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Microsatellite variation and genetic relationship among Rajasthani sheep: Relevance for conservation

R Arora and S Bhatia

National Bureau of Animal Genetic Resources, P.O.Box 129, Karnal-132001, Haryana, India
rejagati@yahoo.co.in

Abstract

The present study illustrates the genetic diversity and relationships within and among Magra, Marwari and Sonadi sheep breeds of northwestern arid and semi arid zone of India, based on microsatellite markers. Parameters of genetic variation viz., allele diversity, observed heterozygosity, gene diversity and population inbreeding estimate were calculated for the three breeds.

 

The values obtained for allele diversity and gene diversity showed that Magra, Marwari and Sonadi breeds possessed substantial amount of genetic diversity.  The pairwise genetic distance (DS), genetic differentiation (FST), gene differentiation (GST) and gene flow (Nm) between Magra, Marwari and Sonadi revealed Marwari and Sonadi to be the closest and highest degree of genetic differentiation was found between Magra and Sonadi. Further, the contribution of each breed to the total diversity of the breeds was quantified by the Weitzman approach, so that the relative importance of individual breeds becomes clearer. The marginal loss of diversity incurred with removal of Magra breed was the highest (64%) whereas, removal of Marwari breed resulted in lowest loss of diversity (36.3%) from the set. Hence, these approaches enable the establishment of the order of priority for conservation and genetic management for the three breeds under consideration.

Keywords: Diversity, Indian sheep, uniqueness


Introduction

Better sustainability of indigenous breeds in less favoured areas and the output gains from them under zero input system has renewed interest in characterization and evaluation of their genetic potential. Indian sheep breeds (Acharya 1982) thriving in such low input systems have adapted to the adverse conditions over the years. These breeds represent a vast gene pool with untapped potential for our future commercial purposes as well as for maintenance of genetic diversity in this species. Safeguarding the genetic diversity is a major focus of conservationists as it represents the essential evolutionary potential for species to adapt to changing environment.

 

Over the past decade there has been a growing interest in using microsatellite markers for diversity analysis in livestock breeds. Although exotic breeds of sheep have been extensively evaluated for genetic diversity using microsatellite markers (Arranz et al 2001, Saitbekova et al 2001) there are still limited reports on Indian Sheep (Arora and Bhatia 2004, 2006, Mukesh et al 2006, Sodhi et al 2003).

 

In the present study the genetic variability of Magra was compared with two other breeds viz., Marwari and Sonadi from the same northwestern arid and semi-arid region with FAO recommended microsatellite markers for ovines. Detailed description of these breeds has been given elsewhere (Acharya 1982, Geerlings 2004, Arora and Bhatia 2006). The purpose of this study was to investigate and compare the contribution of each breed to the total diversity by application of the Weitzman approach (Thaon d’Arnoldi 1998) so that the relative importance of individual breeds becomes clear.

 

Materials and methods 

Random blood samples were obtained from 48 unrelated animals of Magra and 50 each of Marwari and Sonadi breeds of sheep from their respective breeding areas. Genomic DNA was isolated from blood samples following standard procedures (Sambrook et al 1989).

 

A set of thirteen FAO suggested microsatellites (BM757, BM827, BM1314, BM8125, CSSM47, OarAE129, OarCP34, OarFCB128, OarHH35, OarHH64, OarJMP8, OarVH72, RM4) was analyzed on all the individual samples. Polymerase Chain Reaction (PCR) was carried out in a 25 Ál reaction volume containing 100 ng of genomic DNA, 50 ng of each primer, 1.5 mM MgCl2, 200 ÁM dNTPs, 0.5 U Taq polymerase and 1x buffer. The thermal touchdown profile used for amplification was as follows: 3 cycles of 45 sec at 950C, 1 min at 600 C; 3 cycles of 45 sec at 950 C, 1 min at 570 C; 3 cycles of 45 sec at 950 C, 1 min at 540 C; 3 cycles of 45 sec at 950 C, 1 min at 510 C and 20 cycles of 45 sec at 920 C, 1 min at 480 C. Amplified products were checked on 2% agarose gel, genotyped on 6% denaturing polyacrylamide gel and visualized by silver staining (Bassam et al 1991). Allele size was estimated by running a 10 bp DNA molecular weight marker along with the PCR products. To avoid mistyping of alleles, reference samples representing different alleles at one locus in one population were concurrently run with samples of the other populations.

