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

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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References

  1. Aamir S, Sutar S, Singh SK, Baghela A (2015) A rapid and efficient protocol of fungal genomic DNA extraction, suitable for PCR based molecular protocols. Plant Path Quar 5(2):74–81

  2. Arana DM, Prieto D, Roman E, Nombela C, Alonso-Monge R, Pla J (2009) The role of the cell wall in fungal pathogenesis. Microbial Biotechnol 2(3):308–320

  3. Ardui S, Ameur A, Vermeesch JR, Hestand MS (2018) Single molecule real-time (SMRT) sequencing of age: applications and utilities for medical diagnostics. Nucl Acid Res 46(5):2159–2168

  4. Bencina M, Jakopic M, Friedrich J (2007) High-molecular weight genomic DNA isolation for Doratomyces microspores and synthesis of a genomic DNA library. Acta Chim Slov 54:893–899

  5. Bowman SM, Free SJ (2006) The structure and synthesis of the fungal cell wall. Bioessay 28(8):799–808

  6. Chong KP, Lum MS, Foong CP, Wong CMVL, Atong M, Rossall S (2011) First identification of Ganoderma boninense isolated from Sabah based on PCR and sequence homology. Afr J Biotechnol 10(66):14718–14723

  7. Coetzee MPA, Marincowits S, Muthelo VG, Wingfield MJ (2015) Ganoderma species, including new taxa associated with root rot of the iconic Jacaranda mimosifolia in Pretoria, South Africa. IMA Fungus 6(1):249–256

  8. Dentinger BTM, Margaritescu S, Moncalvo J-M (2010) Rapid and reliable high-throughput protocols of DNA extraction for use in barcoding and molecular systematics of mushrooms. Mol Ecol Resour 10:628–633

  9. Goes-Neto A, Loguercio-Leite C, Guerrero RT (2005) DNA extraction from frozen field-collected and dehydrated herbarium fungal basidiomata: performance of SDS and CTAB-based protocols. Biotemas 18(2):19–32

  10. Gow NAR, Latge J-P, Munro CA (2017) The fungal cell wall: structure, biosynthesis and function. Microbiol Spectr 5(3):FUNK-0035-2016

  11. Ho YW, Nawawi A (1985) Ganoderma boninense Pat. from basal stem rot of oil palm (Elaeis guineensis) in Peninsular Malaysia. Pertanika 8(3):425–428

  12. Idris AS (1999) Basal stem rot (BSR) of oil palm (Elaeis guineensis Jacq.) in Malaysia: factors associated with variation in disease severity. Dissertation, Wye College, Wye, UK

  13. Irene LI, Abu-Bakar FD, Idris AS, Murad AMA (2015) Comparison of protocols for isolating high quality genomic DNA and total RNA from the mycelia of an oil palm pathogen, Ganoderma boninense. Malays Appl Biol 44(1):19–24

  14. Jing CJ, Seman IA, Zakaria L (2015) Mating compatibility and restriction analysis of Ganoderma isolates from oil palm and other palm hosts. Trop Life Sci Res 26(2):45–57

  15. Kok SM, Goh YK, Tung HJ, Goh KJ, Wong WC, Goh YK (2013) In vitro growth of Ganoderma boninense isolates on novel palm extract medium and virulence on oil palm (Elaeis guineensis) seedlings. Malays J Microbiol 9(1):33–42

  16. Kushairi A, Singh R, Ong-Abdullah M (2017) The oil palm industry in Malaysia: thriving with transformative technologies. J Oil Palm Res 27(4):431–439

  17. Liao B, Chen X, Han J, Dan Y, Wang L, Jiao W, Song J, Chen S (2015) Identification of commercial Ganoderma (Lingzhi) species by ITS2 sequences. Chin Med 10:22–30

  18. Lim F-H, Fakhrana IN, Rasid OA, Idris AS, Parveez GKA, Ho C-L, Shaharuddin NA (2014) Isolation and selection of reference genes for Ganoderma boninense gene expression study using quantitative real-time PCR (qPCR). J Oil Palm Res 26(2):170–181

