بررسی تنوع ژنتیکی ژنوتیپ‌های نخود با استفاده از نشانگر مولکولی ssr

بهره‌برداری از تنوع ژنتیکی یکی از عوامل مهم در به‏نژادی گیاهان زراعی است. این تحقیق به‌منظور ارزیابی تنوع ژنتیکی 35‌ژنوتیپ نخود با استفاده از 15‌جفت آغازگر ssr، انجام شد. پس از استخراج dna، انجام واکنش‌های pcr و الکتروفورز، آغازگرها در مجموع 49‌آلل را در تمام ژنوتیپ‌ها تکثیر کردند. متوسط تعداد آلل تکثیرشده به ازای هر آغازگر 26/3‌آلل بود. آغازگر ssr35 با پنج ‌آلل، همراه با آغازگرهای ssr14، ssr21، ssr26 و ssr63 با چهار آلل، بیشترین و آغازگرهای ssr61 و ssr7 با 2‌آلل، کمترین آلل را تکثیر نمودند. بر اساس ضرایب تشابه به‌دست‌آمده ارزش‏های تشابه دامنه‏ای از 10/0 تا 80/0 با میانگین 40/0‌درصد را داشتند. میزان اطلاعات چندشکلی (pic)، از 20/0 تا 88/0 متغیر بود (با متوسط 50/0). بیشترین و کمترین میزان pic به‌ترتیب مربوط به آغازگرهای ssr21 و ssr62 بود. تجزیه خوشه‏ای با استفاده از ضریب تشابه جاکارد و روش upgma انجام شد و ژنوتیپ‌ها در هشت گروه قرار گرفتند. گروه‏بندی تا حدودی توانست ژنوتیپ‌های مختلف نخود را از همدیگر تفکیک نماید. تفکیک ژنوتیپ‌ها از طریق تجزیه هماهنگ اصلی و پلات‏های دو و سه‌بعدی نیز تقریباً تایید‌کننده تجزیه خوشه‏ای بود. ضریب همبستگی کوفنتیک بالا بیانگر ارتباط موثر بین داده‏های مولکولی، روش تجزیه خوشه‏ای و صحت گروه‏بندی ژنوتیپ‌ها بود. از تنوع موجود می‏توان در برنامه‏های اصلاح نخود استفاده نمود.

Investigation of genetic diversity among chickpea genotypes using SSR markers

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 twoweek old plants of each genotype following the protocol of CTABmethod. 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 (BioRad 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 5065°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 BioRad Gel Doc. Molecular data was analyzed using the NTSYSpc 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 coordinate 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.