Amplifying and Sequencing Analysis the Internal Transcribed Spacer (ITS) Regions of Olpidium Viciae Kusano’s Ribosomal DNA in Broad Bean
Plasmodiophora fire of broad bean is responsible for Olpidium Viciae Kusano, which is a kind of Fungi subdivided into bacteria flagellum amon. We have developed a polymerase chain reaction based method for the rapid identification internal transcribed spacer (ITS) regions of productionally significant fungi Olpidium Viciae Kusano from areas of 2500~3000 metres above sea level. Sequences of the nuclear internal transcribed spacer (ITS) regions ITS1 and ITS4 have been used widely in molecular characteristic studies because of their relatively high variability and facility of amplification. A universal quickly SDS micro-DNA extraction method was used combining a RNaseA pretreatment step to remove PCR interferential RNA. Target sequences in ITS regions genomic were amplified by PCR and sequenced. Using Hanpanchun lesion and healthy bean leaves as template and ITS1, ITS4 as primer to amplify ITS region, the results revealed ITS gene of broad bean genome could be amplified with size of 750bp from healthy leaves, it could be amplified two fragments of 750bp and 500bp from the DNA template extracted from Hanpanchun lesion tissue. The ITS sequence of Olpidium Viciae Kusano is 99% homoeology with Cercospora (grey speck) pathogen. This may lay the foundation for research about classification and analyze evolutionary relationships of Olpidium Viciae Kusano.
J. M. Yan et al., "Amplifying and Sequencing Analysis the Internal Transcribed Spacer (ITS) Regions of Olpidium Viciae Kusano’s Ribosomal DNA in Broad Bean", Advanced Materials Research, Vols. 271-273, pp. 507-513, 2011