Genetic Diversity of Indigenous Chicken (Gallus Gallus domesticus) from Ecozones of Egypt and Kingdom of Saudi Arabia

Genetic diversity of two chicken ecotypes from Ismailia-Egypt (ISM) and Taif-Saudi Arabia (TA) was evaluated using 39 microsatellites. DNA was extracted from blood of 25 chickens/ecotype. The number of alleles was 157 and 138, the number of alleles/locus averaged 4.2±2.2 and 3.6±1.6, and the highest number of private alleles was 9 and 5 for ISM and TA, respectively. Percentage of shared alleles between the two ecotypes was 45%. This panel of markers is reasonably informative as the mean polymorphic information content for ISM and TA was 0.47±0.21, and 0.41±0.2. Similar average of observed heterozygosity was attained for both ecotypes. Conversely, averages of expected heterozygosity differed between two ecotypes, 0.52±0.23 vs. 0.45±0.21 for ISM and TA. 8 and 12 loci have significantly deviated from HWE of ISM and TA. Estimate of genetic distance was 0.2 and FST index was 0.29. Results showed only 6% of genetic diversity is shared between these two ecotypes.


Introduction
Chickens' ecotypes are being utilized in rural and backyard farming of developing countries all over the world. The significance of these ecotypes in the rustic economy is tremendous in various counties [1]- [7]. This significance is due to affordable low production cost, scavenging competency as well as adaptability to harsh and stressful environmental conditions [8] [9]. Moreover, chicken ecotypes supply work security to the family as well as getting the accessibility of food. Jobless youth and ladies can likewise acquire pay through poultry cultivating [10]. Still, local ecotypes have low egg productivity and light mature body size [11] [12].
The poultry industry is globally dominated by commercial chicken breeds, where breeding strategy is based on few chicken genotypes. Such a strategy has the shortcoming of erosion of valuable genetic resources (ecotypes) [13]. Their low productivity does not meet the global demands of intensive production systems, as such ecotypes could barely compete with commercial breeds. Therefore, ecotypes are negatively selected regardless of their good quality of egg and meat, disease resistance as well as adaptation to the local environment [13]. The establishment of a framework for preserving these genetic resources is of need [8].
Investigating the genetic makeup of indigenous chicken breeds was carried out in several studies [11] [14] [15] [16] and has been used as a sampling guide in genetic diversity research [17].
At Taif governorate (~1.7 km above sea) local chickens are acclimated to the rough environment of high altitudes, such as low oxygen tension, as well as daily temperature fluctuations [18] [19] [20] [21].
Microsatellites (MS) markers are normally used because they are plentiful, codominant, randomly spread throughout the genome, and very polymorphic, also because of their reproducibility [22] [23]. This coherence has made MS markers a dependable tool for genetic diversity evaluation and assessment in many commercial chicken breeds and ecotypes [13] [18] [19] [20]. Quite a few MS markers are available in chickens and have been utilized to develop linkage maps in numerous chicken breeds [22] [24]. These markers introduced efficient machinery to QTL research and have additionally been effectively utilized to consider the hereditary associations among and within chicken populations [23].
Osaman et al., [25] utilized the complete sequence of mitochondrial DNA D-loop to explain the genetic descent of Egyptian indigenous chicken and Asian chicken. Results of this study revealed that both Egyptian native chicken and West and Central Asian chicken are sharing the same common ancestor as they branched together in the same clade. However, this work did not include native chicken strains from Saudi Arabia. Therefore, the current study was carried out as an attempt to introduce a better understanding of the genetic characterization of 2 ecotypes sampled from two different ecozones at Taif (Saudi Arabia) and Ismailia (Egypt). A dense microsatellites panel of 39 markers was genotyped for two ecotypes.

Sample Collection
Chicken samples were collected from individual framers in local village either in

DNA Extraction
Collected blood samples were used for DNA isolation using DNA extraction kit (QIAGEN) according to manufacture manual. Extracted DNA quality checked by electrophoresis in a minigel while the quantity checked spectrophotometrically using NanoDrop 2000C (Spectronic Genesys, Thermo Electron Corporation). DNA samples were stored at −20˚C until microsatellites analysis.

Microsatellite Genotyping
Thirty nine MS markers were utilized to assess DNA polymorphism of these 2 ecotypes. These MS markers were chosen based on reports by the International Society of Animal Genetics (ISAG)-FAO to think about the hereditary assorted variety of chickens [26] and additionally 150 microsatellites were investigated for the segregation of five thoroughbred KNC lines [20]. Names of each of the 39 MS markers, chromosome number and respective allelic size are appeared in Table 1.