  19. Manak MM (1993) Sample preparation. In: Keller GH, Manak MM (eds) DNA probes. Stockton Press, New York, pp 27–68

  20. Merciere M, Laybats A, Carasco-Lacombe C, Tan JS, Klopp C, Durand-Gasselin T, Alwee SSRS, Camus-Kulandaivelu L, Breton F (2015) Identification and development of new polymorphic microsatellite markers using genome assembly for Ganoderma boninense, causal agent of oil palm basal stem rot disease. Mycol Progress 14:103–113

  21. Moller EM, Bahnweg G, Sadermann H, Geiger HH (1992) A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies and infected plant tissues. Nucleic Acids Res 20(22):6115–6116

  22. Moncalvo JM, Wang H, Hseu RS (1995) Gene phylogeny of the Ganoderma lucidum complex based on ribosomal DNA sequences. Comparison with traditional taxonomic characters. Mycol Res 99:1489–1499

  23. MPOB (2016) Malaysian oil palm statistics 2016, 36th edition

  24. Nasim G, Ali M, Mehmood N (2010) Molecular analysis of Ganoderma lucidum isolates from Lahore. Pak J Bot 42(5):3307–3315

  25. Noor-Adila AK, Farah-Diba AB, Zamri Z, Wan-Mohtar WY, Aidil AH, Mahadi AM, Murad AMA (2007) Comparison of protocols for isolating high quality DNA and RNA from an oleaginous fungus Cunninghamella bainieri strain 2a1. Mal J Microbiol 3(1):7–13

  26. Raeder U, Broda P (1985) Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol 1:17–20

  27. Rajesh K, Dhanasekaran D, Panneerselvam A (2014) Isolation and taxonomic characterization of medicinal mushroom Ganoderma spp. Acad J Microbiol Res 2(2):61–70

  28. Rakib MRM, Bong CFJ, Khairulmazmi A, Idris AS, Jalloh MB, Wahida NH (2017) Ganoderma species of basal and upper stem rots in oil palm (Elaeis guineensis) in Sarawak, Malaysia. J Acad UiTM Negeri Sembilan 5:27–35

  29. Shao J, Chen H, Yang D, Jiang M, Zhang H, Wu B, Li J, Yuan L, Liu C (2017) Genome-wide identification and characterization of natural antisense transcripts by strand-specific RNA sequencing in Ganoderma lucidum. Sci Rep 7:5711–5725

  30. Umesha S, Manukumar HM, Raghava S (2016) A rapid protocol for isolation of genomic DNA from food-borne fungal pathogens. Biotech 6:123

  31. Utomo C, Werner S, Nieplod F, Deising HB (2005) Identification of Ganoderma, the causal agent of basal stem rot disease in oil palm using a molecular method. Mycopathologia 159:159–170

  32. van Peer AF, de Bekker C, Vinck A, Wosten HAB, Lugones LG (2009) Phleomycin increases transformation efficiency and promotes single integrations in Schizophyllum commune. Appl and Environ Microbiol 75(5):1243–1247

  33. Voigt K, Cigelnik E, O’Donnell K (1999) Phylogeny and PCR identification of clinically important Zygomycetes based on nuclear ribosomal-DNA sequence data. J Clin Microbiol 37(12):3957–3964

  34. Zheng L, Jia D, Fei X, Luo X, Yang Z (2009) An assessment of the genetic diversity within Ganoderma strains with AFLP and ITS PCR-RFLP. Microbiol Res 164:312–321

  35. Zhou X, Li Q, Zhao J, Tang K, Lin J, Yin Y (2007) Comparison of rapid DNA extraction protocols applied to PCR identification of medical mushroom Ganoderma spp. Prep Biochem and Biotechnol 37(4):369–380

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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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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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Keywords

  • DNA extraction
  • RNA extraction
  • Basidiomycetes
  • Mycelium
  • PacBio