 

Genotyping was done manually from the silver stained gels. Allele frequencies were determined by direct counting. The genetic variability measures for each breed as allele diversity (mean number of alleles), observed heterozygosity, gene diversity (mean expected heterozygosity) and within breed heterozygosity deficit (Fis), genetic differentiation (Fst), gene differentiation (Gst) were estimated using POPGENE ver 1.31 (Yeh et al 1999), and FSTAT ver 2.9.3.2 (Goudet 1995), software packages respectively. The genetic relationships between the breeds studied were inferred from Nei’s genetic distance (Nei 1972). The distances between breeds were used for tree construction based on unweighted pair group methods with arithmetic averages (UPGMA) using the PHYLIP ver. 3.6 software package (Felsenstein 1993). The relative contribution of each breed to the total genetic diversity of the three breeds put together was evaluated by the Weitzman approach (Weitzman 1992, Thaon d’Arnoldi 1998). The Nei’s DS distances were used to compute the marginal loss of genetic diversity using this approach.

 

Results and discussion 

All the microsatellite loci amplified well and were observed to be polymorphic in the all three breeds (≥2 alleles per locus). Allele frequencies at the 13 amplified loci in Magra, Marwari and Sonadi breeds ranged from 0.014 to 0.847 and are depicted in Figure 1.


 


Figure 1.  Allele frequency histograms at 13 microsatellite loci in Magra, Marwari and Sonadi breeds of sheep


Allelic variation at the studied loci was high. Table 1 summarizes the estimates of various measures of diversity at the breed level.


Table 1.  Genetic variability measures – Observed allele number (Na), observed heterozygosity (Ho), gene diversity (He) and within breed heterozygote deficiency (FIS) across Magra, Marwari and Sonadi breeds.

Locus

Breed

Na

Ho

He

FIS

BM757

Magra

4

0.61

0.62

0.04

 

Marwari

6

0.65

0.79

0.05

 

Sonadi

6

0.82

0.79

-0.01

BM827

Magra

5

0.61

0.72

0.17

 

Marwari

6

0.76

0.82

0.10

 

Sonadi

5

0.90

0.77

-0.13

BM1314

Magra

9

0.88

0.86

-0.01

 

Marwari

9

0.64

0.82

0.25

 

Sonadi

7

0.77

0.76

0.03

BM8125

Magra

6

0.39

0.63

0.40

 

Marwari

6

0.62

0.76

0.21

 

Sonadi

3

0.65

0.53

-0.20

CSSM47

Magra

4

0.32

0.37

0.13

 

Marwari

4

0.35

0.31

-0.12

 

Sonadi

3

0.30

0.27

-0.11

OarAE129

Magra

6

0.77

0.76

0.01

 

Marwari

4

0.33

0.56

0.43

 

Sonadi

3

0.23

0.52

0.58

OarCP34

Magra

4

0.40

0.69

0.43

 

Marwari

5

0.76

0.72

-0.03

 

Sonadi

5

0.86

0.75

-0.12

OarFCB128

Magra

4

0.73

0.73

0.03

 

Marwari

4

0.50

0.69

0.30

 

Sonadi

6

0.48

0.76

0.39

OarHH35

Magra

8

0.95

0.84

-0.10

 

Marwari

9

1.00

0.84

-0.17

 

Sonadi

9

0.96

0.83

-0.13

OarHH64

Magra

5

0.56

0.71

0.22

 

Marwari

8

0.29

0.80

0.65

 

Sonadi

8

0.18

0.82

0.79

OarJMP8

Magra

6

0.61

0.80

0.25

 

