Microsatellite Analysis of Three Poultry Breeds of India

: The genetic variability of three poultry breeds namely Aseel, Miri and Nicobari taken from different geographical locations of India were evaluated using 15 microsatellite loci. No. of alleles varied from 3 to 9 in Aseel, 3 to 8 in Miri and 2 to 7 in Nicobari. Mean PIC values in Aseel, Miri and Nicobari breeds were 0.64, 0.66 and 0.63, respectively. Average unbiased heterozygosity and direct count heterozygosity were 0.65 and 0.59, 0.68 and 0.61, and 0.64 and 0.57 in Aseel, Miri and Nicobari breeds, respectively. High heterozygosity values revealed in this study are indicative of low level of inbreeding, large population size and no or low selection pressure for commercial trait in all three populations. The estimate of genetic distances using Nei's standard, Nei's minimum and Reynold's distance revealed Aseel and Nicobari to be more closely related than Miri breed of poultry. (Asian-Aust. J. Anim. Sci. 2002. Vol 15, No.


INTRODUCTION
There are 17 defined breeds of domestic fowl (Gallus domesticus) in India (Acharya and Bhat, 1984). The three diverse breeds from different geographical location Aseel, Miri and Nicobari were identified for the study. These breeds are of local importance in their regions and are reared as backyard poultry. Several techniques like RAPD, AFLP and Microsatellite analysis are utilized to assess the population relationships. Among these the microsatellite analysis provide even distribution in the genome, and are highly polymorphic and thus are markers of choice for biodiversity analysis. In this study 15 highly polymorphic unlinked microsatellite markers have been utilized for genetic relationship among three native breeds of India.

MATERIALS AND METHODS
Blood samples about 2 ml were collected aseptically from wing vein of three Indian poultry breeds. The samples of Aseel were collected from Bastar district of Chattisgarh and district Khammam of Andhra Pradesh, Miri samples from Dhimaji and North Lakhimpur district of Assam and samples of Nicobari breed were collected from CARI, Portblair (Andaman and Nicobar Islands). Due care was taken that the samples were from unrelated birds to ensure that the samples were random and represent the population. A total of 94 samples were collected, 20 samples of Miri from upper Assam, 38 of Aseel from Bastar region of Chhatisgarh state and 36 of Nicobari from Andaman and Nicobar group of Islands. All the three poultry breeds are predominantly kept by local tribes of the regions. Samples were collected from wing vein using heparin as an anticoagulant. The blood samples were transported to lab at 0-5°C in heparinzed vacutainers. DNA was isolated using standard protocol (Sambrook et al., 1989) PCR reactions were carried out in a volume of 25 µl containing 50-100 ng genomic DNA 1.5 mM MgCl 2 , 200 µM of each primer, 200 µM dNTP, one Unit of Taq DNA polymerase. The annealing temperature and Magnesium Chloride concentration were standardized to get the optimal product. Denaturation, annealing and extension steps for 30 cycles were carried out using PTC-200 PCR machine (MJ Thermal Cycler).
Amplified PCR products were separated on 6% denaturing polyacrylamide gels along with standard DNA markers for sizing. The PAGE was run for sufficiently long period for proper resolution of alleles. The gels were silver stained following standard protocol (Bassam et al., 1991) The size of the alleles were estimated by making a standard curve taking log 10 of the size of standard marker on X-axis and mobility of the DNA on Y-axis. The sizes of the alleles were estimated from the standard curve.
The statistical analysis was carried out using POPGENE software (Yeh et al., 1999). The heterozygosity was calculated using the following formulae given by (Nei, 1978). In order to estimate the genetic variation the observed/direct count and unbiased heterozygosities were calculated for all microsatellite loci in three breeds of poultry. 1. The observed/ direct count heterozygosity was calculated as: 2. The unbiased heterozygosity k=no. of alleles X i =frequency of i th allele X j =frequency of j th allele The PIC values (polymorphic information content) was calculated using the formula given by (Botstein et al., 1980) PIC=1- The used distance measure was developed by (Nei, 1972) called as Nei's Standard genetic distance Ds was estimated. This was calculated as Ds=-ln I Where I is the identity. Identity is estimated from √ Where Jxy and Jx and Jy are the means for all loci of Σ x i y i , Σx i 2 , and Σ y i 2 for each locus. The Nei's minimum genetic distance was calculated (Nei, 1973) as D m =(J x +J y )/2-J xy The effective number of alleles was calculated as given by (Kimura and Crow, 1964). The genetic distances were calculated using Nei's standard (Nei, 1972) and unbiased (Nei, 1978) distance and identity measures. Nei's minimum distances (Nei, 1973;Nei, 1978) were also calculated. The coancestory identity/distance were calculated using Reynold et al., 1983. The phylogenetic tree construction was done using UPGMA algorithm (Swofford and Olsem, 1990). Bootstrapping was used to generate increased confidence in the tree constructed using the original data. The relative lengths of the nodes produced by UPGMA analysis were tested for consistency indices for each node generated by the original data set. The relative strength of the node was tested to find out if the alternative topologies existed (Backeljau et al., 1996). The time of divergence was estimated based on the equation D=2 a t, where a is the estimated microsatellite mutation rate and t is time in generations.

