Mycological Progress

, Volume 13, Issue 2, pp 439–444 | Cite as

Development of polymorphic microsatellite markers for the genetic characterisation of Knoxdaviesia proteae (Ascomycota: Microascales) using ISSR-PCR and pyrosequencing

  • Janneke Aylward
  • Léanne L. Dreyer
  • Emma T. Steenkamp
  • Michael J. Wingfield
  • Francois Roets
Short Communication


Knoxdaviesia proteae is one of the first native ophiostomatoid fungi discovered in South Africa, where it consistently occurs in the infructescences of the iconic Cape Biome plant, Protea repens. Although numerous studies have been undertaken to better understand the ecology of K. proteae, many questions remain to be answered, particularly given its unique niche and association with arthropods for dispersal. We describe the development and distribution of microsatellite markers in K. proteae through Interspersed Simple Sequence Repeat-Polymerase Chain Reaction (ISSR-PCR) enrichment and pyrosequencing. A large proportion of the 31492 sequences obtained from sequencing the enriched genomic DNA were characterised by microsatellites consisting of short tandem repeats and di- and tri-nucleotide motifs. Seventeen percent of these microsatellites contained flanking regions sufficient for primer design. Twenty-three primer pairs were tested, of which 12 amplified and 10 generated polymorphic fragments in K. proteae. Half of these could be transferred to the sister species, K. capensis. The developed markers will be used to investigate the reproductive system, genetic diversity and dispersal strategies of K. proteae.


ISSR-PCR Knoxdaviesia Microsatellites Ophiostomatoid Pyrosequencing 



We thank the National Research Foundation (NRF) and the Department of Science and Technology (DST)/NRF Centre of Excellence in Tree Health Biotechnology (CTHB) for financial support and the Western Cape Nature Conservation Board for issuing the necessary collecting permits.


