ارزيابي تنوع ژنتيكي اكوتيپ‌هاي كلكسيون عناب ايران (Ziziphus spp.) با استفاده از نشانگر مولكولي ISSR

Evaluation of Genetic Diversity in Collection from Iranian Jujube Ecotypes (Ziziphus spp.) using ISSR-molecular Marker

عناب (Zizyphus jujuba Mill.) به عنوان یك میوه ارزشمند و مقاوم به شرایط مختلف آب و هوایی در بسیاری از مناطق ایران گسترش دارد. از عناب علاوه بر صادرات بعنوان گیاه دارویی نیز استفاده می‌شود لذا ارزش اقتصادی قابل توجهی دارد. ارزیابی تنوع ژنتیكی در مواد گیاهی از اهمیت بالایی برخوردار بوده و گام اولیه برای شناسایی و نگهداری ذخایر ژنتیكی و از اصول اولیه اصلاح گیاهان محسوب می‌شود. استفاده از نشانگرهای DNA از بهترین روش‌های موجود برای بررسی تنوع ژنتیكی می‌باشند. در این بررسی از نشانگر ISSR به منظور بررسی تنوع ژنتیكی 31 اكوتیپ عناب جمع‌آوری شده از هشت استان عناب‌خیز كشور استفاده گردید. تعداد 13 آغازگر در واكنش PCR آزمایش شدند كه شش آغازگر DNA‌ی الگو را بخوبی تكثیر و در بین نمونه‌ها چندشكلی را آشكار نمودند. از میان 84 مكان ژنی شناسایی شده، تعداد 70 مكان (83‌ درصد) چندشكلی نشان دادند. تجزیه خوشه‌ای اكوتیپ‌ها بر اساس ضریب تشابه دایس و به روش UPGMA انجام گرفت. در تجزیه خوشه‌ای، نمونه‌ها در سطح تشابه 85/0 در هفت گروه اصلی جای گرفتند. بیشترین تشابه ژنتیكی (95/0) بین نمونه‌های كلاله و درح و همچنین نوغاب و دوستیران و كمترین تشابه ژنتیكی (48/0) بین نمونه‌های برزادران و كنگان بدست آمد. آغازگرهای(AC)8YT و (GA)8Aمناسب‌ترین آغازگرها برای مطالعات بعدی تشخیص داده شد. نتایج این مطالعه نشان داد كه تنوع كل نمونه‌های كشور تقریباً در تنوع ناحیه خراسان‌جنوبی خلاصه شده است. همچنین در اكوتیپ‌های متعلق به استان‌های اصفهان و مازندران، سطح قابل ملاحظه‌ای از تنوع مشاهده شد.

Introduction Jujube (Zizyphus jujuba Mill.) as a valuable medicinal plant and adapted to different climatic conditions is widespread in many parts of Iran. Nowadays, beside the export of its fruit, jujube is also used as an herbal medicine to treat the diseases, so it has a high economic value. Study on genetic diversity is the first step to identify and preservation of germplasm. It is also considered as the basic principles of plant breeding. DNA markers seem to be the best way in determination of the genetic diversity. Inter simple sequence repeats (ISSR) markers are highly polymorphic and combine most benefits of Simple Sequence Repeats (SSRs) and amplified fragment length polymorphism (AFLP) to the generality of random amplified polymorphic DNA (RAPD). Materials and Methods In order to study of the genetic diversity among 31 ecotypes collected from eight Jujube-rich provinces, including South Khorasan, Razavi Khorasan, Mazandaran, Golestan, Qom, Isfahan, Lorestan and Fars. Genomic DNA was extracted by CTAB method and polymerase chain reaction (PCR) was performed with 13 ISSR primers in which six most efficient primers were selected. Cluster analysis based on Dice similarity coefficient and Unweighed Pair Group Method with Arithmetic Mean (UPGMA) was carried out and POPGENe.3.2 software was used to determine the similarity of populations with each other. Results and Discussion 84 loci were amplified and 70 of them (83%) revealed a proper polymorphism with the size between 200 and 2000 base pair. The average number of amplified and polymorphic bands per primer was 14 and 11.6 respectively. Primers with di-nucleotide repeats produced more polymorphic bands than ones with tri-nucleotide repeats. It seems that this is due to a higher frequency of sequences containing di-nucleotide repeats in intergenic regions and higher possibility of mutation revealed in more diversity in comparison to gene coding regions. Anchored primers with 1 or 2 nucleotides at the 5’ end make sure annealing only to the ends of SSRs in template DNA, so avoiding internal priming and smear formation. In addition, the anchor lets only a subset of the microsatellites to serve as priming sites. Primers (AC)8YT and (GA)8A with the higher percentage of polymorphism is recommended for further analysis. According to the cluster analysis, the ecotypes could be classified into seven main groups at the 0.85 level of genetic similarity. The most genetic similarity (0.95) was observed between ecotypes from Kalaleh and Doroh and also Noghab and Dustiran and the least genetic similarity (0.48) observed between Kangan and Borzaderan. POPGENe.3.2 software data indicated that populations of Isfahan and South Khorasan had the slightest difference while populations of Isfahan and Razavi Khorasan showed the most difference. Conclusions In general results demonstrated that the total diversity of jujube ecotypes in Iran is summarized in the area of South Khorasan province. Given data showed that South Khorasan has been an original place of cultivation of this medicinal plant, this area could be considered as one of the important centers of jujube diversity. In addition, significant levels of diversity were observed among ecotypes belonging to Isfahan and Mazandaran provinces.