Genetic variability in Jalauni sheep of India inferred from microsatellite data

R Arora, S Bhatia, A Sehrawat, S B Maity* and S S Kundu*

National Bureau of Animal Genetic Resources, P.O.Box129, Karnal-132001, Haryana, India
*Indian Grassland and Fodder Research Institute, Jhansi-284003, Uttar Pradesh, India
rejagati@yahoo.co.in

Abstract

This paper examines the genetic variability in Jalauni - an important sheep of northwestern arid and semi arid region of India, at 25 microsatellite loci covering 19 chromosomes, in order to determine the genetic diversity available in this mutton and wool producing indigenous breed of the country.  The allele diversity, gene diversity and the statistics that express polymorphism, inbreeding and genetic bottleneck within the breed were determined.

A total of 148 alleles were identified with an allele diversity of 5.92. The effective allele number (3.71) was lower than the allele diversity. The mean observed heterozygosity (0.58) and gene diversity (0.68) estimates elucidated substantial genetic diversity within Jalauni breed. The used set of microsatellite loci which exhibited high genetic polymorphism (PIC=0.64) also represented a useful panel of markers for population genetic studies in Indian sheep breeds. The typical L-like distribution of the allele frequencies obtained in the Mode shift test implied that reduction in effective population size or a recent genetic bottleneck (40-80 generations) was very unlikely in this indigenous breed of sheep.

The present study suggested that  breeding strategies designed for conservation and improvement of Jalauni breed under field conditions must involve education of farmers for frequent exchange of breeding rams between the populations, in order to curb adverse effects of inbreeding (F_IS =0.12). Moreover, subsequent utilization of generated data for calculation of between breeds diversity would further allow selection of priority breeds at national level to maximize diversity conserved for the benefit of future human generations.

Keywords: conservation, genetic variability, Indian sheep, microsatellite

Introduction

The role of local/indigenous sheep breeds for the upliftment of the rural economy needs to be given greater emphasis as these breeds are able to thrive in zero/low input system. Over the years, farming communities across the globe have directly or indirectly selected and improved indigenous sheep breeds to meet the needs of the harsh and adverse conditions they dwell in. Complete characterization of these indigenous breeds is imperative as they are the only remaining sources of putative alleles of economic values that might be lost through unchecked increase, uncontrolled intermixing amongst them, infusion of exotic germplasm due to haphazard crossbreeding, absence of planned strategies for their conservation and sheer negligence (Bhatia and Arora 2005). Most of the ovine genetic resources in India either lack phenotypic or genetic documentation, it is therefore, germane to measure the genetic indices of all those ovine populations whose information is scanty, phenotypically as well as at the DNA level so that generated results can be utilized for breeding and conservation programmes /policies.

Jalauni is one of the recognized sheep breeds of the Bundelkhand region, mostly prevalent in Jalaun, Jhansi and Lalitpur districts of Uttar Pradesh and the Tikamgarh and Datia districts of Madhya Pradesh (Figure1).

Jalauni sheep, well adapted to the local agro-climatic conditions of this region - maintained for mutton and wool are small to medium in size with straight nose line, white coloured body and light brown or black head in most animals. Jalauni sheep are reared by the ‘Pal’ community which comprises small, marginal or landless, mostly illiterate farmers/labourers. In view of declining status of the breed (Sahana et al 2004) due to decrease in grazing area as a result of increased crop production in the breeding tract, there is a vital need for its genetic characterization, to enable subsequent identification of indigenous populations/breeds of high conservation priority.

The microsatellite markers, due to their high levels of polymorphism, ubiquitous nature, codominant inheritance, ease and accuracy of typing have been successfully applied in population genetic studies (Buchanan et al 1994; Machugh et al 1998; Forbes et al 1995) parentage testing (Coppieters et al 1993), and linkage analyses (Barendse et al 1994). Status on genetic characterization using microsatellites have so far been focused across the world on various sheep populations viz., Spanish (Arranz et al 2001), Swiss (Saitbekova et al 2001), Baltic (Grigaliunaite et al 2003), Brazilian (Paiva et al 2005), African (Wafula et al 2005), Austrian (Baumung et al 2006) etc. However, meager information available on microsatellite data in Indian sheep has emerged mainly from authors laboratory (Sodhi et al 2003, 2006, Arora and Bhatia 2004, 2006, Mukesh et al 2006), though populations from the Indian subcontinent account for 5.32 % of the world’s sheep population (FAO 1998). The aim of the present study was to examine patterns of microsatellite variation in Jalauni, a domestic sheep population from India as a part of ongoing programme on “Molecular genetic characterization of indigenous breeds of sheep” at NBAGR, Karnal, to enable evaluation of genetic structure for identification of sheep populations of high conservation priority and subsequent development of appropriate breeding strategies for their improvement.