  1. Agapow P-M, Burt A (2001) Indices of multilocus linkage disequilibrium. Mol Ecol Notes 1(1–2):101–102CrossRefGoogle Scholar
  2. Benjamini Y, Yekutieli D (2001) The Control of the False Discovery Rate in Multiple Testing under Dependency. Ann Stat 29(4):1165–1188. doi: 10.2307/2674075 CrossRefGoogle Scholar
  3. Chakraborty R, Kimmel M, Stivers DN, Davison LJ, Deka R (1997) Relative mutation rates at di-, tri-, and tetranucleotide microsatellite loci. Proc Natl Acad Sci 94(3):1041–1046PubMedCentralPubMedCrossRefGoogle Scholar
  4. Crous PW, Summerell BA, Shivas RG, Burgess TI, Decock CA, Dreyer LL, Granke LL, Guest DI, Hardy GESTJ, Hausbeck MK, Hüberli D, Jung T, Koukol O, Lennox CL, Liew ECY, Lombard L, McTaggart AR, Pryke JS, Roets F, Saude C, Shuttleworth LA, Stukely MJC, Vánky K, Webster BJ, Windstam ST, Groenewald JZ (2012) Fungal Planet description sheets: 107-127. Persoonia 28(1):138–182PubMedCentralPubMedCrossRefGoogle Scholar
  5. De Beer ZW, Seifert KA, Wingfield MJ (2013a) A nomenclature for ophiostomatoid genera and species in the Ophiostomatales and Microascales. In ‘Ophiostomatoid fungi: expanding frontiers.’ (Eds KA Seifert, ZW De Beer, MJ Wingfield.) CBS Biodiversity Series 12 pp. 1-19Google Scholar
  6. De Beer ZW, Seifert KA, Wingfield MJ (2013b) The ophiostomatoid fungi: their dual position in the Sordariomycetes. In ‘Ophiostomatoid fungi: expanding frontiers.’ (Eds KA Seifert, ZW De Beer, MJ Wingfield.) CBS Biodiversity Series 12 pp. 1-19Google Scholar
  7. Dettman JR, Taylor JW (2004) Mutation and Evolution of Microsatellite Loci in Neurospora. Genetics 168(3):1231–1248. doi: 10.1534/genetics.104.029322 PubMedCentralPubMedCrossRefGoogle Scholar
  8. Dutech C, Enjalbert J, Fournier E, Delmotte F, Barrès B, Carlier J, Tharreau D, Giraud T (2007) Challenges of microsatellite isolation in fungi. Fungal Genet Biol 44(10):933–949PubMedCrossRefGoogle Scholar
  9. Goldstein DB, Clark AG (1995) Microsatellite variation in North American populations of Drosophila melanogaster. Nucleic Acids Res 23(19):3882–3886PubMedCentralPubMedCrossRefGoogle Scholar
  10. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  11. Hantula J, Dusabenyagasani M, Hamelin RC (1996) Random amplified microsatellites (RAMS) — a novel method for characterizing genetic variation within fungi. Eur J For Pathol 26(3):159–166CrossRefGoogle Scholar
  12. Karaoglu H, Lee CMY, Meyer W (2005) Survey of Simple Sequence Repeats in Completed Fungal Genomes. Mol Biol Evol 22(3):639–649PubMedCrossRefGoogle Scholar
  13. Katti MV, Ranjekar PK, Gupta VS (2001) Differential Distribution of Simple Sequence Repeats in Eukaryotic Genome Sequences. Mol Biol Evol 18(7):1161–1167PubMedCrossRefGoogle Scholar
  14. Kolařík M, Hulcr J (2009) Mycobiota associated with the ambrosia beetle Scolytodes unipunctatus (Coleoptera: Curculionidae, Scolytinae). Mycol Res 113(1):44–60PubMedCrossRefGoogle Scholar
  15. Kruglyak S, Durrett RT, Schug MD, Aquadro CF (1998) Equilibrium distributions of microsatellite repeat length resulting from a balance between slippage events and point mutations. Proc Natl Acad Sci 95(18):10774–10778PubMedCentralPubMedCrossRefGoogle Scholar
  16. Lim S, Notley-McRobb L, Lim M, Carter DA (2004) A comparison of the nature and abundance of microsatellites in 14 fungal genomes. Fungal Genet Biol 41(11):1025–1036PubMedCrossRefGoogle Scholar
  17. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen Y-J, Chen Z, Dewell SB, Du L, Fierro JM, Gomes XV, Godwin BC, He W, Helgesen S, Ho CH, Irzyk GP, Jando SC, Alenquer MLI, Jarvie TP, Jirage KB, Kim J-B, Knight JR, Lanza JR, Leamon JH, Lefkowitz SM, Lei M, Li J, Lohman KL, Lu H, Makhijani VB, McDade KE, McKenna MP, Myers EW, Nickerson E, Nobile JR, Plant R, Puc BP, Ronan MT, Roth GT, Sarkis GJ, Simons JF, Simpson JW, Srinivasan M, Tartaro KR, Tomasz A, Vogt KA, Volkmer GA, Wang SH, Wang Y, Weiner MP, Yu P, Begley RF, Rothberg JM (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437(7057):376–380PubMedCentralPubMedGoogle Scholar
  18. Möller EM, Bahnweg R, Sandermann 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):6115–6116PubMedCentralPubMedCrossRefGoogle Scholar
  19. Niu B, Fu L, Sun S, Li W (2010) Artificial and natural duplicates in pyrosequencing reads of metagenomic data. BMC Bioinforma 11(1):187CrossRefGoogle Scholar
