29, YWLL36, LCA56) to 19 (CVRL01). AR is a major decisive factor to measure genetic diversity, and this parameter is of key relevance especially in conservation programs (Foulley and Ollivier, 2006). AR over pooled population per locus was measured at between 2 (LCA56, YWLL29 and YWLL36) and 13.94 (CVRL01) and 5.795 across all loci. A Mean frequency of 0.0846 was obtained for private alleles. 3.2. Diversity estimation Table 1 depicts various genetic parameters estimated for the three populations based on 25 polymorphic markers. The mean observed heterozygosity (Ho) for Kachchhi (0.446 70.039), Kharai (K) (0.272 70.040) and Kharai (A) (0.423 70.044) where less than
Table 1 Genetic diversity indices across 25 microsatellite markers in camel breeds. Breed
Kachchhi
Kharai-K
Kharai-A
Locus
Na
Ne
Ho
He
PIC
Na
Ne
Ho
He
PIC
Na
Ne
Ho
He
PIC
VOPL03 LCA66 LCA66 YWLL44 VOPL08 VOPL32 YWLL59 YWLL38 VOPL67 LCA59 LCA56 YWLL29 YWLL36 VOPL10 LCA33 CMS13 CMS121 LCA90 CMS50 LCA18 CMS16 CVRL04 CVRL07 CVRL05 CVRL01 Mean SE
7 7 5 4 4 2 2 6 5 3 2 2 2 8 3 8 8 7 9 6 4 3 9 7 15 5.185 0.618
1.424 4.233 3.954 1.977 1.538 1.785 1.676 2.910 2.127 1.764 1.540 1.609 1.301 3.806 2.166 4.193 3.513 4.558 7.080 3.754 1.987 2.851 4.103 1.970 6.271 2.818 0.303
0.713 0.560 0.760 0.440 0.360 0.467 0.453 0.600 0.107 0.427 0.293 0.400 0.240 0.507 0.440 0.693 0.622 0.693 0.627 0.587 0.459 0.676 0.400 0.465 0.597 0.446 0.039
0.298 0.764 0.747 0.494 0.350 0.440 0.403 0.656 0.530 0.433 0.351 0.378 0.231 0.737 0.538 0.762 0.715 0.781 0.859 0.734 0.497 0.649 0.756 0.492 0.841 0.535 0.045
0.2874 0.7263 0.7010 0.4149 0.3113 0.3432 0.3219 0.5885 0.4272 0.3501 0.2891 0.3068 0.2044 0.6978 0.4677 0.7293 0.6861 0.7472 0.8429 0.6935 0.3977 0.5761 0.7186 0.4620 0.8228 NA NA
3 4 5 3 3 5 2 6 1 2 2 2 1 7 3 6 8 5 8 2 2 6 13 4 9 4.222 0.566
1.598 2.512 3.021 1.530 1.208 2.370 1.806 2.264 1.000 1.580 1.753 1.882 1.000 3.463 1.768 4.229 3.789 3.101 4.288 1.906 1.117 2.842 6.344 2.419 3.269 2.373 0.245
0.391 0.000 0.469 0.422 0.188 0.000 0.422 0.359 0.000 0.422 0.344 0.000 0.000 0.267 0.234 0.571 0.641 0.136 0.317 0.365 0.079 0.453 0.180 0.541 0.548 0.272 0.040
0.374 0.602 0.669 0.346 0.172 0.578 0.446 0.558 0.000 0.367 0.430 0.469 0.000 0.711 0.434 0.764 0.736 0.678 0.767 0.475 0.105 0.648 0.842 0.587 0.694 0.461 0.051
0.3149 0.5488 0.6090 0.3075 0.1619 0.4941 0.3466 0.5111 0.000 0.2997 0.3374 0.3589 0.0000 0.6616 0.3466 0.7263 0.6954 0.6144 0.7317 0.3623 0.0994 0.6028 0.8246 0.5449 0.6493 NA NA
3 4 4 3 3 2 2 4 3 2 2 2 2 4 4 4 4 4 10 5 2 4 5 5 8 3.593 0.378
2.016 1.955 3.323 1.776 1.366 1.946 1.647 2.918 1.699 1.480 1.670 1.857 1.624 3.500 3.772 2.078 1.852 2.721 5.982 1.457 1.097 2.855 3.954 1.771 4.148 2.313 0.224
0.444 0.500 0.759 0.389 0.315 0.463 0.389 0.642 0.100 0.333 0.222 0.426 0.259 0.615 0.519 0.444 0.444 0.667 0.926 0.296 0.560 0.593 0.389 0.444 0.778 0.423 0.044
0.504 0.489 0.699 0.437 0.268 0.486 0.393 0.657 0.411 0.324 0.401 0.461 0.384 0.714 0.735 0.519 0.460 0.633 0.833 0.314 0.088 0.650 0.747 0.435 0.759 0.474 0.043