Materials and methods

Sample collection and DNA extraction

The blood samples of 50 unrelated animals of Jalauni breed were collected from Indian Grassland and Fodder Research Institute’s (IGFRI) farm at Jhansi. These animals had been purchased from different villages of its distribution area. Attempts were made, as far as possible, to avoid sampling of related animals. DNA was extracted from the white blood cells using a standard phenol/chloroform/isoamyl alcohol extraction protocol (Sambrook et al 1989).

Genotyping

Twenty five microsatellites from the list of MoDAD (FAO) by Bradley et al (1997) were genotyped on 50 DNA samples of Jalauni breed. Amplifications for the loci was performed in a 25µl final reaction volume containing at least 100ng of genomic DNA, 50ng of each primer, 1.5mM MgCl₂, 200µM dNTPs, 0.5U Taq polymerase and 1x buffer. The thermal touchdown profile for PCR 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. Amplification was confirmed on 2% agarose gel and the products were size separated on 6% denaturing polyacrylamide gel and visualized by silver staining (Bassam et al 1991). Estimation of allele size was done by running a 10bp DNA molecular weight marker along with the PCR products. Genotyping was done manually from the silver stained gels.

Computation and statistical analysis

Genetic variation was quantified by calculating observed and effective number of alleles, observed heterozygosity, expected heterozygosity and within breed heterozygotes deficiency (F_is) following the POPGENE program version 1.31 (Yeh et al 1999). The polymorphism information content (PIC) of microsatellite loci was estimated using the formula given by Botstein et al (1980). An exact test was used to determine deviations from the Hardy Weinberg Equilibrium (HWE) using the GENEPOP software package version 3.2a (Raymond and Rousset 1995). HWE tests were performed for each locus and over all loci. Genetic bottleneck effect was inferred for the population using the qualitative graphical method (mode shift analysis) under the assumption of two phase microsatellite mutation model (TPM), implemented in the programme BOTTLENECK ver 1.2.02 (Cornuet and Luikart 1996). This programme is based on the principle that any population that has experienced a recent reduction in its effective population size exhibits a correlative reduction in the allele numbers and gene diversity at polymorphic loci.

Results and discussion 

Microsatellite profiles for 25 loci located on 19 chromosomes were recorded for 50 animals of Jalauni sheep. Allele frequencies ranged from 0.02 to 0.86. Table 1 summarizes various genetic diversity measures estimated for each locus in Jalauni sheep viz., observed number of alleles (No), effective number of alleles (Ne), observed heterozygosity (Ho) and expected heterozygosity (He).

In total 148 alleles were detected across 25 microsatellite loci that were typed and the actual number of observed alleles at each locus ranged from 2 (BM8125) to 9 (CSSM31, OMHC1 and TGLA137) with a mean of 5.92. Apart from the loci BM8125 where only two alleles were detected, a fairly high degree of genetic variation was observed in terms of number of alleles per locus (>2). The effective number of alleles, being distinctly lower than the observed number of alleles ranged between 1.3 (OarCP20) to 6.3 (TGLA137) with an average of 3.7 in Jalauni.

The number of alleles observed at a locus is an indication of genetic variability at that locus having direct impact on differentiation of breeds within a species (Buchanan et al 1994) and the mean allele number (allele diversity) provides a reasonable indicator of the levels of variability present within a breed assuming that the population is in mutation drift equilibrium (MacHugh et al 1998). The allele diversity measure (5.92) reflected substantial level of genetic variability in Jalauni sheep. Previous studies on other Indian ovine breeds viz., Garole, Nali, Chokla (Sodhi et al 2003, 2006), Muzzafarnagri, Magra, Kheri (Arora and Bhatia 2004, 2006, Bhatia et al 2005a, 2005b) and a few exotic breeds (Arranz et al 1998, Paiva et al 2005, Wafula et al 2005) reported allele diversity estimates comparable with those obtained in this study for Jalauni sheep (Table 2).

The average number of alleles found in the investigated breed was, however, lower than most of the Baltic (Grigaliunaite et al 2003), Austrian (Baumung et al 2006) and Caucasian (Hirbo et al 2006) sheep breeds. Nevertheless, due to a different set of markers used in this study, a direct comparison could not be made. The number of genotypes per locus varied from 3 (BM8125) to 17 (OMHC1). The high genotypic values could be attributed to the high number of alleles in Jalauni sheep, which also suggested the existence of heterozygous genotypes in this population.

