Estimation of Genetic Diversity in Four Genetic Groups of Local Kurdish Chicken Using RAPD- PCR

Keywards: Kurdish Chicken, Molecular Genetics, Characterization, RAPD. يوونلا ضماحلل يئاوشعلا ميخضتلا ةينقت مادختسأب يلحملا يدروكلا جاجدلا نم ةيثارو تاعومجم عبرا يف يثارولا عونتلا ريدقت لاكشلاا ددعتم

in order to satisfy their genetic characteristics and know what genes they have. Therefore the progress of genetic science especially by development of molecular techniques, it has been possible to make selection (4), Identification (5), characterization (6), and genetic improvement (7) of livestock.
Up to date, there is no research on the genetic convergence between these lines, which stated as the main objective of this experiment between the production lines in the province of Kurdistan region.

Chickens lines:
The current study was carried out in July 2019. Blood samples were collected from a total of (52) local chickens bred at the farm of animal production department, college of Agriculture, University of Salahaddin Erbil, and at animal production department, college of Agriculture, University of Sulaimani, in association with the ministry of Agriculture in KGR-Iraq. The chickens were representing Black (B=12), Black with brawn neck (BBN=18), white with shank feather (WSF=12), and white non-feathering shank (WNFS= 10) as it shown in Table 1.

Blood samples collection and DNA extraction:
At least 2.5 ml of fresh blood sample was withdrawn from wing vein from each of the chicken in EDTA tube, (23) gauge needle. The blood was gently mixed with anticoagulant, and kept on ice. Subsequently the blood samples were transported to the laboratory and stored at -20 C° until the isolation of genomic DNA.
Genomic DNA was isolated using a commercial kit, AccuPrep Genomic DNA Extraction kit (Bioneer, South Korea) with slight modifications. [20 μl of proteinase K, total volume 200 μl of 20 μl whole blood and 180 μl of PBS, and 200 μl of Binding buffer (GC)] were added to 1.5-μl Eppendorf tube, the samples mix immediately by vortex mixer and then incubated at 60 °C for 10 min in boiling water bath. After incubation, 100 μl isopropanol was added and gently pipetted for a few min, then samples spin down at 10.000 rpm for 1 min. The cleared lysates were transferred into binding column tube (2 ml) then centrifuged at 12.000 rpm for 2 min and then the binding column tube transferred to new (2 ml) tube.
The samples were concentrated and the purity of the extracted DNA examined using Nanodrop (Nano-Drop2000, Delaware USA) based on 260 and 280 nm absorbance and agarose gel electrophoresis analysis (29).
RAPD-PCR condition and electrophoresis: The RAPD bands were scored visually from the Ethidium bromide staining agarose gel. The analysis was based on the generated date from the detection of polymorphic fragments. The genotypes were analyzed in the form of binary variable by scoring the presence as (1) and absence (0). The genetic distance and genetic identity among the genetic groups in both males and females were calculated using Nei equations (31) by using (pop gene) software Table (2) was shown all of primers that were used with their information. A total of 10 primers were screened for RAPD analysis. One hundred twenty four polymorphic bands were amplified, the bands size ranged between 100 bp and 2500 bp. The primer (OPA-05, OPA-18) produced maximum number of polymorphic bands, while primers named (OPA-6) produced minimum number of polymorphic bands. The detected polymorphisms were varied among the four genetic groups (black, black brown neck, white shank feather, and white non-feathering shank). The (WNFS) genetic group showed the highest number of amplified fragments (55) for both male and female (30,25) respectively. While (BBN) genetic group showed the lowest number of amplified fragments (20) for both male and female (7, 13) respectively as we showed in (Table 3).

Results and discussion
Table (4) shows the Na and Ne, h, I for the two groups of male and female Kurdish local chicken was analyzed using ten RAPD markers.
The genetic distance in male groups found highest (D=1.2040) between B and WSF and lowest (D=0.1054) between (B) and (WNFS). While in female groups it was found that the highest genetic distance (D=0.9163) was observed between (WSF) and (WNFS) and the lowest (D=0.2231) was between (BBN) and (WNFS). In addition, the males genetic identity was higher (I=0.900) between (B) and (WNFS), and lower (I=0.3000) was between both (B), and (WSF), and between (BBN) and (WSF). In females genetic identity the highest (I=0.8000) was between (BBN) and (WNFS) and the lowest (I=0.4000) was between (WSF) and (WNFS) ( Table 5). Dendrogram was constructed to show the phylogenetic relationships among the four lines Figure 1 was shown the male phylogenetic relationship that indicates the (WSF) genetic group to be the most distance from the other genetic groups, while B and BBN were closely related. As for female phylogenetic relationship ( Figure 2) indicate that (WNFS) line recorded most distance from the other genetic groups, while (BBN) and (WSF) were closely related.
It is obvious that the project of breeding local chicken in Kurdistan region government-Iraq was started 15 years ago. The four mapped genetic groups (B, BBN, WSF, WNFS) were bred and selected for 12 generation, in the directorate of agricultural research in Erbil, moreover it is selected according to the feathering color and appearance of shank feather (12). The results of genetic distance in male genetic groups of this study supporting the findings of (21) who recorded that there was no significant difference between genetic groups (B) and (WNFS) in their egg length, which both inherited by roosters (32). Moreover the (WSF) genetic group was higher in their egg length while the lower recorded for the (BBN) genetic group. Furthermore the genetic distance of the hen genetic groups was linked to the breadth traits, which already inherited by hen. According to the correlation results found by (21), there is Vol. 13 Issue:2, (2020) ISSN: P-1999:6527 E-2707:0603 no significant difference between genetic groups (BBN) and (WSF), moreover the (WNFS) genetic group was the higher and the (B) genetic group was the lowest in egg breadth trait. (33) used the RAPD marker in their study to characterize Five Egyptian indigenous chicken strains and they found that RAPD marker is a valuable tool to evaluate genetic diversity of chicken.

Conclusions
According to the results obtained from the current study, it can be conclude that the four lines differ genetically. Moreover the results will help the breeders to study new selection strategies between the four genetic groups. Further studies are needs to provide the genetic map of these genetic groups based on microsatellite markers.