  20. Rebelo T (1995) Proteas: A field guide to the Proteas of Southern Africa. Fernwood Press, Vlaeberg, South AfricaGoogle Scholar
  21. Roets F, Dreyer LL, Crous PW (2005) Seasonal trends in colonisation of Protea infructescences by Gondwanamyces and Ophiostoma spp. S Afr J Bot 71(3):307–311Google Scholar
  22. Roets F, de Beer ZW, Dreyer LL, Zipfel R, Crous PW, Wingfield MJ (2006) Multi-gene phylogeny for Ophiostoma spp. reveals two new species from Protea infructescences. Stud Mycol 55:199–212PubMedCentralPubMedCrossRefGoogle Scholar
  23. Roets F, Wingfield MJ, Crous PW, Dreyer LL (2007) Discovery of Fungus-Mite Mutualism in a Unique Niche. Environ Entomol 36(5):1226–1237PubMedCrossRefGoogle Scholar
  24. Roets F, Crous PW, Wingfield MJ, Dreyer LL (2009a) Mite-mediated hyperphoretic dispersal of Ophiostoma spp. from the Infructescences of South African Protea spp. Environ Entomol 28(1):143–152CrossRefGoogle Scholar
  25. Roets F, Wingfield MJ, Crous PW, Dreyer LL (2009b) Fungal radiation in the Cape Floristic Region: An analysis based on Gondwanamyces and Ophiostoma. Mol Phylogenet Evol 51(1):111–119PubMedCrossRefGoogle Scholar
  26. Roets F, Theron N, Wingfield MJ, LaL D (2011a) Biotic and abiotic constraints that facilitate host exclusivity of Gondwanamyces and Ophiostoma on Protea. Fungal Biol 116(1):49–61PubMedCrossRefGoogle Scholar
  27. Roets F, Wingfield MJ, Wingfield BD, Dreyer LL (2011b) Mites are the most common vectors of the fungus Gondwanamyces proteae in Protea infructescences. Fungal Biol 115(4–5):343–350PubMedCrossRefGoogle Scholar
  28. Rousset F (2008) Genepop’007: a complete re-implementation of the genepop software for Windows and Linux. Mol Ecol Resour 8(1):103–106PubMedCrossRefGoogle Scholar
  29. Santana QC, Coetzee MPA, Steenkamp ET, Mlonyeni OX, Hammond GNA, Wingfield MJ, Wingfield BD (2009) Microsatellite discovery by deep sequencing of enriched genomic libraries. BioTech 46(3):217–223CrossRefGoogle Scholar
  30. Spatafora JW, Blackwell M (1994) The polyphyletic origins of ophiostomatoid fungi. Mycol Res 98(1):1–9CrossRefGoogle Scholar
  31. Thurston MI, Field D (2005) Msatfinder: detection and characterisation of microsatellites. Distributed by the authors at CEH Oxford, Mansfield Road, Oxford OX1 3SR
  32. Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JAM (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 35(suppl 2):W71–W74PubMedCentralPubMedCrossRefGoogle Scholar
  33. Van der Linde JA, Six DL, Wingfield MJ, Roux J (2012) New species of Gondwanamyces from dying Euphorbia trees in South Africa. Mycologia 104(2):574–584PubMedCrossRefGoogle Scholar
  34. White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, San Diego, California, pp 315–322Google Scholar
  35. Wingfield MJ, Van Wyk PS (1993) A new species of Ophiostoma from Protea infructescences in South Africa. Mycol Res 97(6):709–716CrossRefGoogle Scholar
  36. Wingfield MJ, Wyk PSV, Marasas WFO (1988) Ceratocystiopsis proteae sp. nov., with a new anamorph genus. Mycologia 80 (1):23-30Google Scholar
  37. Wingfield BD, Viljoen CD, Wingfield MJ (1999) Phylogenetic relationships of ophiostomatoid fungi associated with Protea infructescences in South Africa. Mycol Res 103(12):1616–1620CrossRefGoogle Scholar
  38. Xu X, Peng M, Fang Z (2000) The direction of microsatellite mutations is dependent upon allele length. Nat Genet 24(4):396–399PubMedCrossRefGoogle Scholar
  39. Zietkiewicz E, Rafalski A, Labuda D (1994) Genome Fingerprinting by Simple Sequence Repeat (SSR)-Anchored Polymerase Chain Reaction Amplification. Genomics 20(2):176–183PubMedCrossRefGoogle Scholar

Copyright information

© German Mycological Society and Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Janneke Aylward
    • 1
    • 3
  • Léanne L. Dreyer
    • 1
    • 3
  • Emma T. Steenkamp
    • 2
    • 3
  • Michael J. Wingfield
    • 3
  • Francois Roets
    • 3
    • 4
  1. 1.Department of Botany and ZoologyStellenbosch UniversityMatielandSouth Africa
  2. 2.Department of Microbiology and Plant PathologyUniversity of PretoriaPretoriaSouth Africa
  3. 3.Department of Science and Technology (DST)/National Research Foundation (NRF) Centre of Excellence in Tree Health Biotechnology (CTHB)University of PretoriaPretoriaSouth Africa
  4. 4.Department of Conservation Ecology and EntomologyStellenbosch UniversityMatielandSouth Africa

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