0.3860 0.4381 0.6426 0.3933 0.2367 0.3680 0.3157 0.5919 0.3448 0.2718 0.3207 0.3550 0.3103 0.6637 0.6856 0.4580 0.4274 0.5584 0.8121 0.2838 0.0844 0.5798 0.7083 0.4035 0.7199 NA NA
Na-Observed number of alleles; Ne-Effective number of alleles; Ho-Observed heterozygosity; He-Expected heterozygosity; PIC-Polymorphism Information Content; NA-Not Applicable
A.C. Patel et al. / Livestock Science 181 (2015) 85–88
the mean expected heterozygosity (He). The mean effective numbers of alleles, Ne (2.8187 0.303, 2.373 70.245 and 2.3137 0.224), were less than the mean observed numbers of alleles, MNA (5.1857 0.618, 4.222 70.566 and 3.5937 0.378) for Kachchhi, Kharai (K) and Kharai (A) respectively. The MNA and Ne for the pooled population were 6.5197 0.846 and 3.412 70.382 respectively. The lower mean number of alleles and lower heterozygosity estimates suggested existence of relatively lower genetic variability in dromedary camels. 3.3. F-statistics The mean estimates of F statistics were FIS 0.233, FIT 0.414, and FST 0.237. The within population inbreeding estimates (FIS) (0.173 for Kachchhi, 0.417 for Kharai (K) and 0.118 for Kharai (A)), which represents the nonrandom union of gametes and deviation from Hardy–Weinberg equilibrium (HWE), revealed that 12 loci in Kachchhi, 10 loci in Kharai (K) and 18 loci in Kharai (A) were not in HWE (P o0.01). Departure from HWE was also confirmed by significant difference between OH and EH as tested by Fisher's exact test. 3.4. Genetic distances and clustering Pair wise Nei's genetic distance between Kachchhi and Kharai (K) was 0.328, between Kachchhi and Kharai (A) was 0.270 and that of between Kharai (K) and Kharai (A) it was 0.571. All the three populations were shown to be distinct. This assembly pattern was further supported by PCA analysis which indicated towards three distinct clusters (Fig. 1). Breed differentiation via Bayesian cluster analysis also revealed a similar type of genetic structure (Supplementary file).
4. Discussion The overall and average numbers of alleles were not very high, reflecting low genetic variability in these camel breeds. The effective numbers of alleles are significantly less than the number of alleles observed, revealing large number of alleles at low frequency. Such low values have also been reported in other Dromedarian camel breeds of India (Vijh et al., 2007) and in four Camelus dromedarius populations of Saudi Arabia (Mahmoud et al., 2012). All previous reports on Indian camel (Gautam et al., 2004; Mehta et al., 2007; Vijh et al., 2007) also show such lower number of alleles than in other livestock breeds e.g. cattle (MNA ¼5.6 to 6.52) (Maudet et al., 2002), buffalo (MNA ¼5.6 to 9.1) (Van Hooft et al., 2000), sheep (MNA ¼7.5 to 9.9) (Arranz et al., 1998), goat
Fig. 1. Principal Coordinates Analysis (PCA) of the breeds. Kachchhi breed Kharai K population (Kharai at Kachchh region) Kharai A population (Kharai at Aliabet region).
