MATERIALS AND METHODS

Indigenous Chicken Population
One hundred indigenous chickens were collected from the geographical areas of Northern (includes Irbid, Ajloun and Jerash governorates), Eastern (includes Mafraq and Zarqa governorates), Western (includes Amman, Al-Salt and Madba governorates) and Southern (includes Al-Karak and Al-Tafyleh governorates) provinces of Jordan. To avoid bias, 25 indigenous chickens were randomly collected from each province in order to cover a wide range of indigenous chicken ecotypes during the summer period of year 2006. Where relevant throughout this study, indigenous chicken population will be referred to indigenous chicken ecotype of each geographical area.

Blood and Tissue Samples Collection
Samples were collected in two forms; blood and tissue. The blood sample was collected from the wing vein and transferred into vacutainer tube. This method was used to sample all young chickens, while tissue sampling was applied to the chicken of old ages by taking a punch of ~0.5 cm of Wattle tissue using an animal punch applicator and then transferred into Eppendorf tube. Blood and tissue samples were immediately stored at a recommended temperature till the DNA has been extracted. All work for this research using animals was performed with the permission of and in accordance with the guidelines set by Animal Ethics Committee of Mutah University.

DNA Extraction
A commercial kit, Wizard^® Genomic DNA purification kit-Promega^® was used as a simple and convenient technique to isolate high quality genomic DNA from blood and tissue samples (Technical Manual, 2007). The recommended protocols in the technical manual were used for isolation genomic DNA from 10 mL blood volume and 0.5 cm animal tissues. After extraction step, precipitation of DNA pellet was dried for 30 min in a 37°C incubator, resuspended in 100 μL TE buffer and then incubated at 65°C for 5 min to aid solubilisation. Finally, the DNA sample was stored at 4°C (Sambrook et al., 1989).

DNA Quantification
Agarose gel electrophoresis of 1% was used to quantify DNA and check the integrity of genomic DNA. Figure 1 is an example of quantifying DNA by using 1% agrose gel for some DNA samples. All DNA samples were also quantified by spectrophotometer. The measurements were taken at λ 260 and 280 nm. The purity of DNA samples were ranged from 1.2 up to 1.9. Samples were then diluted to 10 ng μL^–1 for use in subsequent PCR (PTC-200 programmable Thermal Controller, MJ Research Inc.) reactions.

RAPD Markers Genotyping
Ten RAPD primers, as shown in Table 1, have been selected from Operon Technologies company, USA for their ability to be useful in studies of biodiversities, taxonomic identities and systematic relationships. At the beginning, 20 RAPD primers of genotyping process were screened to find high polymorphic primers. The 10 highly polymorphic identified RAPD primers were used in PCR reaction and further study (Table 1). PCR reaction for 20 μL volume conducted under the following conditions: 10 ng template DNA, 250 nM of each primer, 200 nM dNTPs, 1U Taq polymerase and 1.5 mM MgCl₂. The reaction was performed for each type of primer pair following the programs recommended in protocols of Sambrook et al. (1989). As an example, allele information collected from genotyping of some RAPD markers are shown in Fig. 2. All gel photographs were scored for the presence or absence of RAPD bands.

Fig. 1:	Agarose gel of 1% loaded with DNA ladder of 1 kb and DNA samples of 1 and 10 μL

Table 1:	Operon codes and DNA sequences and fragment size of the 10 random amplified polymorphic DNA primers used in polymorphic analysis of the Jordan indigenous chicken ecotypes

Fig. 2:	Agarose gel of 1% loaded with DNA ladder of 1 kb and three RAPD primers (OPZ-11, OPA-05 and OPT-07, consequently)

Population Genetic Parameters
Population genetic parameters of the studied chicken ecotypes were investigated using genotypic data of RAPD by utilizing POPGENE^® software (Yeh and Boyle, 1996). Using the calculated allele frequencies for RAPD markers in each chicken ecotypes, effective number of alleles (A_e), expected heterozygosity (H_e) (Nei, 1973), Shannon's Information index (I) or gene diversity (Shannon and Weaver, 1949) and measures of genetic identity and genetic distance were calculated. This program also constructed UPGMA dendrogram that showed the genetic distance among ecotypes constructed according to Nei (1978).

