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Improved nucleic acid extraction protocols for Ganoderma boninense, G. miniatocinctum and G. tornatum

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Abstract

The first and most crucial step of all molecular techniques is to isolate high quality and intact nucleic acids. However, DNA and RNA isolation from fungal samples are usually difficult due to the cell walls that are relatively unsusceptible to lysis and often resistant to traditional extraction procedures. Although there are many extraction protocols for Ganoderma species, different extraction protocols have been applied to different species to obtain high yields of good quality nucleic acids, especially for genome and transcriptome sequencing. Ganoderma species, mainly G. boninense causes the basal stem rot disease, a devastating disease that plagues the oil palm industry. Here, we describe modified DNA extraction protocols for G. boninense, G. miniatocinctum and G. tornatum, and an RNA extraction protocol for G. boninense. The modified salting out DNA extraction protocol is suitable for G. boninense and G. miniatocinctum while the modified high salt and low pH protocol is suitable for G. tornatum. The modified DNA and RNA extraction protocols were able to produce high quality genomic DNA and total RNA of ~ 140 to 160 µg/g and ~ 80 µg/g of mycelia respectively, for Single Molecule Real Time (PacBio Sequel® System) and Illumina sequencing. These protocols will benefit those studying the oil palm pathogens at nucleotide level.

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Acknowledgements

The authors would like to thank the Director General of MPOB, Dr. Ahmad Kushairi Din for the permission to publish this paper. A special appreciation is extended to the Biomarker Discovery Group members of MPOB and Dr. Ravigadevi Sambanthamurthi who gave valuable insights on the protocols. We also thank Shamala Sundram from the GanoDROP Unit of MPOB for providing the Ganoderma samples for this study.

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Authors and Affiliations

  1. Malaysian Palm Oil Board, No. 6, Persiaran Institusi, Bandar Baru Bangi, 43000, Kajang, Selangor, Malaysia

    Jayanthi Nagappan, Lee Pei Lee Angel & Eng-Ti Leslie Low

  2. School of Biosciences, Faculty of Science, University of Nottingham Malaysia Campus, Jalan Broga, 43500, Semenyih, Selangor, Malaysia

    Jayanthi Nagappan & Chiew Foan Chin

  3. Nottingham Arabidopsis Stock Centre, Division of Plant and Crop Sciences, School of Biosciences, University of Nottingham, Loughborough, UK

    Sean T. May

  4. Department of Biology & Biochemistry, University of Bath, Bath, BA2 7AY, UK

    Richard M. Cooper

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  1. Jayanthi Nagappan
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  2. Chiew Foan Chin
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  3. Lee Pei Lee Angel
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  4. Richard M. Cooper
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  5. Sean T. May
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  6. Eng-Ti Leslie Low
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Correspondence to Jayanthi Nagappan.

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Nagappan, J., Chin, C.F., Angel, L.P.L. et al. Improved nucleic acid extraction protocols for Ganoderma boninense, G. miniatocinctum and G. tornatum. Biotechnol Lett 40, 1541–1550 (2018). https://doi.org/10.1007/s10529-018-2603-7

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  • DOI: https://doi.org/10.1007/s10529-018-2603-7

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