بررسی چند شکلی DNA در جمعیت‌های AG-4 Rhizoctonia solani با استفاده از نشانگر مولکولی rDNA RFLP
محورهای موضوعی : دو فصلنامه تحقیقات بیماریهای گیاهی
فرزانه بادپا
1
(دانش آموخته کارشناسی ارشد، گروه زیست شناسی، دانشگاه اصفهان، اصفهان، ایران.)
غلامرضا بلالی
2
(دانشیار، گروه زیست شناسی، دانشکده علوم، دانشگاه اصفهان، اصفهان، ایران.)
بهرام شریف نبی
3
(استاد، گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه صنعتی اصفهان، اصفهان، ایران)
کلید واژه: تنوع ژنتیکی, نواحی فاصله ساز داخلی(ITS), واکنش زنجیره‌ای پلی‌مراز, Rhizoctonia solani گروه آناستوموزی 4,
چکیده مقاله :
توالیهای DNA ریبوزومی بهدلیل اینکه دارای کپیهای زیادی از ژنهای rRNA و نیز درجه بالایی از تغییر میباشند، بهمنظور بررسی روابط فیلوژنی در محدوده وسیعی از سطوح تاکسونومی مورد استفاده قرار میگیرند. نواحی ITS و فواصل داخل ژنی DNA ریبوزومی هستهای، واحدهای تکراری تکامل یافته ثابت میباشند و ممکن است در بین گونه های داخل یک جنس یا در بین افراد یک جمعیت متغیر باشند. بهمنظور ارزیابی چند شکلی نواحی حد فاصل ژنهای s28 و s18 در ژنوم قارچ Rhizoctonia solani گروه آناستوموزی 4 (AG-4)، 28جدایه از میزبانهای مختلف مورد ارزیابی قرار گرفتند. از جدایهها DNA استخراج و از آنها به عنوان الگو در واکنشهای PCR استفاده شد. در تکثیر نواحی فاصلهساز داخلی بین ژنهای ریبوزومی با استفاده از آغازگرهای ITS1 و ITS4 یک قطعه DNA به اندازه 700 جفت باز ردیابی شد. قطعه مذکور با آنزیمهای برشیTaq I Xba I, Hae III, Bam HI, Hind III, Sac I, Pst I, Nde I, Xho I, Hinc II, Hinf I, Eco RI, هضم شد. ولی آنزیمهای Xba I, Bam HI, Hind III, Sac I, Pst I, Nde I, Xho I فاقد محل برش بودند. نتایج حاصله نشان داد که جدایههای Rhizoctonia solani AG4 هتروژن بوده و ITS-RFLP با استفاده از آنزیم Hinc II تفاوت بین زیرگروههای AG4-HG I و AG4-HG II رااز نظر الگوی برش آنزیمی نشان داد.
Ribosomal DNA (rDNA) sequences have been widely used to study the phylogenetic relationships in different fungi. Fungal nuclear rRNA genes are arranged as tandem repeats with several hundred copies per genome. These spacer regions are considerably more variable than the subunit sequences and have been widely used in studies on the relationships among species within a single genus or among intraspecific populations. To evaluate the polymorphism between 18S and 28S genes, 28 isolates of Rhizoctonia solani anastomosis group 4 were examined. Genomic DNA was extracted from the isolates and prepared for PCR reaction. The amplification was performed using internal transcribed spacer (ITS) ITS1 and ITS4 primers. A DNA fragment of 700 bp in size was detected in PCR products of all tested isolates. To assess existence of any further polymorphism in the ITS region, the PCR products were digested with restriction endonucleases. Although there were restriction sites for XbaI, HaeIII, BamHI, HindIII, SacI, PstI, and TaqI endonucleases, there were no restriction sites for NdeI, XhoI, HincII, HinfI, EcoRI and XhoI endonucleases. The endonuclease HincII recognized a restriction site on PCR products that discriminated the isolates belonging to AG4-HGII form isolates of AG4-HGI. Based on the results, it has been concluded that AG-4 isolates of Rhizoctonia solani wereheterogenic.
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