87
(MNA ¼2 to 19) (Saitbekova et al., 1999). We strongly believe that there is lower genetic variability in Kachchhi and Kharai camel breeds compared to other livestock species which is also supported by low Ho and lesser Na and Ne. The breed we analyzed revealed low Ho and He. The values of observed heterozygosity were lower than the expected heterozygosity values in both the breeds of camel. In the population undergoing genetic bottleneck, the rare alleles are lost first leading to excess of observed heterozygosity (Allendorf, 1986). Similar trend of higher expected than observed heterozygosity was also reported in other camel breeds (Gautam et al., 2004; Vijh et al., 2007; Wei. et al., 2009; Banerjee et al., 2012). Although AR per locus varied over a wide range (2 to 13.94), the mean AR over all loci was substantially low (5.795 only), particularly Aliabet Kharai population showed very low AR of 3.69 only. Though these populations show moderate level of differentiation as reflected by their mean FST value as well by breed differentiation via Bayesian Cluster Analysis, the mean number of private alleles were very low, only 0.0846 per population. Number of private alleles for Kachchhi, Kharai (K) and Kharai (A) populations were 28 (1.12 per locus), 23 (0.92) and 7 (0.28) respectively. The values of PIC are generally lower than heterozygosity values for the corresponding markers. Based on PIC values, nearly 32% of the markers were observed to be highly informative (PIC4 0.60) (Kawka et al., 2012). The PIC values reported in New World Camelidae are relatively higher due to more number of alleles at these loci (Penedo et al., 1999). The results of F statistics in the camel populations showed that majority of the loci deviated from HWE (P o0.01). This might be due to inbreeding in population, since as the camel population is declining, available breeding males become limited. Although the traditional breeders try to avoid mating among relatives by changing the breeding males every 3–4 year from outside herds (Anonymous, 2011), inbreeding cannot be avoided due to small population size. Here we obtained comparatively high positive FIS value (0.233), which further indicates deficit of heterozygosity and possibility of inbreeding within population. Since enough care was taken for sampling, and the microsatellite markers used are neutral and not known to be linked with any trait under selection, most probable reason for H–W disequilibrium is inbreeding within populations. Large extent of genetic exchange between the breeds was evident from a high number of migrants from one population to another (Nm ¼0.805), mainly due to sharing of a common breeding tract. This assumption is substantiated by breed wise FIS values significantly different from zero, 0.173 for Kachchhi, 0.417 for Kharai (K) and 0.118 for Kharai (A). FIS values in similar range (0.12–0.21) were earlier reported in Bikaneri, Jaisalmeri, Kachchhi, Mewari camel breeds (Vijh et al., 2007). The mean FST which is the relative measure of gene differentiation among breeds was 0.237 suggesting moderate breed differentiations among the breeds. There are limited studies available on genetic differentiation among camel breeds. The present study on microsatellite investigation of three rapidly declining populations suggested the genetic differentiation. Earlier, moderate genetic differentiation in Indian camel breeds (Vijh et al., 2007) was revealed. Interestingly another study covering wider geographic region of African, Asian and Arabian Peninsula using microsatellite markers did not support the present breed differentiation (Mburu et al., 2003). Principle Component Analysis (PCA) revealed three distinct clusters indicating their distinctness with respect to each other. As per the local literature, some families of traditional camel breeders migrated some 300–400 years back with their animals from coastal region of Kachchh to mangrove rich Aliabet, a Narmada delta south to Bay of Cambay. However, they still maintain cultural and social relationship with their Kachchh counterpart and exchange animals too. Although the camel population was much
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higher (around 2000) in the past Aliabet as revealed on local inquiry, currently the Aliabet population enumerates around 800 animals only. Probably small and partially isolated population contributes to its genetic differentiation from other camel populations of Gujarat (Kharai K and Kachchhi in order of magnitude). Estimation of genetic similarity within and between breeds and genetic distance among different breeds of livestock is an important application of the DNA based genetic markers. These camel breeds are the source of livelihood for nomadic tribes. Microsatellite profiling of rapidly declining population of camel restricted in small geographical area suggests genetic distinctness of these breeds which earlier were believed to be a single breed. This may help Government in taking decision on their conservation considering them as separate breeds rather than a metapopulation. Establishment of separate breeding farms as source of purebred males to the breeders could be a good in situ conservation strategy.
Conflict of interest statement None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.
Acknowledgment We are thankful to Sahajeevan Trust, Bhuj,; Unt Uchherak Maldhari Sanghthan, Bhuj and to the animal owners for providing camel blood samples.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.livsci.2015.10.007.
