HarYana Vet.47 (December, 2008), pp 77-83 Research Article GENETIC CHARACTERIZATION AND BOTTLENECK STUDIES IN KATHIAWARI HORSE BREED OF INDIA P. G. KORINGA' , C. G. JOSHI, J. V. SOLANKF and D. N. RANK2 Department of Animal Biotechnology, College of Veterinary Sciences AnandAgricultural University,Anand (Gujarat) ABSTRACT To assist the conservation efforts and breeding strategies, the first genetic characterization of the Kathiawari breed of horse was undertaken and 24 microsatellite markers were used to genotype 50 unrelated Kathiawari horses. The observed heterozygosity for the Kathiawari breed was 0.6509 ± 0.1874 with the expected heterozygosity of 0.6448 ± 0.1544. The average number ofalleles per locus was 5.166 ± 1.493 while polymorphism information content was 0.591. Wright's fixation index, F,s (0.1155 ± 0.0252) indicated high level of heterozygote deficiency which suggested a high level of inbreeding. This basic study indicated the existence of substantial genetic diversity in the Kathiawari horse popula- tion. Significant genotypic linkage disequilibrium was detected across the population, suggesting evidence of linkage between loci. A normal 'L' shaped distribution of mode-shift test, non-significant heterozygote excess on the basis of different models, as revealed from Sign, Standardized differences and WiJcoxon sign rank tests as well as non-significant M ratio value suggested that there was no recent bottleneck in the existing Kathiawari breed population, which is important information for equine breeders. This study also revealed that the Kathiawari, a Indian horse breed can be differentiated from some other exotic breeds of horses on the basis of these microsatellite primers. Key words: Kathiawari horse, microsatellite, bottleneck, breed characterization India has a rich biodiversity of equines in the genetic diversity within the breed, an the form of six distinct indigenous horse (Equus objective breed classification based on genetic cabal/us) breeds, namely Kathiawari, Marwari, uniqueness is of priority (May, 1990, Hall and Spiti, Zanskari, Bhutia and Manipuri, in addition Bradly, 1995). Characterization at the to indigenous donkeys and wild asses (Yadav et morphological and genetic levels is the first step al., 2001). These horse breeds are well adapted towards formulating breeding policies and to different agroclimatic regions and possess prioritising the breeds for conservation in an certain unique characteristics. Kathiawari breed effective and meaningful way. Recently an array of horse is found in Saurashtra region ofGujarat of DNA based markers has been developed to State. The animals of this breed are well known carry out studies on genetic variation {Bradley for their canter gait popularly known as et al., 1996, Canon et al., 2000). Among these, 'Rawaal'. Kathiawari is well known for its pace microsatellites are considered to be the most and speed, relative disease resistance, possesses suitable marker system for evaluating breeds for good endurance power and faithfulness to owner genetic diversity, owing to their abundance in which attracts the royal community of India. the mammalian genome, high level of However, owing to indiscriminate breeding polymorphism, codominant inheritance and and lack of sound breeding policies, the breed's amenability for automation (Takezaki and Nei characteristics are being diluted and presently 1996). Except for phenotypic characterization, only few thousand true Kathiawari horses are no genetic characterization studies have been in existence (Singhvi, 2001). To avoid further carried out in Kathiawari horses. The present loss of potential unique genes, and to preserve study involved molecular characterization based on microsatellite markers to detect historical 'Corresponding author population bottlenecks, if any, in the Kathiawari 2Department of Animal Genetics and Breeding horse breed. MATERIALS AND METHODS package (Yeh et al. 1999). Polymorphism information content (PlC) values were Sample collection and DNA isolation: calculated by using the method described by Vacutainers tubes containing ethylenediamine- Botstein et al (1980). The probability of random tetra acetic acid (EDTA) as an anticoagulant mating in the population was estimated by Chi- were used to collect blood samples from 50 square (Xl) and likelihood ratio (G2) tests to unrelated horses from four different districts of examine Hardy-Weinberg equilibrium (HWE) at Gujarat. Genomic DNA was isolated and purified each locus. The tests for departure from Hardy- from white blood cells using method of proteinase Weinberg proportions and linkage disequilibrium K digestion and standard phenol/chloroform! were performed using exact probability tests isoarnyl alcohol extraction and absolute ethanol