Population Structure and Genetic Bottleneck Analysis of Ankleshwar Poultry Breed by Microsatellite Markers

Genetic variation at 25 microsatellite loci, population structure, and genetic bottleneck hypothesis were examined for Ankleshwar poultry population found in Gujrat, India. The estimates of genetic variability such as effective number of alleles and gene diversities revealed substantial genetic variation frequently displayed by microsatellite markers. The average polymorphism across the studied loci and the expected gene diversity in the population were 6.44 and 0.670±0.144, respectively. The population was observed to be significantly differentiated into different groups, and showed fairly high level of inbreeding (f = 0.240±0.052) and global heterozygote deficit. The bottleneck analysis indicated the absence of genetic bottleneck in the past. The study revealed that the Ankleshwar poultry breed needs appropriate genetic management for its conservation and improvement. The information generated in this study may further be utilized for studying differentiation and relationships among different Indian poultry breeds. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 7 : 915-921)


INTRODUCTION
The ankleshwar poultry breed (Gallus gallus) is known to be a breed of Gujarat, India and its native tract is Bharuch (21.41 N latitude and 73.01 E longitude) and Narmada districts (21.38 N latitude and 73.02 E longitude).The name of the breed is derived from the area 'Ankleshwar' of Bharuch district of Gujarat.The breeding area is also extended to Jumbusar, Zagadia, Bharuch, Hansot and Valia of Bharuch and Dediapada, Rajpipla, Tilakwada and Nadod of Narmada district of Gujarat.These poultry birds are being kept mainly by tribes under backyard poultry farming as a livelihood source of income.These birds are maintained without vaccination and medication, and have reasonable feed efficiency as they survive on 25-30 gms of grains, scavenging and maintain excellent fertility (GAU Report, 2003).In spite of its unique characteristics, there is a lack of concern for the conservation and improvement of this breed under field conditions.However, there is worldwide recognition of the need for the conservation of livestock diversity (FAO, 1995) and for characterization of breeds and populations including their genetic differentiation and relationships.These unique characteristics are result of evolutionary forces and their interactions over longer period of time.However, these adaptation and unique characteristics might have been diluted due to intermixing, sub-structuring and/or consequent genetic drift in the population over time.Therefore, an investigation for genetic variation within the breed, and its structure may help to evaluate how likely these factors are operating, and present genetic information to be used in the conservation and improvement of this unique poultry breed.
Of the many genetic markers now available, microsatellite loci are best suited for answering these questions (Goldstein and Pollock, 1997) because of their high variability, high mutation rate, large number, distribution throughout the genome, codominant inheritance and neutrality with respect to selection (Boyce et al., 1996).They are very useful to analyze the degree and pattern of genetic variability within and differences betweenpopulations (Chenyambuga et al., 2004;Li et al., 2004;Wang et al., 2004), genetic differentiation between closely related species (Sun et al., 2004) and parentage verification (Cho and Chol, 2004).The objectives of this study were to study the estimates of genetic variability in the Ankleshwar breed of poultry, its population structure, and to evaluate the genetic bottleneck hypothesis in this breed.The estimated population of this poultry breed is approximately 5,000 (GAU Report, 2003).method described by Sambrook et al. (1989) and Pandey et al. (2002).A battery of 25 microsatellite markers (Table 1) was selected based on the guidelines of ISAG & FAO's DADIS programme to generate data in a panel of 40 birds.Polymerase Chain Reaction (PCR) was carried out on about 50-100 ng genomic DNA in a 25 µl reaction volume.The reaction mixture consisted of 200 µM each of dATP, dCTP, dGTP and dTTP, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 1.5 mM MgCl 2 , 0.75 unit Taq DNA polymerase and 4 ng/µl of each primer (Sigma Genosys) using PTC-200 PCR machine (M J Research).At the end of the reaction, 5.0 µl of stop dye (95% formamide, 0.25% bromophenol blue and 0.25% xylene cyanol) was added and 6 µl of PCR products were loaded on to a 2% agarose gel, electrophoresed and visualized over UV light after ethidium bromide staining to detect the amplification.
The PCR products were resolved on 6% denaturing polyacrylamide gels (Sequi-Gen GT System, Bio-Rad), φX174 Hinf-I digest was used as a size standard for sizing PCR products.To visualize the PCR products gels were stained using silver staining (Bassam et al., 2001) and dried between sheets of cellophane paper.The genotypes were scored manually.The size of the alleles was calculated online using 'INCHWORM' programme which estimates the length of the molecule, based on the electrophoretic mobility (http://www.molecularworkshop.com/programs/inchworm.html).

