Characterizing Nali and Chokla sheep differentiation with microsatellite markers

Small Ruminant Research 65 (2006) 185–192

Characterizing Nali and Chokla sheep differentiation with microsatellite markers M. Sodhi, M. Mukesh, S. Bhatia ∗ Molecular Genetics Laboratory, Animal Genetics Division, National Bureau of Animal Genetic Resources, Karnal 132001, Haryana, India Received 11 February 2004; received in revised form 17 March 2005; accepted 8 April 2005 Available online 15 August 2005

Abstract Nali and Chokla thin tailed, brown-faced reputed carpet wool sheep breeds from Northwestern arid and semi-arid region of India were characterized for population structure and genetic variability using 25 Food and Agriculture Organization (FAO) proposed ovine specific microsatellite markers. The results revealed high level of genetic variability in each of the two investigated breeds (allele diversity: Nali = 5.520, Chokla = 5.320; gene diversity: Nali = 0.651, Chokla = 0.657). Low level of genetic differentiation between Nali and Chokla sheep was evident from low genetic differentiation estimates (FST = 0.083); high number of shared alleles (70.4%) and Nei’s genetic distances (DS = 0.229 and DA = 0.168). Gene flow (Nm = 3.896) could have played an important role for close genetic similarity in the two investigated sheep of narrow geographical vicinity. Population inbreeding estimates (FIS , Nali = 0.397, Chokla = 0.299; FIT = 0.40) indicated considerable level of inbreeding and high genetic homogeneity in the investigated sheep populations (p < 0.05). Close genetic identity of these sheep breeds based on microsatellite quantification is in agreement to the earlier arrangement of these Rajasthani sheep in a Bikaneri group. The present information is important for meeting the demands of future breeding programmes as well as for formulating effective conservation strategies. © 2005 Elsevier B.V. All rights reserved. Keywords: DNA microsatellite; Characterization; Genetic diversity; Nali and Chokla sheep; Indian native breeds

1. Introduction Recent awareness of the value of genetic resources has encouraged studies on the genetic diversity present in different livestock breeds. Presently, it is not possible to characterize differences between breeds at the ∗ Corresponding author. Tel.: +91 184 2267153x60/2267918; fax: +91 184 2267654. E-mail address: [email protected] (S. Bhatia).

0921-4488/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2005.04.032

level of genes of agricultural interest, therefore, general genetic variability within and among extant breeds has to be taken into account (Moazami-Goudarzi et al., 1997). Several studies have successfully used microsatellite markers, to characterize the general genetic variation among breeds of various livestock species (Buchanan et al., 1994; MacHugh et al., 1998; Saitbekova et al., 1999; Diez-Tascon et al., 2000; Li et al., 2000; Arranz et al., 1998, 2001; Barker et al., 2001; Kim et al., 2002; Mukesh et al., 2004). How-

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ever, very little information is currently available on the genetic quantification of native Indian sheep to assess level of genetic diversity and relationships among these indigenous sheep breeds (Sodhi et al., 2003; Arora and Bhatia, 2004). In addition, detailed knowledge of population structure and genetic relationship among similar appearing indigenous breeds of the same group/region is not well understood. The Government of India uses a geographical break down as regards the distribution of sheep according to fleece type and productivity and sees the type of sheep in Rajasthan as a breed group named Bikaneri with several varieties, such as Bagri, Chokla, Nali, Magra, Pugal, Sonadi Malpura, Marwari and Jaiselmari. Gatenby (1991) also uses the word Bikaneri to describe the types of sheep found in Rajasthan and distinguishes between them as different strains of the Bikaneri breed. FAO (Acharya, 1982; FAO, 2001) categorized these Rajasthani sheep as different breeds. According to similarity in appearance and origin, Mason (1991) arranged these Rajasthani breeds in a Bikaneri group too. Nali and Chokla, Rajasthani sheep of Bikaneri group are important thin tailed, brown-faced carpet wool sheep mainly confined to Northwestern arid and semi-arid agro climatic zone of the country. Nali, a light brown-faced sheep also known as Hisar type, is native of Ganganagar, Churu and Jhunjhunu districts of Rajasthan and southern parts of Hisar and Rohtak districts of Haryana (Fig. 1), and is a good carpet wool type with the densest and heaviest fleeces among the sheep of Rajasthan. Need is being felt to conserve this breed in lights of its involvement in largescale cross-breeding programmes. Chokla also known as Shekhawati/Chapper is found around Sikar and bordering areas of Bikaner, Jaipur and Nagaur districts of Rajasthan (Fig. 1). As an admixture it is also seen in Jhunjhunu and Churu districts. Sheep farmers generally name this breed as “Ratomunda”, means the sheep with dark brown/tan face. Chokla grows the finest wool of all the Rajasthani breeds and is, therefore, also known as Rajasthan Merino. Natural reasons and indiscriminate cross-breeding with exotic sheep (Rambouillet and Russian Merino) for converting Chokla into an apparel wool type has led to the declining numbers of this important ovine genetic material. In this study, we present an analysis based on individual genotypes at 25 microsatellite loci with a view