References www.nbagr.res.in/regcamel.html. Allendorf, F., 1986. Genetic drift and the loss of alleles versus heterozygosity. Zoo Biol. 5, 181–190. Anonymous, 2011. Sahjeevan Trust Progress Report April-June. In, City. Arranz, J.J., Bayon, Y., San Primitivo, F., 1998. Genetic relationships among Spanish sheep using microsatellites. Anim. Genet., 435–440. Banerjee, P., Joshi, J., Sharma, U., Vijh, R.K., 2012. Population differentiation in Dromedarian camel: a comparative study of camel inhabiting extremes of geographical distribution. Int. J. Anim. Vet. Adv. 4, 84–92. Felsenstein, J., 1993. Department of Genetics. University of Washington, Seattle, PHYLIP: Phylogeny Inference Package. Version 3.5p. Foulley, J.L., Ollivier, L., 2006. Estimating allelic richness and its diversity. Livest. Sci., 150–158. Gautam, L., Mehta, S.C., Gahlot, R., Gautam, K., 2004. Genetic chracterisation Of Jaisalmeri camel using microsatellite markers. Indian J. Biotechnol. 3, 457–459. Goudet, J., 2002. FSTAT (version 2.9.3.2): A program to estimate and test gene diversities and fixation indices. F-statistics. J. Hered., 485–486. John, A.J., Weitzner, G., Rozen, R., Scriver, C.R., 1991. A rapid procedure for extracting genomic DNA from leukocytes. Nucleic Acids Res. 19, 408. Kawka, M., Parada, R., Jaszczak, K., Horbanczuk, J.O., 2012. The use of microsatellite polymorphism in genetic mapping of the ostrich (Struthio camelus). Mol. Biol. Rep. 39 (3), 3369–3374. Mahmoud, A., Alshaikh, M., Aljumaah, R., O., M., 2012. Genetic variability of camel (Camelus dromedarius) populations in Saudi Arabia based on microsatellites analysis. Afr. J. Biotechnol. 11, 11173–11180. Maudet, C., Luikart, G., Taberlet, P., 2002. Genetic diversity and assignment tests among seven French cattle breeds based on microsatellite DNA analysis. J. Anim. Sci., 942–950. Mburu, D.N., Ochieng, J.W., Kuria, S.G., Jianlin, H., Kaufmann, B., Rege, J.E.O., Hanotte, O., 2003. Genetic diversity and relationships of indigenous Kenyan camel (Camelus dromedarius) populations: implications for their classification. Anim. Genet., 26–32. Mehta, S.C., Goyal, A., Sahani, M.S., 2007. Microsatellite markers for genetic characterization of Kachchhi camel. Indian J. Biotechnol. 6, 336–339. Penedo, M.C., Caetano, A.R., Cordova, K., 1999. Eight microsatellite markers for South American camelids. Anim. Genet. 30, 166–167. Rousset, F., 2008. Genepop'007: a complete reimplementation of the Genepop software for Windows & Linux. Mol. Ecol. Resour. 8, 103–106. Saitbekova, N., Gaillard, C., Obexer-Ruff, G., Dolf, G., 1999. Genetic diversity in Swiss goat breeds based on microsatellite analysis. Anim. Genet. 30, 36–41. Van Hooft, W.F., Groen, A.F., Prins, H.H.T., 2000. Microsatellite analysis of genetic diversity in African buffalo (Syncerus caffer) populations throughout Africa. Mol. Ecol. 9 (12), 2017–2025. Vijh, R.K., Tantia, M.S., Mishra, B., Kumar, S.T., 2007. Genetic diversity and differentiation of dromedarian camel of India. Anim. Biotechnol. 18, 81–90. Wei., hong., G., Jing, W., JunXia, H., LuYong, C., Ji, R., 2009. Analysis on the genetic diversity and evolution in the Camelus bactrianus using SSR markers. J. Shanghai Jiaotong Univ.- Agric. Sci. 27, 89–95.
Contents lists available at ScienceDirect
Livestock Science journal homepage: www.elsevier.com/locate/livsci
Short communication
Molecular characterization of camel breeds of Gujarat using microsatellite markers A.C. Patel a, T.K. Jisha a, Disha Upadhyay a, Rakesh Parikh a, Maulik Upadhyay a, Riddhi Thaker a, S. Das b, J.V. Solanki a, D.N. Rank a,b,n a b
Department of Animal Genetics and Breeding, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand 388001, Gujarat, India Sahjeevan Trust, Vijay Nagar, Bhuj 370001, Gujarat, India
art ic l e i nf o
a b s t r a c t
Article history: Received 2 May 2014 Received in revised form 3 October 2015 Accepted 6 October 2015
Camels are the economic backbone of many nomadic tribes of the world including India. Camel population in India is restricted to western part of the country and is represented by eight breeds. The western most state of the country, Gujarat, possesses two camel breeds, Kachchhi and Kharai inhabiting in the same area. Populations of both are facing severe decline which calls for their immediate conservation. In the present study we examined genetic variability and structure in Kachchhi and two populations of Kharai (n ¼193) using 27 microsatellite markers. A total of 138, 112 and 95 alleles were observed in Kachchhi, Kharai (K) and Kharai (A) respectively with the mean effective number of alleles per locus 2.818 70.303; 2.373 7 0.245 and 2.313 7 0.224. The mean observed heterozygosity was 0.4467 0.039 for Kachchhi, 0.272 7 0.040 for Kharai (K) and 0.423 7 0.044 for Kharai (A), which was lower than expected heterozygosity 0.53570.045, 0.461 7 0.051 and 0.474 7 0.043 respectively. The average inbreeding coefficient (FIS) 0.2337 0.037 was substantially high in both the breeds. The test for Hardy–Weinberg equilibrium showed significant deviations at most of loci. The mean multilocus FST value (0.237) suggested significant population differentiation. This was also supported by AMOVA (Analysis of Molecular Variance), Principal component analysis and Bayesian cluster analysis. The genetic distinctness of these camel breeds as revealed by microsatellite analysis may have significant impact on issues concerning conservation and biodiversity. & 2015 Elsevier B.V. All rights reserved.