The observed heterozygosity (Ho) ranged from 0.26 (OarHH47) to 0.95 (TGLA137) and the expected heterozygosity (He) from 0.24 (OarCP20) to 0.84 (TGLA137) in Jalauni sheep. The mean observed heterozygosity (0.58) and gene diversity (mean expected heterozygosity, 0.68) values were in accordance to earlier studies (Saitbekova et al 2001, Sodhi et al 2003, Arora and Bhatia 2004, 2006,  Bhatia et al 2005, Paiva et al 2005, Wafula et al 2005, Table 2). The gene diversity estimate was, however, much higher than the wild Mouflon sheep (He=0.45) probably due to close captive relatedness in this wild sheep flock (Saitbekova et al 2001). In the present study, the mean observed heterozygosity value, though lower than the gene diversity value was, however, not observed to be significantly different from gene diversity using ANOVA test (p>0.05), which is an indicator of the maintenance of random mating structure by the investigated breed. Moreover, heterozygosity values further showed that Jalauni sheep breed possessed a considerable amount of genetic diversity.

The Jalauni breed showed significant (P<0.05) deviation from HWE at six loci, which might represent sub-structure present in the form of localized heterozygote deficiencies suggestive of localized inbreeding. Departure from HW proportions may possibly be attributed to the presence of null alleles. Nevertheless, it was not possible to estimate the extent of null alleles as no pedigree records were available for analysis and care was taken to collect blood samples from unrelated animals only. However, due to absence of significant deviations from HWE across all the loci (p>0.05) within this population, subsequent analysis was carried out on the basis that Hardy-Weinberg equilibrium prevailed in the investigated sheep population (Marshall et al 1999).

The polymorphic information content (PIC) is a parameter indicative of the degree of informativeness of a marker. The PIC value may range from 0 to 1. Loci with many alleles and a PIC value of 1 are most desirable (Botstein et al 1984). Hence, the degree of informativeness of a marker reveals its usefulness in diversity analysis of a breed. A higher value would mean more alleles and greater polymorphism at that locus. In the present study polymorphism information content revealed an average of 0.64 with a range of 0.24 (OarCP20) to 0.82 (TGLA137). The high mean PIC value displayed by panel of 25 microsatellites in Jalauni supported suitability of the used set of markers for genetic diversity analysis in Indian sheep too (Kemp et al 1995). Similar results were obtained from the same set of markers in earlier reported indigenous breeds of sheep by the authors (Sodhi et al 2003, Arora and Bhatia 2004, 2006, Mukesh et al 2006). Following the criteria of Botstein et al (1980), 80% of the markers were observed to be highly informative (PIC >0.5), 16% reasonably informative (0.25<PIC<0.5) and 4% were slightly informative (PIC<0.25) across Jalauni breed, which also suggested 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.

Population inbreeding estimate (F_IS), which indicates heterozygote deficiency was observed to be 0.12 with a range from -0.32 (BM8125) to 0.61 (OarHH47). The shortage of heterozygotes (12.3%) and excess of homozygotes might be attributed to a number of factors viz., Wahlund effect if population subdivision is occurring, assortative mating (sample relatedness), linkage with loci under selection (genetic hitchhiking), population heterogeneity, null alleles (non-amplifying alleles) or inbreeding. Positive F_IS value suggested inbreeding to be one of the main causes for lack of heterozygotes in Jalauni sheep. In the absence of detailed past information about this breed it is, however, difficult to identify precisely which past demographic factors affected the current genetic structure of this breed.

Sahana et al (2004) reported a declining trend of 0.04 % per annum over the years from 1977 to 1997 in the Jalauni sheep, except in Jhansi district where sheep population increased during this period. The bottleneck analysis was, therefore, performed to assess whether population decline had an impact on the maintenance of genetic variation within Jalauni breed. The typical L-like distribution of the allele frequencies obtained in the Mode shift test (Figure2) strongly indicated that reduction in effective population size or a recent genetic bottleneck (40-80 generations) was very unlikely in this indigenous breed of sheep (Luikart et al 1998). The absence of genetic bottleneck in the experimental population is consistent with the results of similar studies on the declining Magra sheep breed of the same agro ecological region of India (Arora and Bhatia 2006).

 The long term management and ex situ as well as in situ restoration efforts for a breed require the understanding of its genetic structure, hence the characterization of Jalauni sheep will facilitate the recognition and maintenance of this unique indigenous gene pool for current and future generations. Further, integrating genetic improvement programmes for this breed with market oriented production strategies will raise the economy of its rearers and thereby ensure its sustainable conservation.

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 departmental help from Plant Animal Relationship Division, Indian Grassland and Fodder Research Institute, Jhansi, in collection of blood samples is greatly acknowledged. The authors wish to thank Mr. Rakesh Kumar for technical assistance in blood sample collection and DNA isolation during the study.

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Received 21 September 2007; Accepted 17 October 2007; Published 1 January 2008

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