to obtain a deeper insight into genetic differentiation within and between Nali and Chokla breeds of Indian sheep. The information derived from this analysis will allow us to describe levels of genetic variability and estimate the degree of inbreeding for retaining the maximum amount of ancestral genetic diversity in order to carry out the programme of conservation and genetic improvement in these Bikaneri sheep.

2. Materials and methods Fresh blood samples were randomly collected from 100 genetically unrelated animals comprising 50 animals each of Nali and Chokla sheep from their respective breeding tracts in line with MoDAD recommendations (FAO, 1998). Genomic DNA was extracted using standard phenol chloroform procedure of Sambrook et al. (1989). A panel of 25 ovine specific microsatellite markers recommended in MoDAD project of FAO (1996) for sheep genetic diversity studies was selected (Table 1). Primer pairs for these microsatellite loci were synthesized by M/s Sigma–Aldrich, USA. Polymerase chain reaction (PCR) was carried out in 25 ␮l reaction volume containing ∼60 ng of template DNA, 50 ng of each primer, 200 ␮M of each dNTP (Promega Corporation, Madison, WI, USA), 0.5 units of Taq DNA polymerase (Promega) and 1.5 mM MgCl2 using PTC-100 thermocycler (MJ Research Inc., MA, USA). A common “Touchdown” PCR programme used for amplification of all the 25 markers involved 3 cycles of 45 s at 95 ◦ C, 1 min at 60 ◦ C; 3 cycles of 45 s at 95 ◦ C, 1 min at 57 ◦ C; 3 cycles of 45 s at 95 ◦ C, 1 min at 54 ◦ C; 3 cycles of 45 s at 95 ◦ C, 1 min at 51 ◦ C; 20 cycles of 45 s at 92 ◦ C, 1 min at 48 ◦ C (FAO, 1996). The amplified PCR products were analyzed on ethidium bromide (1.5 ␮g/ml) stained 2% agarose gel and mixed with 5 ␮l of formamide-based dye for denaturation at 95 ◦ C for 5 min. The denatured samples were electrophoresed on standard 6% ureaPAGE denaturing sequencing gel of 30 cm × 38 cm (Biorad Sequi: Gen GT apparatus) at 75 W. The resolved bands of DNA (alleles) were visualized by silver staining procedure of Bassam et al. (1991). Allelic size range (Table 1) was estimated using 10 bp sequencing ladder (Gibco BRL, Life Technologies, TM). Genotype of individual animal of the two

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Fig. 1. Geographical distribution of Nali and Chokla breeds of sheep.

breeds at 25 microsatellite loci was recorded by direct counting. POPGENE software package (Yeh et al., 1999) was used to calculate allele frequencies, observed number of alleles (na ), effective number of alleles (ne ), expected heterozygosity (He ) and estimates of gene flow (Nm ). Polymorphism information content (PIC) value for each locus was calculated as per Botstein et al. (1980). Pair-wise allele sharing was calculated to find out the occurrence of common alleles in the

two indigenous sheep breeds that are geographically close to each other. Population structure of the two breeds was assessed using the F-statistics parameters of F (FIT , total inbreeding estimate), theta (FST , measurement of population differentiation) and f (FIS , within-population inbreeding estimate) proposed by Weir and Cockerham (1984). These were computed using FSTAT Version 2.9.3.2 computer programme (Goudet, 2002). The level of significance (p < 0.05) was determined from permutation tests with the sequen-