Keywords: Camel Genetic variability Microsatellite markers Population differentiation
1. Introduction Camels belong to the family Camiladae. Genus Camelus consists of Camelus dromedarius, dromedary (one hump) camel and Camelus bactrianus, Bactrian (two humped) camel. India inhabits mainly dromedary camels and its distribution is restricted to the western part of the country particularly in Rajasthan and Gujarat states with eight recognized camel breeds (www.nbagr.res.in/regcamel.html). Camels in India are mainly reared by landless nomadic or semi-nomadic tribes as an ancestral business. India has experienced recently a sharp decline in camel population. According to recent Livestock Census, the population of Kachchhi camel has declined from 10,477 in 2003 to 8575 in 2007 and that of Kharai just 2173 in 2007 thus, registering approximately 20 percent decline in Kachchhi camel population in last four years. Hence their conservation assumes national priority. n Corresponding author at: Department of Animal Genetics and Breeding, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand388001, Gujarat, India. Fax: þ 91 2692 261486. E-mail address: [email protected] (D.N. Rank).
http://dx.doi.org/10.1016/j.livsci.2015.10.007 1871-1413/& 2015 Elsevier B.V. All rights reserved.
Characterization at morphological and genetic level is the first step towards formulating breeding policies and prioritizing the breeds for conservation in an effective and meaningful way. Recent studies have established the usefulness of microsatellite loci as genetic tools for the study of dromedary and Bactrian camelids (Mburu et al., 2003). Gujarat possesses two breeds, Kachchhi and Kharai (a recently recognized breed). Kachchhi breed inhabit Kachchh and Banaskantha, dry and semi-arid districts of north Gujarat. Kharai (meaning saline adapted) mainly thrive on mangroves and marine vegetation. They are restricted to coastal areas of Kachchh district (Kharai K). A small population of Kharai camel was translocated in the past (300–400 years before) at Aliabet (an erstwhile Narmada river delta) in Bharuch district (Kharai A). Though both, Kachchhi and Kharai camels share many characters they are not only morphologically distinct but differ with respect to milk and wool quality (Anonymous, 2011). The present study was undertaken to evaluate the genetic diversity and to estimate its relationship among camel population using 27 microsatellite markers.
86
A.C. Patel et al. / Livestock Science 181 (2015) 85–88
2. Materials and methods Blood samples were collected from the Jugular vein in 9 ml capacity vacutainer (EDTA, K3) from non-related animals of Kachchhi breed (n ¼75), Kharai (K) (n ¼64) and Kharai (A) populations (n ¼ 54). Not more than five samples were collected per herd (22 herds per breed were sampled). The collected blood samples were brought to the laboratory on ice. Genomic DNA was extracted manually (John et al., 1991) or by using Kit (HiPurATM Mammalian Genomic DNA Purification Spin Kit, HiMedia). PCR amplification of DNA samples, using 27 microsatellite markers was carried out in seven multiplex panels. Samples were genotyped by capillary electrophoresis on automated DNA sequencer (ABI PRISMs 310 Genetic Analyzer) using GSLiz500 as size standard. Further analysis of the samples was done using Gene mapper 4.0 version. 2.1. Statistical analysis Allele frequency, observed and effective number of alleles, observed and expected heterozygosity estimates and Pair wise Nei's genetic distance, mean number of migrants (Nm) were computed using GenAlEx6.41 software. F-statistics (FIS, FIT, and FST) and Allelic Richness (AR) were computed using FSTAT v 2.9.3.2 program (Goudet, 2002) with Jackknifing procedure applied over loci in deriving their significance levels. AR was calculated using a rarefied sample size of 40 diploid individuals per breed. Polymorphism Information Content (PIC) was measured using SAS software. A Fisher's exact test was performed to determine possible deviations from the HWE and genotype linkage disequilibrium for all pairs of loci using GENEPOP v 4.1.4 (Rousset, 2008). The same software was also used for finding the frequency of private alleles. Pair wise distance matrix based on the
proportion of shared alleles with individuals as taxonomic unit was utilized to construct neighbor-joining tree using PHYLIP version 3.5 (Felsenstein, 1993). Pair-wise chord distances between individual animals were utilized to perform principal component analysis using SPSS version 13.0.