RESULTS

The ten studied RAPD markers molecular size showed very large number of bands within wide range from 200 to 4000 bp across all studied chicken ecotypes (Table 1). Table 2 shows the results of RAPD markers of the A_e, H_e and I in the indigenous chicken ecotypes for each studied locus. The mean A_e locus was the lowest for the Southern chicken ecotype (1.47), when compared with the other populations; 1.54, 1.64 and 1.7 for Eastern, Northern and Western chicken ecotypes, respectively. The OPZ-11 marker showed the highest value (1.84) of A_e for all chicken ecotypes. Despite the OPZ-11 showed low A_e (1.47) in the Northern chicken ecotypes, it was relatively very high A_e for Southern, Eastern and Western chicken ecotypes (ranged 1.81-2.00). Therefore, the OPZ-11 can be considered more effective in assessing chicken genetic polymorphism in the present population than other studied RAPD markers. This can be also concluded from its highest H_e and I across all chicken ecotypes over the other markers (Table 2).

In fact, large variation range of H_e and I was detected for all chicken ecotypes at different loci, from 0.28 to 0.41 (mean = 0.39) and from 0.42 to 0.60 (mean = 0.58), respectively (Table 2). The lowest H_e and I values were found in the Southern chicken ecotype (H_e of 0.28 and I of 0.42) and the greatest in the Western ecotypes (H_e of 0.41 and I of 0.60). The results also indicated that similar genetic diversity values were shown for the Northern (H_e = 0.36, I = 0.53) and Eastern (H_e = 0.33 and I = 0.51) ecotypes. On the other hand, there were large differences in the level of genetic diversity for the other two ecotypes; being the Western ecotype of highest and the South ecotypes of lowest. Particularly, the Western chicken ecotype showed higher levels of A_e, H_e and I than the other ecotypes. These may be partly due to that the Western ecotype carried the highest genetic variation between its individuals indicating higher level of genetic polymorphism, whereas the Southern ecotype showed the least genetic diversity or higher similarity among its individuals indicating lower genetic polymorphism. Table 2 also shows that the ten RAPD markers had an average H_e of 0.41, which is considered very high, ranging from 0.31 (OPC-13) to 0.46 (OPZ-11) for all indigenous chicken ecotypes.

Table 2:	The effective number of alleles, expected heterozygosity and polymorphism in the indigenous chickens ecotypes population using RAPD markers
Ae: Effective No. of alleles, He: Expected heterozygosity, I: Shannon's information index or gene diversity

Table 3:	Genetic identity* and distances of the four indigenous chicken ecotypes
*: Genetic identities are above the diagonal and genetic distances are below the diagonal

Fig. 3:	Dendrogram of genetic relationships among the four Jordan indigenous chicken ecotypes

The same was observed for the I; with the lowest (0.49) found for OPC-13 and the greatest for (0.65) for OPZ-11 (Table 2). The most notable results regarding inter-ecotype comparisons was that the highest A_e, H_e and I found at 5 RAPD markers (OPA-05, OPN-16, OPF-14, OPT-07 and OPR-09) in Northern chicken ecotypes compared with the lowest values in the same markers detected values in the Southern ecotype.

The inter-ecotype similarity indices were expressed as genetic identity and shown in Table 3. The largest value of genetic identity (0.97) was between the East and the West ecotypes followed by 0.96 between the North and the East ecotypes and 0.95 between the North and the West ecotypes. The lowest value of genetic identity (0.69) was between the North and the South ecotypes. Table 3 also shows the values of genetic distance between the studied ecotypes that ranged from 0.04 between the East and the North to 0.37 between the South and the North. The UPGMA dendrogram, based on the genetic distances, constructed the genetic relationship among the studied indigenous chicken ecotypes (Fig. 3). The dendrogram grouped the ecotypes genotypes into two main clusters. The first cluster was divided into two sub-clusters; the first included the East and the West ecotypes, while the second sub-cluster included only the North ecotypes. The second cluster included the only the South.