Marwari

6

0.55

0.79

0.33

 

Sonadi

7

0.59

0.82

0.30

OarVH72

Magra

6

0.66

0.70

0.06

 

Marwari

5

0.35

0.53

0.36

 

Sonadi

5

0.78

0.71

-0.08

RM4

Magra

5

0.44

0.69

0.38

 

Marwari

8

0.74

0.80

0.10

 

Sonadi

7

0.96

0.82

-0.15

Mean

Magra

5.5

0.61

0.70

0.15

 

Marwari

6.1

0.58

0.71

0.19

 

Sonadi

5.6

0.65

0.70

0.10


In total 106 alleles were detected across 13 microsatellite loci that were typed in these breeds. The number of alleles per locus varied from 3 (BM8125, CSSM47, OarAE129) to 9 (BM1314, OarHH35). The allele diversity (mean number of alleles) was 5.5, 6.1 and 5.6 respectively for Magra, Marwari and Sonadi samples. These relatively high values are comparable with those reported earlier for other sheep breeds (Sodhi et al 2003, 2006, Arora and Bhatia 2004, 2006, Mukesh et al 2006)

 

The observed heterozygosity (Ho) ranged from 0.32 (CSSM47) to 0.95 (OarHH35); 0.29 (OarHH64) to 1.0 (OarHH35) and 0.18 (OarHH64) to 0.96 (OarHH35, RM4) in Magra, Marwari and Sonadi respectively. The expected heterozygosity (He) per locus varied from 0.37 (CSSM47) to 0.86 (BM1314) in Magra, 0.31 (CSSM47) to 0.84 (OarHH35) in Marwari and 0.27 (CSSM47) to 0.83 (OarHH35) in Sonadi sheep. The estimates of observed heterozygosity averages and gene diversity obtained in the present study were in line with those reported in other domestic and exotic sheep breeds investigated earlier (Saitbekova et al 2001, Sodhi et al 2003, Arora and Bhatia 2004).

 

Table 2 presents the different estimators of genetic differentiation /relationship when the breeds were considered in pairs.


Table 2.  Pairwise Genetic distance (Nei’s 1972, DS), genetic differentiation (FST), the coefficient of gene differentiation (GST), and gene flow (Nm) measures for Magra, Marwari and Sonadi

 

Magra-Marwari

Magra-Sonadi

Marwari-Sonadi

DS

0.42

0.47

0.27

FST

0.14

0.15

0.06

GST

0.14

0.15

0.06

Nm

2.4

2.1

3.8


The value of genetic differentiation (FST) among the analyzed breeds ranged from 6.2% for Marwari – Sonadi breed pair to 15% for Magra – Sonadi breed pair. The GST values of breed differentiation were also similar to the FST estimates and ranged from 6.3 % (Marwari-Sonadi pair) to 15.1% (Magra-Sonadi pair). The present values of genetic differentiation are lower than those reported for Swiss sheep breeds (GST =18%, Saitbekova et al 2001).   The FST estimates obtained in this study suggested moderate to low level of genetic differentiation between the three Indian sheep populations of northwestern arid and semi arid region (rates up to 15%, Hartl and Clark 1997) in comparison to high genetic distinctness reported earlier among sheep breeds from two different geographical locations of the country (FST =18. 3% and GST =16.5%, Mukesh et al 2006).

 

The highest degree of genetic differentiation found between Magra - Sonadi was further supported by relatively low level of gene flow between these two breeds (Table 2). Considerably higher level of gene flow was observed between Marwari and Sonadi (Nm=3.8), in comparison to Magra – Marwari (Nm=2.4) or Magra - Sonadi (Nm=2.1) breed pairs. These estimates were corroborated by the Nei’s (1972) genetic distance (DS), another parameter for estimating the genetic relationships among breeds, which revealed a close relationship between Marwari and Sonadi breeds (0.27). The distances between Magra-Sonadi (0.47) and Magra-Marwari (0.42) were comparatively greater which further supported highest degree of divergence between these breed pairs and substantial genetic similarity between Marwari and Sonadi breeds. These values were of the same order as those reported earlier for closely related sheep breeds (Sodhi et al 2006, Mukesh et al 2006).