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Table 1 Number of alleles (na , observed and ne , effective), expected heterozygosity (He ), polymorphism information content (PIC) and size range at each microsatellite locus in Nali and Chokla Loci

BM757 BM827 BM6506 BM6526 BM8125 CSSM31 CSSM47 HUJ616 ILSTS002 OarAE129 OarCP20 OarCP34 OarFCB48 OarFCB128 OarHH35 OarHH41 OarHH47 OarHH64 OarJMP8 OarJMP29 OarVH72 OMHC1 RM004 TGLA137 TGLA377

Size range (bp)

178–198 210–228 194–206 146–172 106–122 136–168 128–166 124–160 122–134 138–164 122–136 114–128 144–176 96–120 118–144 110–140 132–144 116–134 132–144 132–154 122–140 190–206 210–244 136–164 75–105

Mean

Nali

Chokla

No. of alleles

Heterozygosity

na

ne

He

4.000 6.000 3.000 5.000 6.000 9.000 5.000 3.000 4.000 4.000 5.000 6.000 10.00 5.000 9.000 3.000 6.000 6.000 7.000 6.000 7.000 4.000 5.000 6.000 4.000

2.321 3.955 1.290 1.493 4.283 3.559 1.563 2.690 3.462 2.354 3.480 3.406 6.613 2.286 5.482 2.689 4.122 2.476 3.140 3.879 4.523 2.852 3.470 5.355 2.703

0.569 0.747 0.225 0.330 0.767 0.719 0.360 0.628 0.711 0.575 0.713 0.706 0.849 0.563 0.818 0.628 0.757 0.596 0.682 0.742 0.779 0.649 0.712 0.813 0.630

5.52

3.338

0.651

tial Bonferroni procedures (Hochberg, 1988) applied over all loci. Genetic distances among populations, namely DS : Nei’s (1972) standard genetic distance and DA : distance of Nei et al. (1983) was estimated from the allele frequencies data using the DISPAN computer programme (Ota, 1993). The software GeneClass V.1.0.02 (Cornuet et al., 1999) was used to compute the proportion of the individuals correctly assigned to their population of origin by applying Bayesian statistical approach with 10,000 simulated individuals (Rannala and Mountain, 1997).

3. Results and discussion This study on genetic variability of Rajasthani Nali and Chokla sheep is the first report on their

PIC

No. of alleles

Heterozygosity

PIC

na

ne

He

0.530 0.708 0.210 0.317 0.729 0.693 0.344 0.551 0.657 0.514 0.666 0.675 0.831 0.525 0.794 0.551 0.720 0.556 0.655 0.713 0.749 0.581 0.675 0.787 0.586

3.000 6.000 3.000 6.000 7.000 6.000 4.000 6.000 4.000 5.000 6.000 6.000 8.000 6.000 4.000 5.000 5.000 7.000 7.000 6.000 7.000 4.000 4.000 6.000 2.000

2.066 3.620 2.082 3.702 2.473 3.920 1.625 3.670 2.950 3.281 1.826 2.923 4.809 4.971 1.984 3.984 3.514 5.803 4.135 2.923 3.495 3.522 2.423 4.278 1.800

0.516 0.724 0.520 0.730 0.596 0.745 0.385 0.728 0.661 0.695 0.453 0.658 0.792 0.799 0.496 0.749 0.715 0.828 0.758 0.658 0.714 0.716 0.587 0.766 0.444

0.456 0.685 0.406 0.693 0.572 0.704 0.359 0.685 0.595 0.638 0.433 0.618 0.766 0.768 0.459 0.712 0.672 0.605 0.733 0.620 0.685 0.666 0.504 0.734 0.346