3. Results 3.1. Microsatellite polymorphism and alleles Our observation on 193 samples showed that except two loci (YWLL40 and YWL08) all other loci were polymorphic in nature. The number of alleles per polymorphic locus ranged from 2 to 15 in Kachchhi breed, 2 to 13 in Kharai population (K) and 2 to 10 in Kharai population (A). Out of 27 markers, 14 markers in Kachchhi, 11 markers in Kharai (K) and 5 markers in Kharai (A) showed high polymorphism (having more than 4 observed alleles). In the pooled population (all population together), the number of alleles in polymorphic markers ranged from 2 (YWLL29, YWLL36, LCA56) to 19 (CVRL01). AR is a major decisive factor to measure genetic diversity, and this parameter is of key relevance especially in conservation programs (Foulley and Ollivier, 2006). AR over pooled population per locus was measured at between 2 (LCA56, YWLL29 and YWLL36) and 13.94 (CVRL01) and 5.795 across all loci. A Mean frequency of 0.0846 was obtained for private alleles. 3.2. Diversity estimation Table 1 depicts various genetic parameters estimated for the three populations based on 25 polymorphic markers. The mean observed heterozygosity (Ho) for Kachchhi (0.446 70.039), Kharai (K) (0.272 70.040) and Kharai (A) (0.423 70.044) where less than
Table 1 Genetic diversity indices across 25 microsatellite markers in camel breeds. Breed
Kachchhi
Kharai-K
Kharai-A
Locus
Na
Ne
Ho
He
PIC
Na
Ne
Ho
He
PIC
Na
Ne
Ho
He
PIC
VOPL03 LCA66 LCA66 YWLL44 VOPL08 VOPL32 YWLL59 YWLL38 VOPL67 LCA59 LCA56 YWLL29 YWLL36 VOPL10 LCA33 CMS13 CMS121 LCA90 CMS50 LCA18 CMS16 CVRL04 CVRL07 CVRL05 CVRL01 Mean SE
7 7 5 4 4 2 2 6 5 3 2 2 2 8 3 8 8 7 9 6 4 3 9 7 15 5.185 0.618
1.424 4.233 3.954 1.977 1.538 1.785 1.676 2.910 2.127 1.764 1.540 1.609 1.301 3.806 2.166 4.193 3.513 4.558 7.080 3.754 1.987 2.851 4.103 1.970 6.271 2.818 0.303
0.713 0.560 0.760 0.440 0.360 0.467 0.453 0.600 0.107 0.427 0.293 0.400 0.240 0.507 0.440 0.693 0.622 0.693 0.627 0.587 0.459 0.676 0.400 0.465 0.597 0.446 0.039
0.298 0.764 0.747 0.494 0.350 0.440 0.403 0.656 0.530 0.433 0.351 0.378 0.231 0.737 0.538 0.762 0.715 0.781 0.859 0.734 0.497 0.649 0.756 0.492 0.841 0.535 0.045
0.2874 0.7263 0.7010 0.4149 0.3113 0.3432 0.3219 0.5885 0.4272 0.3501 0.2891 0.3068 0.2044 0.6978 0.4677 0.7293 0.6861 0.7472 0.8429 0.6935 0.3977 0.5761 0.7186 0.4620 0.8228 NA NA
3 4 5 3 3 5 2 6 1 2 2 2 1 7 3 6 8 5 8 2 2 6 13 4 9 4.222 0.566
1.598 2.512 3.021 1.530 1.208 2.370 1.806 2.264 1.000 1.580 1.753 1.882 1.000 3.463 1.768 4.229 3.789 3.101 4.288 1.906 1.117 2.842 6.344 2.419 3.269 2.373 0.245
0.391 0.000 0.469 0.422 0.188 0.000 0.422 0.359 0.000 0.422 0.344 0.000 0.000 0.267 0.234 0.571 0.641 0.136 0.317 0.365 0.079 0.453 0.180 0.541 0.548 0.272 0.040
0.374 0.602 0.669 0.346 0.172 0.578 0.446 0.558 0.000 0.367 0.430 0.469 0.000 0.711 0.434 0.764 0.736 0.678 0.767 0.475 0.105 0.648 0.842 0.587 0.694 0.461 0.051
0.3149 0.5488 0.6090 0.3075 0.1619 0.4941 0.3466 0.5111 0.000 0.2997 0.3374 0.3589 0.0000 0.6616 0.3466 0.7263 0.6954 0.6144 0.7317 0.3623 0.0994 0.6028 0.8246 0.5449 0.6493 NA NA
3 4 4 3 3 2 2 4 3 2 2 2 2 4 4 4 4 4 10 5 2 4 5 5 8 3.593 0.378
2.016 1.955 3.323 1.776 1.366 1.946 1.647 2.918 1.699 1.480 1.670 1.857 1.624 3.500 3.772 2.078 1.852 2.721 5.982 1.457 1.097 2.855 3.954 1.771 4.148 2.313 0.224