 

Phylogenetic tree constructed for genetic distances to elucidate relationships among these breeds also showed that Marwari and Sonadi breeds clustered together whereas Magra appeared separated from both the breeds. These results suggested a marked differentiation of Magra from Marwari and Sonadi breeds which appeared to be genetically closer to each other. The grouping of Marwari and Sonadi (Figure 2) in our study is in agreement with behaviour and geographical propinquity of the studied breeds rather than the morphological differences between them, since morphological variations between populations are not taken into account by neutral markers such as microsatellites (Hartl and Clark 1997).


 


Figure 2.  Dendrogram of genetic relationship between Magra, Marwari and Sonadi sheep breeds
using Nei┤s genetic distance (1972), and UPGMA algorithm, from 13 m icrosatellite loci


In the present study moderate variability (FST upto 15%) between sheep breeds is most likely attributed to genetic drift/admixture between the investigated breeds. Since no herd books are established till date and hence even today admixture of neighboring breeds is a common phenomenon.  The high level of genetic exchanges depicted by gene flow between Marwari and Sonadi (Nm=3.8 ) may be ascribed to the highly migratory nature of Marwari breed and cross breeding with Sonadi or other nearby breeds in better years in view of higher productive potential of Sonadi under years with better foliage availability (Geerlings 2004). Further, more and more Sonadi sheep breeders are introducing Marwari rams for breeding their native ewes since Marwari rams are not only hardy but are also superior to Sonadi as regards to wool production traits (Mehta et al 1995).The close relationship elucidated by dendrogram between Marwari (good fleece) and Sonadi (milk, meat, inferior carpet wool) may be explained on the basis of highly migratory nature of Marwari.  Following a transhuman system of management in Marwari it has left greatest impact on other breeds, especially those with very coarse and hairy fleece like Sonadi.  Nevertheless, high genetic similarity between these two sheep breeds may also be ascribed to their occurrence as an admixture in some states of northwestern arid and semi arid agro-ecological region viz., Rajasthan (Udaipur) and Gujarat (North Gujarat).

 

The Weitzman approach quantifies the relative loss of genetic diversity caused by loss of one specific breed and can be regarded as a measure of uniqueness of individual breeds in comparison to the complete set (Thaon d’Arnoldi 1998). The marginal loss of diversity attached to each breed based on Nei’s standard distance is shown in Table 3.


Table 3.  Marginal losses of Weitzman’s diversity in Indian sheep breeds

Name(i)

V(S-i)

dV(i)

dV(i), %

V(S)

0.74

 

 

Magra

0.27

0.47

63.7

Marwari

0.47

0.27

36.3

Sonadi

0.42

0.32

43.5

V(S)- diversity  of  set; V(S-i)- diversities of set without  element  i;  
dV(i)- marginal diversity; dV(i) %-marginal diversity in percent.


In the investigated set, the highest loss of diversity would be incurred with removal of Magra breed (64%) and the lowest from the removal of Marwari breed (36.3%). Hence, this approach enables the establishment of the order of priority for conservation for a set of breeds under consideration.
 

 

Conclusions

 

Acknowledgements 

This work was financially supported by Indian Council of Agricultural Research (ICAR, New Delhi). We are grateful to Director, NBAGR for providing laboratory facilities. The authors thank Dr. Teja Ram (Deputy Director, Animal Husbandry, Nagaur), Dr. Bhatti (Senior Veterinary Officer, Nagaur); Dr. Lakshman Rathore (Deputy Director, Animal Husbandry, Udaipur) for their help in collection of blood samples and Mr. Rakesh Kumar for technical assistance during the study.

 

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Received 21 September 2007; Accepted 13 September 2008; Published 5 December 2008

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