0.613

5.320

3.271

0.657

0.605

population structure and extent of genetic variability using microsatellite genotype data. All the 25 loci, which have been documented to be polymorphic in ovine species, amplified successfully and produced unambiguous banding patterns from which individual genotypes could be assessed. Various genetic variability measures estimated for each locus in Nali and Chokla sheep, viz., number of observed alleles, effective number of alleles and expected heterozygosity are shown in Table 1. Allele frequencies (not presented) for the 25 microsatellites in the investigated sheep can be obtained from the authors on request. All the loci exhibited polymorphism in both the sheep populations (Crawford et al., 1995). The size range of allelic PCR products was in accordance with that reported previously (FAO, 1996). A total of 159 microsatellite alleles were identified across the 25 analyzed microsatellite

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loci. The number of observed alleles (na ) ranged from 3.00 (BM6506, HUJ616, OarHH41) to 10.00 (OarFCB48) in Nali and 2.00 (TGLA377) to 8.00 (OarFCB48) in Chokla with means of 5.52 and 5.32, respectively. Effective number of alleles (ne ) in two breeds were distinctly less than the observed values and varied from 1.29 (BM6506) to 6.61 (OarFCB48) with an average of 3.34 in Nali and 1.63 (CSSM47) to 5.80 (OarHH64) with a mean of 3.27 in Chokla sheep. The comparison of ne with na at each locus provides information about the predominance of certain alleles in each breed (Arranz et al., 2001). The mean values of observed and effective number of alleles per locus in Nali and Chokla sheep were similar to that of earlier report of Sodhi et al. (2003) in Garole—other Indian sheep population (na = 6.20 and ne = 3.73). Unique alleles observed at 21 loci (84%) constituted only 29.5% (47/159) of the total number of alleles. These are unlikely to be used as genetic markers due to their low frequencies (average ∼ 0.12) in the investigated sheep populations (Kim et al., 2002). The presence of unique alleles at such low frequencies (0.10–0.20) has also been reported for Swiss and Spanish sheep breeds (Saitbekova et al., 2001; Arranz et al., 2001). In contrast, wild Ibex goats and Mouflon sheep displayed unique alleles at much higher frequencies 1.00 and 0.92, respectively (Saitbekova et al., 1999, 2001). Expected heterozygosity that is considered to be a better estimator of the genetic variability present in a population (Kim et al., 2002) showed variations from 0.225 (BM6506) to 0.849 (OarFCB48) in Nali and 0.385 (CSSM47) to 0.828 (OarHH64) in Chokla with an overall average of 0.651 and 0.697, respectively. Polymorphism information content values ranged from 0.210 (BM6506) to 0.831 (OarFCB48) and 0.346 (TGLA377) to 0.768 (OarFCB128) with mean of 0.613 and 0.605 in Nali and Chokla, respectively. The most polymorphic locus appeared to be OarFCB48 with PIC value of 0.831 in Nali and 0.766 in Chokla. The high average values for na (6.36), He (0.697) and PIC (0.660) displayed by panel of 25 microsatellites across Nali and Chokla (data not given) further supported suitability of the used set of markers for genetic diversity analysis in Indian sheep too (Barker, 1994; Botstein et al., 1980). Based on the PIC values, nearby 92% of the markers were observed to be highly informative (PIC > 0.50) and only a few (8%) were