0.444 0.500 0.759 0.389 0.315 0.463 0.389 0.642 0.100 0.333 0.222 0.426 0.259 0.615 0.519 0.444 0.444 0.667 0.926 0.296 0.560 0.593 0.389 0.444 0.778 0.423 0.044
0.504 0.489 0.699 0.437 0.268 0.486 0.393 0.657 0.411 0.324 0.401 0.461 0.384 0.714 0.735 0.519 0.460 0.633 0.833 0.314 0.088 0.650 0.747 0.435 0.759 0.474 0.043
0.3860 0.4381 0.6426 0.3933 0.2367 0.3680 0.3157 0.5919 0.3448 0.2718 0.3207 0.3550 0.3103 0.6637 0.6856 0.4580 0.4274 0.5584 0.8121 0.2838 0.0844 0.5798 0.7083 0.4035 0.7199 NA NA
Na-Observed number of alleles; Ne-Effective number of alleles; Ho-Observed heterozygosity; He-Expected heterozygosity; PIC-Polymorphism Information Content; NA-Not Applicable
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the mean expected heterozygosity (He). The mean effective numbers of alleles, Ne (2.8187 0.303, 2.373 70.245 and 2.3137 0.224), were less than the mean observed numbers of alleles, MNA (5.1857 0.618, 4.222 70.566 and 3.5937 0.378) for Kachchhi, Kharai (K) and Kharai (A) respectively. The MNA and Ne for the pooled population were 6.5197 0.846 and 3.412 70.382 respectively. The lower mean number of alleles and lower heterozygosity estimates suggested existence of relatively lower genetic variability in dromedary camels. 3.3. F-statistics The mean estimates of F statistics were FIS 0.233, FIT 0.414, and FST 0.237. The within population inbreeding estimates (FIS) (0.173 for Kachchhi, 0.417 for Kharai (K) and 0.118 for Kharai (A)), which represents the nonrandom union of gametes and deviation from Hardy–Weinberg equilibrium (HWE), revealed that 12 loci in Kachchhi, 10 loci in Kharai (K) and 18 loci in Kharai (A) were not in HWE (P o0.01). Departure from HWE was also confirmed by significant difference between OH and EH as tested by Fisher's exact test. 3.4. Genetic distances and clustering Pair wise Nei's genetic distance between Kachchhi and Kharai (K) was 0.328, between Kachchhi and Kharai (A) was 0.270 and that of between Kharai (K) and Kharai (A) it was 0.571. All the three populations were shown to be distinct. This assembly pattern was further supported by PCA analysis which indicated towards three distinct clusters (Fig. 1). Breed differentiation via Bayesian cluster analysis also revealed a similar type of genetic structure (Supplementary file).
4. Discussion The overall and average numbers of alleles were not very high, reflecting low genetic variability in these camel breeds. The effective numbers of alleles are significantly less than the number of alleles observed, revealing large number of alleles at low frequency. Such low values have also been reported in other Dromedarian camel breeds of India (Vijh et al., 2007) and in four Camelus dromedarius populations of Saudi Arabia (Mahmoud et al., 2012). All previous reports on Indian camel (Gautam et al., 2004; Mehta et al., 2007; Vijh et al., 2007) also show such lower number of alleles than in other livestock breeds e.g. cattle (MNA ¼5.6 to 6.52) (Maudet et al., 2002), buffalo (MNA ¼5.6 to 9.1) (Van Hooft et al., 2000), sheep (MNA ¼7.5 to 9.9) (Arranz et al., 1998), goat
Fig. 1. Principal Coordinates Analysis (PCA) of the breeds. Kachchhi breed Kharai K population (Kharai at Kachchh region) Kharai A population (Kharai at Aliabet region).
