بررسي چند شكلي DNA در جمعيت‌هاي AG-4 Rhizoctonia solani با استفاده از نشانگر مولكولي rDNA RFLP

Study on DNA polymorphism in populations of Rhizoctonia solani anastomosis group 4 using rDNA RFLP

توالي­هاي DNA ريبوزومي به ­دليل اينكه داراي كپي­هاي زيادي از ژن­هاي rRNA و نيز درجه بالايي از تغيير مي­باشند، به ­منظور بررسي روابط فيلوژني در محدوده وسيعي از سطوح تاكسونومي مورد استفاده قرار مي­گيرند. نواحي ITS و فواصل داخل ژني DNA ريبوزومي هسته اي، واحد­هاي تكراري تكامل يافته ثابت مي­باشند و ممكن است در بين گونه هاي داخل يك جنس يا در بين افراد يك جمعيت متغير باشند. به­ منظور ارزيابي چند شكلي نواحي حد فاصل ژن­هاي s28 و s18 در ژنوم قارچ Rhizoctonia solani گروه آناستوموزي 4 (AG-4)، 28جدايه از ميزبان­هاي مختلف مورد ارزيابي قرار گرفتند. از جدايه­ها DNA استخراج و از آن­ها به عنوان الگو در واكنش­هاي PCR استفاده شد. در تكثير نواحي فاصله­ساز داخلي بين ژن­هاي ريبوزومي با استفاده از آغازگر­هاي ITS1 و ITS4 يك قطعه DNA به اندازه 700 جفت باز رديابي شد. قطعه مذكور با آنزيم­هاي برشيTaq 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. ولي آنزيم­ هاي Xba I, Bam HI, Hind III, Sac I, Pst I, Nde I, Xho I فاقد محل برش بودند. نتايج حاصله نشان داد كه جدايه­ هاي Rhizoctonia solani AG4 هتروژن بوده و ITS-RFLP با استفاده از آنزيم Hinc II تفاوت بين زيرگروه­هاي AG4-HG I و AG4-HG II رااز نظر الگوي برش آنزيمي 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.