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reasonably informative (0.50 > PIC > 0.25), which also indicated high utility of these markers for population assignment (MacHugh et al., 1998) and genome mapping (Kayang et al., 2002) studies in addition to genetic diversity analysis in Indian sheep too. The observed estimates of allele diversity (mean number of observed alleles per locus) and gene diversity (mean expected heterozygosity per locus) revealed the presence of abundant genetic diversity in both the indigenous sheep of India (Table 1). The high values of various diversity measures observed in Nali and Chokla sheep were in accordance with that of other domestic sheep breeds (Saitbekova et al., 2001; Forbes et al., 1995; Sodhi et al., 2003; Arora and Bhatia, 2004). In contrast, low gene diversity was observed in wild Mouflon and Bighorn sheep herds with values of 0.45 and 0.429, respectively (Saitbekova et al., 2001; Forbes et al., 1995). A small effort for breeding performed in the investigated sheep breeds is presumed to be the possible reason for the high genetic diversity in these two populations, since high-inbred lines reflect very low genetic variability (Zhou and Lamout, 1999). Population differences examined by fixation indices FIT and FST for each of 25 microsatellite loci across two sheep populations are summarized in Table 2. The overall estimates of F-statistics were significantly (p < 0.05) different from zero. From the global analysis, a significant deficit of heterozygotes of 37.0% (p < 0.05) was observed for the whole population. A significant coefficient of multilocus genetic differentiation (FST = 0.083, p < 0.05) showed a certain degree of differentiation between two breeds contributed by 19 loci. It is clear that most of total genetic variation corresponds to differences among individuals (91.7%) and only 8.3% is the result of differences among breeds. The present values of genetic differentiation are lower than those reported for Korean and Chinese domestic goats (FST = 20.2%, Kim et al., 2002), Swiss goat breeds (FST = 17%, Saitbekova et al., 1999), Asian goat populations (FST = 14.3%, Barker et al., 2001), European (FST = 11.2%, MacHugh et al., 1998) and Indian (FST = 11.3%, Mukesh et al., 2004) cattle breeds. However, FST values obtained in this study is similar to those reported for other species, viz., three major human groups (FST = 8.8%, Nei and Roychaudhary, 1982), Spanish dog breeds (FST = 9.9%, Jordana et al., 1992), Spanish horse breeds (FST = 7.8%, Ca˜no´ n

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Table 2 F-statistics analysis and gene flow (Nm ) estimates for each of 25 microsatellite loci across Nali and Chokla sheep breeds

Table 3 With in population in breeding estimates (FIS = f) in Nali and Chokla breeds of sheep

S.no.

Nm

S.no.

Locus

Nali

Chokla

1.344 (0.510) 8.421 (0.905) 1.455 (0.488) 3.501 (0.079) 2.549 (0.269) 13.988 (2.018) 30.643 (5.349) 14.705 (2.161) 3.010 (0.177) 10.351 (1.291) 1.520 (0.475) 22.337 (3.688) 20.186 (3.257) 3.140 (0.151) 2.138 (0.351) 2.573 (0.264) 5.599 (0.341) 4.785 (0.177) 38.734 (6.967) 17.197 (2.661) 1.978 (0.383) 3.214 (0.136) 3.147 (0.149) 13.303 (1.881) 2.795 (0.221)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

BM757 BM827 BM6506 BM6526 BM8125 CSSM31 CSSM47 HUJ616 ILSTS002 OarAE129 OarCP20 OarCP34 OarFCB48 OarFCB128 OarHH35 OarHH41 OarHH47 OarHH64 OarJMP8 OarJMP29 OarVH72 OMHC1 RM004 TGLA137 TGLA377

−0.107 0.238* −0.081 0.395* 0.257 0.263* −0.020 0.835* 0.556* 0.731* 0.661* 0.114 0.404* 1.000* 0.589* 0.154 0.454* 0.335 0.255* 0.272 0.046 0.482* 0.201* 0.870* 0.410*

0.193* 0.246* −0.291 0.096 −0.228 0.319* 0.261 0.470* 0.302* 0.686* 0.417* −0.035 0.576* 0.478* 0.824* 0.591* 0.159* 0.275* 0.157 0.462* 0.068 0.506* 0.308 0.434* −0.082

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Locus BM757 BM827 BM6506 BM6526 BM8125 CSSM31 CSSM47 HUJ616 ILSTS002 OarAE129 OarCP20 OarCP34 OarFCB48 OarFCB128 OarHH35 OarHH41 OarHH47 OarHH64 OarJMP8 OarJMP29 OarVH72 OMHC1 RM004 TGLA137 TGLA377

Mean estimatesa

FIT

FST 0.251*

0.164 0.250* 0.069 0.292* 0.008 0.282* 0.112 0.632* 0.456* 0.702* 0.615* 0.032 0.477* 0.702* 0.701* 0.428* 0.319* 0.314* 0.189 0.345* 0.141 0.513* 0.284* 0.651* 0.231*

0.038 0.229* 0.095 0.147* 0.005 −0.008 −0.003 0.107* 0.010 0.219* 0.000 −0.008 0.089 0.157* 0.135* 0.052 0.067 −0.012 −0.011 0.182* 0.103* 0.110* −0.008 0.105*