provided in GENEPOP version 3.1 a (Raymond precipitation (Sambrook et al., 1989). and Rousset 1999). A Monte Carlo method (Guo Microsatellite amplification: Samples were and Thompson 1992), with the length of chain genotyped for a set of24 microsatellites (Table set to be 50000 iterations, was used to compute 1) recommended for genetic characterization of unbiased estimates of the exact probabilities equines by the International Society for Animal (P values). Ewens- Watterson neutrality test was Genetics (lSAG). PCR was carried out in 25 III performed to test the neutrality of microsatellite reaction containing 100 ng template DNA, 10 markers, the statistics for the test were pmol each primer, 200 IlM dNTPs, 10 mM Tris calculated using the algorithm given by Manly HCl (pH 9.0), 50 mM KC1, and 0.5 U of Taq (1985), using 1000 simulated samples. polymerase (Bangalore Genei Pvt. Ltd., India) Bottleneck events in the population were using Thermal Cycler (Eppendorf, Germany). tested by three methods. The first method Different primer pairs were used with 1.5-2.0 consisted of three excess heterozygosity tests mM MgCl2 concentration. Thermal conditions developed by Cornuet and Luikart (1996) like for amplification included initial denaturation at sign test, standardized di fferences test and a 95°C for 5 min, followed by 30 cycles of wilcoxon sign-rank test. The probability denaturation at 95°C for 45 sec, annealing at distribution was established using 1000 optimum temperature (58-63°C) for 45 sec, and simulations under three models such as Infinite extension at noc for 45 sec each, with a final allele model (lAM), stepwise mutation model extension at noc for 10 min. The PCR products (SMM) and two-phase model of mutation were electrophoresed at 80 volts in a 2% agarose (TPM). The second method was the graphical gel and stained with ethidium bromide (0.5 mg/ representation of the mode-shift indicator ml). The amplified products observed under ultra originally proposed by Luikart et al. (1998). Loss violet light (300 nrn) transilluminator were further of rare alleles in bottlenecked populations was . resolved in 7% urea polyacrylamide denaturing detected when one or more of the common allele sequencing gel on Sequi-GT system 30 x 38 cm classes have a higher number of alleles than the (Bio-Rad Laboratories, USA). Alleles were rare allele class (Luikart et at. 1998). This test visualized by ~ilver staining, following Bassam was re-scaled so that frequency distribution of et al. (1991). The sizes of amplified products the allele frequency classes in each population were estimated using a 10 bp molecular weight would be based on equal 0.05 increments. These marker (Invitrogen, USA). Genotypes of two methods were conducted using Bottleneck individual horses at the different polymorphic loci v1.2.02 (http://www.ensam.inra.fr/URLB). The were recorded by direct counting. third method was the M ratio conducted by Computation and statistical analysis: Allele applying the mp val.exe program (Garza and frequencies for each locus were calculated with Williamson 2001). The ratio of the number of 2n= 100 for Kathiawari horses and can be alleles present at a locus (k) to the range of allele obtained from authors on request. Heterozygosity sizes in base pairs for the same locus (r) is called (Nei 1978) and other genetic diversity variables the M ratio (M = Y! klr; Garza and Williamson were calculated using POPGENE computer 2001). The M ratio is stable if the population 78 size was stable for a long time. When a ratio tests performed to examine Hardy population experiences a bottleneck event, rare Weinberg Equilibrium (HWE) at each locus alleles are lost more often by drift than the indicates that eighteen loci were deviating from common alleles during a population size reduction HWE. Sixteen loci (NVHEQ79, ASB02, and k is reduced. However, lost alleles do not NVHEQ05, NVHEQ21, NVHEQ54, LEX20, always occur at the extremes of the allele size NVHEQ 11, NVHEQ29, NVHEQ40, HTG07, distribution so the range in allele sizes (r) will HTGI4, AHT04, HTG06, HTGI5,NVHEQ82 not be reduced at the same rate as k. and NVHEQ70) showed higher observed Consequently the M ratio declines in the event heterozygosity than the expected values (Table of a bottleneck event. The M ratio was 2). Heterozygote deficiency analysis revealed calculated for the whole population and significant deviation from HWE (P < 0.05) at contrasted with that under equilibrium. To test some of the loci. It is, however, difficult to whether an M value is lower than expected, envisage the exact basis of this departure, 10000 replicates were simulated. The number although the presence of low frequency null of times that the simulated M is higher than the alleles segregating at these loci may