Initial reseach on the presence of ssDNA Sri Lankan Cassava Mosaic Virus (SLCMV) on cassava (Manihot esculenta Crantz 1766)

Abstract

Investigation of the Sri Lankan Cassava Mosaic Virus (SLCMV) resistant cassava (Manihot esculenta Crantz 1766) varieties requires high-efficiency single-strand viral DNA (ssDNA) extraction and molecular detection procedures. In our study, we tested four different DNA extraction methods to isolate total DNA containing high concentration of ssDNA. In addition, using Polymerase Chain Reaction (PCR) and Real-time PCR (RT-PCR), we can detect SLCMV from infected samples by amplifying a region of the Rep/AC1 viral gene anda cassava’s housekeeping gene, Beta-Actin. As our results, a modified DNA extraction method, from Mason, Caciagli, Accotto, and Noris (2008) and Osena, Nyaboga, and Amugune (2017), namely R-kit, gained high ssDNA content from young lamina tissue (from lobes) leaf tissue. Therefore, R-kit is recommended to be used for further ssDNA-using studies. The infection of SLCMV in cassava samples can be detected by PCR and RT-PCR procedures. Moreover, in RT-PCR optimization processes we figured out that PCR efficiency of SqPCR1 primer set (1.851X) and Beta-Actin primer set (1.896X) is compatiple for being processed and analyzed at once. The RT-PCR procedure showed ability to detect lower level of SLCMV in samples than the PCR procedure. Our results provide some essential ideas for studying on low virus titer circumstances, especially for model plants studies, resistant varieties development, or clean germplasm maintenance.