RESULTS AND DISCUSSION
The selected microsatellite loci fulfilled conditions like-Mendelian inheritance with PIC value more than 0.6 and were located on different chromosomes/linkage groups (Table 1) and assorted independently (not linked to one another). If these conditions are fulfilled each microsatellite loci represents an independent evolutionary history and is thus suitable for biodiversity analysis. Ten out of 15 loci namely ADL 102, ADL 136, ADL 158, ADL 171, ADL 176, ADL 210, ADL 267, MCW 14, MCW 41, and MCW 59 were from recommended list of loci Domestic Animal Diversity Analysis (Barker et al., 1998), the remaining 5 loci (ADL 20, ADL 23, MCW 5, MCW 7 and MCW 49) were selected after screening published literature.
The number of alleles, effective number of alleles, polymorphic information content (PIC) value, unbiased heterozygosity and direct count heterozygosity in the Aseel, Miri and Nicobari breeds are presented in Table 2, 3 and 4, respectively.
The These results are in conformity with (Wimmers et al., 2000). They also reported high heterozygosity values i.e. 0.66±0.22, 0.45±0.32 and 0.71±0.15 in Indian breeds namely Kadaknath, Aseel and Frizzle Fowl, respectively. The lower heterozygosity value in Aseel in their report may be attributed to the fact that samples in their study were taken from organized flock where these bird may have been subjected to selection pressure and subsequent inbreeding, where as the Aseel poultry in this study were taken from the villages and due care was taken for the samples being Σ 2X i 2 X j 2 unrelated but conforming to breed character. However, high inbred lines revealed very high degree of homozygosity with heterozygosity values varying from as low as 0.00 to 0.123 in 23 inbred lines derived from Leghorn, jungle fowl, Fayoumi and Spanish breeds (Zhow and Lamont, 1999).
The use of a mixture of highly variable and less variable microsatellite reduces the risk of overestimating genetic variability, which might occur if only highly variable loci  (Wimmers et al., 2000). All the three populations revealed higher values for unbiased and direct count heterozygosity. The high heterozygosity values far exceed the values estimated for commercial breeds i.e. 0.40, (Crooijmans et al., 1996). The observed heterozygosity values were less than 0.4 only for 3 loci in Aseel, 2 each in Miri and Nicobari poultry. High heterozygosity value can be attributed to low level of inbreeding, large population size, no or low selection pressure for commercial traits and large number of alleles present in the population. The demographic structure of the three populations reveal that the poultry birds are reared by the tribes as backyard poultry and birds are a part of culture of the tribes and are not subjected to selection of any kind except Aseel where birds are selected for fighting qualities and aggressive behavior.
The effective number of alleles maintained in the population was less than the actual number of alleles. If all the alleles were equally frequent the population of homozygotes would be the reciprocal of number of alleles at this locus maintained in the population. If there is variations in the allele frequency the population of homozygotes will be greater than this. A total of six alleles on 4 loci were specific for Miri poultry while four unique alleles on four loci were found only in Aseel poultry. The data analysis over all the 15 microsatellite loci revealed the Nicobari fowl had no specific alleles and all were shared by Miri and Nicobari poultry. These are not being termed as private alleles because of the small sample size used in the study.

Genetic distance
The calculation of a genetic distance between two populations gives a relative estimate of the time that has passed since the populations existed as single cohesive units. Small estimations of distance may indicate population substructure (i.e., subpopulations in which there is random mating but there is a reduced amount of gene flow). However, small estimation of distance may also be present because the populations are completely isolated but have only been separated for a short period of time. When two populations are genetically isolated, the two processes of mutation and genetic drift lead to differentiation in the allele frequencies until each population is completely fixed for separate alleles. A number of methods have been developed which estimate genetic distance from these allele frequency data like Nei's standard distance, Nei's minimum genetic distance and Reynold's conancestory distance.
The UPGMA cluster analysis using Nei's original distance revealed two nodes. The node one further diverged into populations of Nicobari and Aseel while the population of Miri poultry diverged from node two. To generate a confidence of 95% the data was simulated with 1000 permutations using bootstrapping. None of the bootstraps replicates produced tree containing ties. The consistency index for each node revealed six loci were supporting the node 1 while all the 15 loci supported the node 2. The Nei's distance and identity values and their unbiased estimates are presented in Table 5.
The analysis revealed similar dendrogram for standard and minimum genetic distance. A third analysis of the genetic distance was calculated taking an assumption that if the period of differentiation is small then the effect shall be solely due to the coancestery/random genetic drift. The dendrograms produced have also been presented in Figure 1, 2, and 3.
The genetic distances were calculated using allelic frequencies and the dendrograms were constructed. UPGMA clustering using (Nei, 1972) original distance gave two Nodes with distances 0.5899 and 0.8758. The values were 0.5609 and 0.8334 for unbiased distance. The Node 1 had Aseel and Nicobari while Node 2 had all the three populations. The bootstrapping results using 1000 permutations revealed 78.60% similar replicates for Node 1 and 100% for Node 2. No bootstrap replicates produced trees containing ties. The alternative topologies did not exist. Six of the 15 loci (40%) supported Node 1 while all the 15 loci supported Node 2. While 7 of the 15 loci supported node 1 when the procedure of minimum distance was employed. Similar results were also produced when data was analyzed using Reynold's coancestory procedure. The data was further analysed using unbiased values and the genetic distances were obtained for node 1 and node 2. The values obtained were 0.5609 and 0.8334 respectively. Six loci supported the node 1 and all the 15 loci supported the node 2.
All the methods revealed similar phylogenetic tree and has support from the history and geographical location. The closed identity between Nicobari and Aseel supports the view that Aseel poultry birds must have been carried in ships by the traders from the Paradeep seaport (on the east coast), which was an important seaport in earlier times. This is reflected by the fact that these two breeds separated 655 generations ago or approximately 450 years. The geographical location of Miri poultry in Northeastern region of Assam has contiguity with the Aseel breed habitat but separated by more than 1,200 to 1,500 kilometers. The Miri breed might have separated a further 300 generations or 200 years ahead of separation between Aseel and Nicobari breeds.