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(MNA ¼2 to 19) (Saitbekova et al., 1999). We strongly believe that there is lower genetic variability in Kachchhi and Kharai camel breeds compared to other livestock species which is also supported by low Ho and lesser Na and Ne. The breed we analyzed revealed low Ho and He. The values of observed heterozygosity were lower than the expected heterozygosity values in both the breeds of camel. In the population undergoing genetic bottleneck, the rare alleles are lost first leading to excess of observed heterozygosity (Allendorf, 1986). Similar trend of higher expected than observed heterozygosity was also reported in other camel breeds (Gautam et al., 2004; Vijh et al., 2007; Wei. et al., 2009; Banerjee et al., 2012). Although AR per locus varied over a wide range (2 to 13.94), the mean AR over all loci was substantially low (5.795 only), particularly Aliabet Kharai population showed very low AR of 3.69 only. Though these populations show moderate level of differentiation as reflected by their mean FST value as well by breed differentiation via Bayesian Cluster Analysis, the mean number of private alleles were very low, only 0.0846 per population. Number of private alleles for Kachchhi, Kharai (K) and Kharai (A) populations were 28 (1.12 per locus), 23 (0.92) and 7 (0.28) respectively. The values of PIC are generally lower than heterozygosity values for the corresponding markers. Based on PIC values, nearly 32% of the markers were observed to be highly informative (PIC4 0.60) (Kawka et al., 2012). The PIC values reported in New World Camelidae are relatively higher due to more number of alleles at these loci (Penedo et al., 1999). The results of F statistics in the camel populations showed that majority of the loci deviated from HWE (P o0.01). This might be due to inbreeding in population, since as the camel population is declining, available breeding males become limited. Although the traditional breeders try to avoid mating among relatives by changing the breeding males every 3–4 year from outside herds (Anonymous, 2011), inbreeding cannot be avoided due to small population size. Here we obtained comparatively high positive FIS value (0.233), which further indicates deficit of heterozygosity and possibility of inbreeding within population. Since enough care was taken for sampling, and the microsatellite markers used are neutral and not known to be linked with any trait under selection, most probable reason for H–W disequilibrium is inbreeding within populations. Large extent of genetic exchange between the breeds was evident from a high number of migrants from one population to another (Nm ¼0.805), mainly due to sharing of a common breeding tract. This assumption is substantiated by breed wise FIS values significantly different from zero, 0.173 for Kachchhi, 0.417 for Kharai (K) and 0.118 for Kharai (A). FIS values in similar range (0.12–0.21) were earlier reported in Bikaneri, Jaisalmeri, Kachchhi, Mewari camel breeds (Vijh et al., 2007). The mean FST which is the relative measure of gene differentiation among breeds was 0.237 suggesting moderate breed differentiations among the breeds. There are limited studies available on genetic differentiation among camel breeds. The present study on microsatellite investigation of three rapidly declining populations suggested the genetic differentiation. Earlier, moderate genetic differentiation in Indian camel breeds (Vijh et al., 2007) was revealed. Interestingly another study covering wider geographic region of African, Asian and Arabian Peninsula using microsatellite markers did not support the present breed differentiation (Mburu et al., 2003). Principle Component Analysis (PCA) revealed three distinct clusters indicating their distinctness with respect to each other. As per the local literature, some families of traditional camel breeders migrated some 300–400 years back with their animals from coastal region of Kachchh to mangrove rich Aliabet, a Narmada delta south to Bay of Cambay. However, they still maintain cultural and social relationship with their Kachchh counterpart and exchange animals too. Although the camel population was much
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higher (around 2000) in the past Aliabet as revealed on local inquiry, currently the Aliabet population enumerates around 800 animals only. Probably small and partially isolated population contributes to its genetic differentiation from other camel populations of Gujarat (Kharai K and Kachchhi in order of magnitude). Estimation of genetic similarity within and between breeds and genetic distance among different breeds of livestock is an important application of the DNA based genetic markers. These camel breeds are the source of livelihood for nomadic tribes. Microsatellite profiling of rapidly declining population of camel restricted in small geographical area suggests genetic distinctness of these breeds which earlier were believed to be a single breed. This may help Government in taking decision on their conservation considering them as separate breeds rather than a metapopulation. Establishment of separate breeding farms as source of purebred males to the breeders could be a good in situ conservation strategy.
Conflict of interest statement None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.
Acknowledgment We are thankful to Sahajeevan Trust, Bhuj,; Unt Uchherak Maldhari Sanghthan, Bhuj and to the animal owners for providing camel blood samples.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.livsci.2015.10.007.
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