Statistical analysis
For 25 microsatellites loci analyzed, observed and expected heterozygosity estimates were calculated after Levene (1949) and Nei (1973) as implemented in POPGENE software (Yeh et al., 1978).The observed and effective numbers of alleles (Kimura and Crow, 1964) were also calculated using POPGENE software.
The tests for deviation from Hardy-Weinberg equilibrium were derived using the exact tests of POPGENE.Heterogeneity of deviations from Hardy-Weinberg equilibrium among the microsatellite loci was examined by treating the deviations as correlation coefficient and tested accordingly (Barker et al., 2001).As samples were obtained from different localities (two districts), deviations from Hardy-Weinberg equilibrium in the population could be due to genetic differences between subpopulations and a consequent Wahlund effect.Given the observed allele frequencies in each subpopulation, the expected heterozygote deficit due to Wahlund effect can be computed (Li, 1976).Expressing this as a percentage of observed heterozygote deficit then measures the contribution of Wahlund effect to the observed heterozygote deficit.Heterozygote deficiencies were expressed as D = [H 0 -H E ]/H E where H 0 and H E are the observed and expected frequency of hetrozygotes, respectively.
Tests for pair wise linkage (genotypic) disequilibrium among the microsatellite loci were done using FSTAT version 2.9.3 an update version 1.2 (Goudet, 1995) for 25 microsatellite loci whose genotypes were determined directly.F-statistics were determined after Weir and Effective number of alleles [Kimura and Crow (1964)].Shannon's Information index [Lewontin (1972)].a Expected heterozygosity were computed using Levene (1949) and Nei's (1973) expected heterozygosity.b Heterozygote deficiencies were expressed as D = (Ho-He)/He, refer the text for symbols used.c f-values (Weir and Cocheran 1984) given for significant tests after Bonferroni corrections.* Wahlund effects varied from 0.006 to 0.028%; PIC (polymorphic information content).Cockerham (1984) as used in F-Stat software with Jackknifing procedure applied over loci in deriving significance levels.These parameters of population structure are defined as the correlations between pairs of genes (i) within individuals (F) (ii) between individuals in the same population (θ), and (iii) within individuals within populations (f), and are analogous to Wright's (1978) F IT , F ST and F IS , respectively.Finally the bottleneck hypothesis was investigated using BOTTLENECK 1.2.01 (Cornuet and Luikart, 1996).The BOTTLENECK tests for the departure from mutation drift equilibrium based on heterozygosity (not hetrozygote), excess or deficiency.This does not require information on historical population sizes or level of genetic variations.It requires only measurement of allele frequencies from 5-20 polymorphic loci in a sample of approximately 20-30 individuals.The bottleneck compares heterozygosity expected (H E ) at Hardy-Weinberg equilibrium to the heterozygosity expected (H eq ) at mutation drift equilibrium in same sample, that has the same size and the same number of alleles.All the three models of mutation were used to calculate H eq : the strict one stepwise mutation model (Ohta and Kimura, 1973), the infinite allele model (Kimura and Crow, 1964) and two-phase model (Di Rienzo et al., 1994).

RESULTS AND DISCUSSION
Various measures of genetic variation are presented in the Table 2.The F-statistics estimates are presented in Table 3.
The number of alleles observed across the microsatellite loci studied varied from 4(ADL39, ADL20, MCW7, MCW4, MCW59 and ADL40) to 11 (ADL171) with an overall mean of 6.44±2.162(Table 2).The observed number of alleles across the loci was more than the effective number of alleles (1.437 to 6.387) as per expectations.The Shannon information index and polymorphic Information Content (PIC) showed that most of the loci were highly informative indicating the high polymorphism across the loci with an overall mean of 1.400 and 0.623, respectively.Relat, an estimator of the average relatedness of individuals within samples when compared to whole (Queller and Goodnight's, 1989).Relatc estmates the inbreeding corrected relatedness (Pamilo, 1985).a Standard errors: estimate from jackknife over loci and significance from t-test using these estimates, p<0.05.R st an estimator of relative genetic differentiation based on fraction of total variance of allele size between two subpopulations.
The f (F IS ) estimates across 15 out of 25 studied loci were significantly positive (significant heterozygote deficit) based on table wide randomizations (p<0.05).The f estimates ranged from 0.142 to 0.589 with an average of 0.240±0.052(Table 3).
The significant heterozygote deficiency found in ankleshwar population of poultry could be due to one or more of the following reasons: segregation of nonamplifying (null) alleles, Wahlund effects, scoring biases (heterozygotes scored incorrectly as homozygotes) or inbreeding.Distinguishing among these generally is difficult (Christiansen et al., 1974).However, null alleles are most unlikely to be segregating at all the loci.Similarly, scoring bias may be possible for a few loci but not for all loci.Wahlund effects (localities with non-subpopulations) do account for 0.006 to 0.028 of the observed heterozygote deficit for >50% of the loci showing significant heterozygote deficiency.There is strong inbreeding in the population as indicated by high value of f estimate (0.240), presumably resulting from the unplanned and indiscriminate mating prevalent in the breeding tract leading to small effective population size/or mating between relatives and consequent genetic drift.Therefore, inbreeding and Wahlund effects may be most reasonable causes of heterozygote deficit.The non-random association of alleles across the loci was also compatible with genotypic disequilibrium observed in the population.The samples (taken from each of the two districts) were substantially differentiated from each other as indicated by the term theta (θ) and Rst estimates.These estimates are also in accordance with measures of Wahlund effect in the study.F IT estimates revealed significant deviations (heterozygote deficit) from Hardy-Weinberg equilibrium across most of the loci studied.These estimates showed global heterozygote deficit in the population after applying the Bonferroni corrections to different tests.
The results of this study indicated that there is substantial genetic variation and polymorphism across studied loci.The Ankleshwar poultry population was neither in Hardy-Weinberg equilibrium nor in mutation drift equilibrium.The population appears to be divided into significantly differentiated small subpopulations, which resulted in mating among close relatives leading to high level of inbreeding observed in this study.Appropriate breeding strategies should therefore be designed under field conditions for its conservation and improvement of its unique attributes maintained without vaccination and medication, reasonable feed efficiency as they survive on availability of 25-30 gms of grains, scavenging and maintaining excellent fertility 91.34%, (GAU Report, 2003) under the given management and climatic conditions of the region.

Figure 1 .
Figure 1.L-shaped mode-shift graph showing lack of bottleneck in the population.

Table 1 .
Details of the microsatellite used in the study S.N.

Table 2 .
Measures of genetic variation in Ankleshwar poultry population

Table 3 .
F-statistics analyses for 25 microsatellite loci in Ankleshwar poultry population * Refer to the text for F symbols.