0.370

0.083

3.8963

F, total inbreeding estimates; θ, measure of population differentiation. a Mean estimates from Jackknife over loci. Standard deviation in parenthesis. * p < 0.05 from permutation tests in FSTAT programme.

et al., 2000) and South European beef cattle breeds (FST = 6.8%, Jordana et al., 2003). The results thus suggested low level of genetic differentiation between Nali and Chokla sheep as rates up to 15% indicate moderate differentiation between populations (Hartl, 1980). Table 3 shows the within-population inbreeding estimates (f = FIS ) for Nali and Chokla sheep populations at each microsatellite locus. Both the analyzed sheep populations showed significant (p < 0.05) heterozygote deficit, being 39.7% in Nali and 29.9% in Chokla. The average FIS values for most of the loci in both the breeds were significantly different (p < 0.05) from zero. A number of factors, viz., inbreeding, locus under selection (genetic hitchhiking), null alleles (nonamplifying alleles), and presence of population sub-

Mean

0.397 (0.073)

0.299 (0.060)

Mean estimates from Jackknife over loci. Standard deviation in parenthesis. * p < 0.05.

structure (Wahlund effect) may be responsible for lack of heterozygotes in a population (Nei, 1987). However, the main cause of the lack of heterozygotes in the investigated populations can be attributed to inbreeding. In the livestock system considered, rams breed with all the ewes in the flock and, therefore, the heterozygote deficiency is likely to arise from the relationship of individuals used for reproduction. The low level of genetic differentiation detected between the investigated Indian sheep breeds suggested large extent of genetic exchange between both the breeds through inadvertent mating due to sharing of a common breeding tract. This was also evident from a considerable high level of gene flow (Nm = 3.896, Table 2), a most probable cause of great genetic similarity among neighboring breeds (Beja-Pereira et al., 2003). Trexler (1988) showed that if Nm > 1.0, gene flow is enough to attenuate the genetic differentia-

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tion between populations. Similar to present findings, Kim et al. (2002) also reported high genetic similarity between Chinese and Saanen goats (Nm = 3.18). On the other hand, low values of Nm between Korean and Chinese (0.71), Korean and Saanen (0.70) reflected very high genetic differentiation between Korean and other two breeds of goats. Pair-wise comparison at each locus in terms of number of alleles shared (70.44%, 112/159) further reflected genetic similarity between Nali and Chokla sheep. This is in contrast to only 42% of the allele sharing, reported for Swamp and River buffaloes which exhibited significant differentiation between them (Barker et al., 1997). Additionally, low genetic distance values (DS = 0.229 and DA = 0.168) supported high genetic similarity between these two breeds and were in the similar range as those cited by Arranz et al. (1998) for closely related Spanish sheep breeds (DS = 0.21–0.36 and DA = 0.14–0.24). The results based on Bayesian assignment method demonstrated the assignment of simulated genotypes to their respective breeds with accuracies of 88.2%. This observed assignment power could be attributed to the large 2150 genotypic combinations (86 individuals × 25 loci) analyzed in the present study and may not be the indicative of true level of genetic divergence especially for the weekly differentiated populations (Cornuet et al., 1999). Simulated genotypes from Charolais and Friesian cattle breeds have also been reported to be assigned with relatively less accuracies (93% Charolais and 94% Friesian) in comparison to ≥99% accuracies in four British Isle breeds and Swiss Simmental breed (MacHugh et al., 1998). The present study revealed closer genetic similarity between Nali and Chokla sheep breeds that correlates to their phenotypic appearance, origins and geographical propinquity. Extending such studies would be useful to classify other Rajasthani sheep populations for interbreed proximity and formulate their conservation plans. Acknowledgements This study was supported by Indian Council of Agricultural Research. The authors gratefully acknowledge Director, National Bureau of Animal Genetic Resources, for providing necessary facilities to undertake this study. The authors wish to thank Dr. Punia, I/c

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Sheep Breeding Farm, HAU, Hisar; Dr. V.P. Kushwaha, Sr. Scientist, CSWRI, Avikanagar, for their help in collection of blood samples; Mr. Rakesh K., Ms. Parvesh K. for technical assistance during this study.

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