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All Journal HAYATI Journal of Biosciences Indonesian Journal of Biotechnology
Keiko T. Natsuaki
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Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Earth & Planetary Sciences Immunology & microbiology Materials Science & Nanotechnology

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Two random amplified polymorphic DNA (RAPD) markers linked to CMV-B2 resistance gene (Creb-2) in melon cultivar Yamatouri were cloned and sequenced to design sequence characterized amplified region (SCAR) markers for detection of CMV-B2 resistance gene (Creb-2) in melon. SCOPE14 derived from OPE-14 yielded a single DNA band at 541 bp, while SCAPB05 derived from APB-05, yielded a single DNA band at 1,046 bp, respectively. Segregation of SCOPE14 and SCAPB05 markers in bulk of F2 plants demonstrate BUDI SETIADI DARYONO; KENJI WAKUI; KEIKO T. NATSUAKI
HAYATI Journal of Biosciences Vol. 16 No. 4 (2009): December 2009
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.16.4.142

Abstract

Two random amplified polymorphic DNA (RAPD) markers linked to CMV-B2 resistance gene (Creb-2) in melon cultivar Yamatouri were cloned and sequenced to design sequence characterized amplified region (SCAR) markers for detection of CMV-B2 resistance gene (Creb-2) in melon. SCOPE14 derived from OPE-14 yielded a single DNA band at 541 bp, while SCAPB05 derived from APB-05, yielded a single DNA band at 1,046 bp, respectively. Segregation of SCOPE14 and SCAPB05 markers in bulk of F2 plants demonstrated that they were co-segregated with RAPD markers from which the SCAR markers were originated. Furthermore, results of SCAR analysis in diverse melons showed SCAPB05 primers obtained a single 1,046 bp linked to Creb-2 in resistant cultivars Sanuki-shirouri and Kohimeuri. However, SCOPE14 failed to detect Creb-2 in diverse melons. Results of this study revealed that SCAR analysis not only confirmed melons that had been clearly scored for resistance to CMV-B2 by RAPD markers, but also clarified the ambiguous resistance results obtained by the RAPD markers.         Key words: Cucumis melo L., Creb-2, RAPD, SCAR
Application of Multiplex RT-PCR for Detection of Cucurbit-infecting Tobamovirus Budi Setiadi Daryono; Keiko T. Natsuaki
Indonesian Journal of Biotechnology Vol 16, No 1 (2011)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (953.754 KB) | DOI: 10.22146/ijbiotech.7833

Abstract

Cucumber green mottle mosaic virus (CGMMV) and Kyuri green mottle mosaic virus (KGMMV) are seed borne viruses and they are also transmitted mechanically during agricultural practice and through water. Hence, these viruses have potential diseases widely distributed throughout the world. To detect different strains of CGMMV and KGMMV, several specific primers for each virus were designed for single and multiplex RT-PCR. The results of single and multiplex RT-PCR showed that CGMMV was detected in zucchini isolated in Bali-Indonesia, while KGMMV was detected both in zucchini isolated in Bali-Indonesia and Cucumis metuliferus isolated in Thailand. Furthermore, artificial co-infection of these two viruses was prepared and carried out using two different ways of viral RNAs extraction. Based on the results, it could be reported that viral RNAs for cDNA amplification by multiplex RT-PCR could be extracted from a mixture of infected leaves or separate extraction of each viruses infected leaves. In addition, results presented in this study demonstrated the application of multiplex RT-PCR to simultaneously detect CGMMV and KGMMV from cucurbit leaves using a mixture of four primers and its feasibility as a sensitive and rapid laboratory assay. Since, no multiplex RT-PCR technique has been described for the detection of CGMMV and KGMMV, this technique can be a good option for sensitive and reliable tool for detection of two major cucurbit infecting Tobamoviruses.Keywords : Cucurbit infecting Tobamovirus, multiplex RT-PCR, seed borne viruses
Co-Authors BUDI SETIADI DARYONO KENJI WAKUI