DISCUSSION

The importance of the studying genetic polymorphism of indigenous chickens as a genetic resource become a global programs to determine genetic distances among chicken populations and to establish core collections of diversity within each species. The A_e, H_e and I values were calculated to estimate the level of genetic polymorphisms in indigenous chicken ecotypes. The results were indicating that there was high genetic polymorphism in studied indigenous chickens. Earlier reports, using RAPD markers, indicated high gene diversity within Chinese, Russian, European, Indian, Egyptian and Asian indigenous chicken populations (Zhang et al., 2002; Semenova et al., 2002; Sharma et al., 2001; Ahlawat et al., 2004; El-Gendy et al., 2006). Heterozygosity estimates in those studies ranged from 0.02 to 0.48. Gene polymorphism was also detected in these studies within a range from 0.32 to 0.65. In general, the gene diversity and polymorphism estimates found in the present study were within the reported range in the earlier reports. Furthermore, the range of molecular size of detectable RAPD bands (from 200 to 4000 bp) was in agreement with the range (200 to 2000 bp) found by Ahlawat et al. (2004) and (128 to 5467) by Chatterjee et al. (2007).

The high H_e and I for the Northern, Eastern and Western ecotypes were expected because their individuals expressed high genetic variation at the most studied loci (Table 2). On the other hand, lowest genetic diversity estimates, H_e and I, among individuals of Southern ecotype was also expected because the five studied RAPD markers (OPA-05, OPN-16, OPF-14, OPT-07 and OPR-09) were limited in detecting polymorphisms (Table 2). In other words, Southern ecotype showed the least genetic diversity or higher similarity among its individuals indicating lower genetic diversity, whereas Western ecotype showed the highest genetic variation among its individuals indicating higher level of polymorphism. The greater polymorphisms in the Western chicken ecotypes may be due to larger population size, random mating and a wider distribution of geographical areas.

The inter-ecotype similarity found between the pairwaise comparisons ranged from 0.69 to 0.97 (Table 3). This is higher than that reported by Ahlawat et al. (2004), who reported the genetic identity between Indian chicken strains ranged from 0.77 to 0.87. The high identity values (0.96 and 0.97) between the Eastern ecotypes and each of the Northern and Western ecotypes can be supposedly explained by their common ancestors, past gene flow and similar geographical and environmental conditions. However, the lower identity values (0.69-0.85) between the Southern ecotypes and others may partly due to very long time divergence, absence of gene flow and dissimilar of geographical conditions. On the other hand, the Eastern, Northern and Western ecotypes showed the least genetic distances between each other, while the Southern ecotypes appeared most distant from them (Table 3). The genetic closeness between the three ecotypes clustered them into one cluster; while the Southern ecotype was further away in the phytogenic tree (Fig. 3). The first cluster included a sub-cluster of the Eastern and the Western chickens that were raised in similar conditions of moderate temperature in winter and mixed production system of flat areas. The Northern chickens of second sub-cluster were raised in mountains where cold temperature in winter and under semi-intensive production system (Abdelqader et al., 2008). The Southern ecotype was raised in the Southern parts of Jordan under scavenging production system of subtropical conditions.

In conclusion, genetic polymorphism of indigenous chickens was relatively high with high values of A_e, H_e and I values at each studied locus. Furthermore, the genetic similarity between the pairwise comparison of Northern, Eastern and Western ecotypes was very high. The largest genetic distance was found between the Northern and Southern ecotypes and the smallest between the Northern and Eastern ecotypes. The result of present study may prove to be valuable for the future conservation of genetic resources of indigenous chicken breeds in Jordan.

ACKNOWLEDGMENTS

This study was supported by Scientific Research Deanship at Mutah University (MU) (No.BA/120/14/636). The Author would like to thank Dr. Adel Abdl-Ghani and Sameer Abo-Esbah for help in RAPD genotyping from Genetics Lab for Animal and Plant at MU.