Data Analyses
Exploratory data analysis and graphical representations of the results were carried out under R statistical environment R Core Team [27]. Fundamental measures of genetic diversity, such as total number of alleles, allele frequencies, mean number of alleles, observed, and expected heterozygosity (Nei, 1987) were computed using adegenet R statistical package [28]. Deviation from Hardy-Weinberg equilibrium (HWE) using chi square test, and phylogenic analysis using Nei's distance [29] as well as population subdivision was examined using Weir and Cockerham [30] unbiased estimator of Wrights fixation indices (F IT , F ST and F IS ) [31] was carried out using "pegas" package R Population and Evolutionary Genetics Analysis System [32]. The null hypothesis was that the estimates were not significantly different from zero, and the level of significance (P < 0.01) was adjusted using Bonferroni correction. Allelic richness number was estimated using hierfstat package [33]. Phylogenetic analysis was carried out using APE package [34].  locus ranged from 1 to 10 alleles, averaged 4.2 ± 2.2. The TA ecotype exhibited a lower number of alleles sum 138, the number of alleles per locus ranged from 1 to 7 averaged 3.6 ± 1.6. These estimates are lower than the reported on other studies, e.g. Abebe et al., [13] on Swedish native chicken (4.7), Muchadeyi et al., [16] on Zimbabwe ecotypes (9.7), Van-Marle-Koster et al., [35] on South Africa native chicken (6.1), and Ramadan et al., [36] on native Egyptian and commercial chicken breeds (7.7). These lower estimates of the present study could be attributed to utilizing a larger number of loci. The number of alleles per locus could also be represented as "allelic richness". The number of observed alleles is basically a function of sample size. Allelic richness (R t ) was estimated across all loci ( Table 2). One definition of R t is the mean number of alleles per locus [37].

Results and Discussion
The significance of R t is that decrease in R t might result in reducing ability of the population to acclimate to future environmental changes, as such an assorted variety is the rough material for evolutionary forces, for example, common determination [38].
The mean of polymorphic information content (PIC) for ISM and TA ecotype were 0.47 ± 0.21, and 0.41 ± 0.2. This means that this penal of markers are Open Journal of Applied Sciences reasonably informative as PIC exceeds 0.25 Botstein et al., (1980). For ISM ecotype 54% of markers are highly informative as PIC was higher than 50%, where only 38% of TA's markers were highly informative. The mean value of PIC in this study was smaller than the estimates of other studies on different native breeds e.g., Seo et al., [20] estimate of PIC was 0.77 on five native Korean lines, Ramadan et al., [36] reported 0.65 on six Egyptian native breeds, and Choi et al., [39] on commercial Korean native breed reported mean PIC value of 0.68, and Soltan et al., [40] estimation of PIC was 0.84 on Sinai and Norfa Egyptian native chicken. However, lower estimate of 0.55 was reported by Abebe et al., [13] on five local Swedish breeds. Again, these differences in mean value of PIC could be ascribed to the dense panel of markers were used in the present study.
The presence of private alleles was also investigated (   Breaking up the genetic diversity within and among populations is best described through Wright's F-statistics (Wright, 1951). Table 4 shows Wright's fixation indices (F ST , F it , and F IS ) across ISM and TA ecotypes for the genotyped microsatellites markers. Figure 3 shows a boxplot for the estimates of Wright's  [13] on Swedish local breed, Seo et al. [20] on Korean chicken as well as Ramadan et al., [36], Soltan et al., [40] on Egyptian native breeds.
Average value of global heterozygosity index (F IT ) was 0.102, ranged from −0.04 to 0.48. This index could be considered as correlation between alleles within individuals with respect to combined sample [41]. The inbreeding coefficient (F IS ) across the 39 MS loci averaged 0.65 ranged from 0.14 to 1.00. This estimate is higher than the other studies on indigenous chicken breeds e.g. 0.187 on Swedish breeds [13], 0.0093 on Korean indigenous breeds [20] and 0.018 on Egyptian indigenous breeds [36], but Soltan et al., [40] reported somewhat closer estimate on Sinai and Norfa indigenous Egyptian chicken breeds. F IS is also taken as an indicative of endangerment potentiality and devised to assess the conservation priorities [44]. Thus, when F IS is lower than 0.05, breed is not in danger; the range from 0.05 to 0.15, breed is potentially endangered; between 0.15 -0.25, they are slightly endangered; between 0.25 -0.40, they are endangered; and more than 0.40, breed is seriously endangered [36] [40]. Open Journal of Applied Sciences Diversity assessment between population is normally quantified by estimation of genetic distance. Both Nei's genetic distance [29] as well as Nei's pairwise F ST index between all pairs of populations [45] was estimated for the two ecotypes over the 39 MS loci. The estimate of Nei's genetic distance was 0.2 where Nei's pairwise F ST was 0.29. This small value is comfortable with the percentage of shared alleles of the two ecotypes 45%.

Conclusion
The results of the present study are, presumably, the first to recount the genetic diversity between two ecozones Egyptian and Saudi Arabia ecotypes. To be concluded, a small proportion of genetic diversity due to allele frequency differences was between these two ecotypes was only 6%. This result is also supported by small estimate of Nei's genetic distance (0.2) as well as small Nei's pairwise F ST (0.29). The percentage of shared alleles of the two ecotypes was 45%.