بررسي تنوع ژنتيكي ژنوتيپ‌هاي نخود با استفاده از نشانگر مولكولي SSR

Investigation of genetic diversity among chickpea genotypes using SSR markers

بهره‌برداري از تنوع ژنتيكي يكي از عوامل مهم در بهنژادي گياهان زراعي است. اين تحقيق به‌منظور ارزيابي تنوع ژنتيكي 35‌ژنوتيپ نخود با استفاده از 15‌جفت آغازگر SSR، انجام شد. پس از استخراج DNA، انجام واكنش‌هاي PCR و الكتروفورز، آغازگرها در مجموع 49‌آلل را در تمام ژنوتيپ‌ها تكثير كردند. متوسط تعداد آلل تكثيرشده به ازاي هر آغازگر 3/26 آلل بود. آغازگر SSR35 با پنج ‌آلل، همراه با آغازگرهاي SSR14، SSR21، SSR26 و SSR63 با چهار آلل، بيشترين و آغازگرهاي SSR61 و SSR7 با 2‌آلل، كمترين آلل را تكثير نمودند. بر اساس ضرايب تشابه به‌دست‌آمده ارزشهاي تشابه دامنهاي از 0/10 تا 0/80 با ميانگين0/40 درصد را داشتند. ميزان اطلاعات چندشكلي (PIC)، از 0/20 تا 0/88 متغير بود (با متوسط 0/50). بيشترين و كمترين ميزان PIC به‌ترتيب مربوط به آغازگرهاي SSR21 و SSR62 بود. تجزية خوشهاي با استفاده از ضريب تشابه جاكارد و روش UPGMA انجام شد و ژنوتيپ‌ها در هشت گروه قرار گرفتند. گروهبندي تا حدودي توانست ژنوتيپ‌هاي مختلف نخود را از همديگر تفكيك نمايد. تفكيك ژنوتيپ‌ها از طريق تجزيه هماهنگ اصلي و پلاتهاي دو و سه‌بعدي نيز تقريباً تأييد‌كنندة تجزية خوشه اي بود. ضريب همبستگي كوفنتيك بالا بيانگر ارتباط مؤثر بين داده هاي مولكولي، روش تجزيه خوشهاي و صحت گروهبندي ژنوتيپ‌ها بود. از تنوع موجود ميتوان در برنامه هاي اصلاح نخود استفاده نمود.

Introduction Utilization of genetic diversity in crop plants is very important for breeding objectives. Chickpea is the third most important grain legume and its seeds contain protein that is an important energy source for human. It ranks third worldwide among grain legumes. In Iran this crop cultivated at about 473000 hectares with an average annual production of 195000 tons. Genetic diversity of chickpea genotypes using SSR markers were examined in several studies.The objective of this study was investigation of genetic diversity among some chickpea genotypes using molecular data obtained from SSR primers. Materials & Methods Genetic diversity of 35 chickpea genotypes (Including cultivated, local variety and promising line) were assessed using 15 SSR primers. These primers were selected from previous study. DNA was extracted from two-week old plants of each genotype following the protocol of CTAB-method. After DNA extraction, the quantity of DNA was measured under 0.8% agarose gel electrophoresis. DNA concentration was estimated using Picodrop. The final DNA concentration of each template stock was adjusted to 50 ng/μl. The PCR was performed in a Thermal Cycler (Bio-Rad Model thermal cycler) in a volume of 15 μL. The amplification step was as follows: 1 cycle at 94°C for 1 min, then 40 cycles comprising 94°C for 5 min, annealing of primer at 50-65°C (depending on the primer) for 1 min. The final extension was carried out at 72°C for 10 min. The amplification products were electrophoresed on 3.5% agarose gels (combination of 50% Metaphor and 50% LE Agarose), and for staining, 3 μL Gel Red and dye (the 1.5:1.5 ratio) was added to each sample. Photographed was performed using the Bio-Rad Gel Doc. Molecular data was analyzed using the NTSYS-pc software version 2.02. Results & Discussion All of fifteen SSR primers were generated scorable bands. Totally 49 alleles (ranged between 2 to 5 alleles per each locus) with an average 3.26 allele per locus was distinguished. These results are agreement with the results of some study and did not match to other. One of the reasons may be due to the use of different genetic material and SSR markers. Majority of primers identified high level of polymorphism. Jaccard similarity coefficient values among genotypes, ranged from 0.10 to 0.80, average value of similarity coefficient was 0.40. The highest similarity was found between Hashem and Arman genotypes. Polymorphic information content (PIC) ranged from 0.20 to 0.88 (average 0.50). The highest (0.88) and the lowest (0.20) value of PIC was pertained to SSR21 and SSR62 Primers, respectively. A high mean PIC value can be attributed to the use of more informative markers. Unweighted pair group method of the arithmetic average (UPGMA), based on Jaccard similarity clustering form a dendrogram with eight genotypes group. Eight groups can be distinguished by truncating the dendrogram at mean similarity coefficient value of 0.40. Clustering somewhat was distinguished chickpea genotypes, as cultivated genotypes and local were put together. In the present study SSR markers almost succeed in separation of genotypes but this marker could not separate promising chickpea completely. Principal co-ordinate analysis (PCoA) was carried out on the mean pairwise genetic distances to display the genetic relationship of genotypes in the PCoA 2D and 3D plot. 2 and 3D plot were confirmed the results of cluster analysis. Cophenetic correlation showed that molecular data and cluster was corresponded. Conclusion SSR primers that used in this study are more informative in chickpea genotypes. The genotypes almost showed diverse and distinct SSR patterns. It was concluded that SSR marker was suitable for evaluation of genetic diversity in chickpea genotypes and this genetic diversity can be used in chickpea breeding programs. To achieve better results in crossing programs, we recommended also these genotypes get evaluated using morphological and agronomic traits.