Analysis of Arbuscular Mycorrhizal Fungal Communities by Terminal Restriction Fragment Length Polymorphism (TRFLP)

  • Álvaro López-GarcíaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2146)


Terminal restriction fragment length polymorphism (TRFLP) approaches enable the detection and identification of microbial taxa into samples coming from root or soil material DNA extraction. The low taxonomic diversity of arbuscular mycorrhizal (AM) fungi makes this technique a cheap and adequate method for fingerprinting their communities. Here, I describe the TRFLP database approach, a version of the technique in which the AM fungal taxa present in the sample pool is identified for, later, match their presence in the different samples contained in the experiment. A final AM fungal operational taxonomic unit x sample presence–absence matrix is obtained, which allows the subsequent multivariate statistical analysis of the experimental results.

Key words

Community assembly Fingerprinting Operational taxonomic units Phylogenetic analyses R software TRAMPR package 



The author was supported by Spanish government under the Plan Nacional de I+D+I (project CGL2015-69118-C2-2-P-COEXMED-II) and University of Jaén (Spain) under the Plan 6-UJA postdoctoral fellowship.

Supplementary material

470476_1_En_10_MOESM1_ESM.txt (57 kb)
Data 1 (TXT 57 kb)
470476_1_En_10_MOESM2_ESM.txt (57 kb)
Data 2 (TXT 56 kb)
470476_1_En_10_MOESM3_ESM.txt (4 kb)
Data 3 (TXT 4 kb)
470476_1_En_10_MOESM4_ESM.txt (0 kb)
Data 4 (TXT 465 bytes)
470476_1_En_10_MOESM5_ESM.xlsx (17 kb)
Data 5 (XLSX 17 kb)
470476_1_En_10_MOESM6_ESM.txt (139 kb)
Data 6 (TXT 139 kb)
470476_1_En_10_MOESM7_ESM.txt (1 kb)
Data 7 (TXT 1 kb)


  1. 1.
    van der Heijden MGA, Martin FM, Selosse MA, Sanders IR (2015) Mycorrhizal ecology and evolution: the past, the present, and the future. New Phytol 205:1406–1423CrossRefGoogle Scholar
  2. 2.
    Courtney KC, Bainard LD, Sikes BA et al (2012) Determining a minimum detection threshold in terminal restriction fragment length polymorphism analysis. J Microbiol Methods 88:14–18CrossRefGoogle Scholar
  3. 3.
    Martínez-García LB, Richardson SJ, Tylianakis JM et al (2015) Host identity is a dominant driver of mycorrhizal fungal community composition during ecosystem development. New Phytol 205:1565–1576CrossRefGoogle Scholar
  4. 4.
    Varela-Cervero S, López-García Á, Barea JM, Azcón-Aguilar C (2016) Spring to autumn changes in the arbuscular mycorrhizal fungal community composition in the different propagule types associated to a Mediterranean shrubland. Plant Soil 26:107–120CrossRefGoogle Scholar
  5. 5.
    López-García Á, Varela-Cervero S, Vasar M et al (2017) Plant traits determine the phylogenetic structure of arbuscular mycorrhizal fungal communities. Mol Ecol 26:6948–6959CrossRefGoogle Scholar
  6. 6.
    Dickie IA, FitzJohn RG (2007) Using terminal restriction fragment length polymorphism (T-RFLP) to identify mycorrhizal fungi: a methods review. Mycorrhiza 17:259–270CrossRefGoogle Scholar
  7. 7.
    Koch AM, Kuhn G, Fontanillas P et al (2004) High genetic variability and low local diversity in a population of arbuscular mycorrhizal fungi. Proc Natl Acad Sci U S A 101:2369–2374CrossRefGoogle Scholar
  8. 8.
    López-García Á, Palenzuela J, Barea JM, Azcón-Aguilar C (2014) Life-history strategies of arbuscular mycorrhizal fungi determine succession into roots of Rosmarinus officinalis L., a characteristic woody perennial plant species from Mediterranean ecosystems. Plant Soil 379:247–260CrossRefGoogle Scholar
  9. 9.
    Lee J, Lee S, Young JPW (2008) Improved PCR primers for the detection and identification of arbuscular mycorrhizal fungi. FEMS Microbiol Ecol 65:339–349CrossRefGoogle Scholar
  10. 10.
    Schloss PD, Westcott SL, Ryabin T et al (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541CrossRefGoogle Scholar
  11. 11.
    Kumar S, Stecher G, Li M et al (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549CrossRefGoogle Scholar
  12. 12.
    Huson DH, Bryant D (2006) Application of phylogenetic networks in evolutionary studies. Mol Biol Evol 23:254–267CrossRefGoogle Scholar
  13. 13.
    Collins RE, Rocap G (2007) REPK: an analytical web server to select restriction endonucleases for terminal restriction fragment length polymorphism analysis. Nucleic Acids Res 35:W58–W62CrossRefGoogle Scholar
  14. 14.
    Fitzjohn RG, Dickie IA (2007) TRAMPR: AN R package for analysis and matching of terminal-restriction fragment length polymorphism (TRFLP) profiles. Mol Ecol Notes 7:583–587CrossRefGoogle Scholar
  15. 15.
    Oksanen J, Blanchet FG, Kindt R et al (2011) Vegan: community ecology package. R package version 2.0-1. R Project.
  16. 16.
    Lekberg Y, Gibbons SM, Rosendahl S (2014) Will different OTU delineation methods change interpretation of arbuscular mycorrhizal fungal community patterns? New Phytol 202:1101–1104CrossRefGoogle Scholar
  17. 17.
    Huson DH, Rupp R, Scornavacca C (2011) Phylogenetic networks: concepts, algorithms and applications. Cambridge University Press, CambridgeGoogle Scholar
  18. 18.
    Öpik M, Vanatoa A, Vanatoa E et al (2010) The online database MaarjAM reveals global and ecosystemic distribution patterns in arbuscular mycorrhizal fungi (Glomeromycota). New Phytol 188:223–241CrossRefGoogle Scholar
  19. 19.
    Kohout P, Sudova R, Janoušková M et al (2014) Comparison of commonly used primer sets for evaluating arbuscular mycorrhizal fungal communities: is there a universal solution? Soil Biol Biochem 68:482–493CrossRefGoogle Scholar
  20. 20.
    Gollotte A, Van Tuinen D, Atkinson D (2004) Diversity of arbuscular mycorrhizal fungi colonizing roots of the grass species Agrostis capillaris and Lolium perenne in a field experiment. Mycorrhiza 14:111–117CrossRefGoogle Scholar
  21. 21.
    Ohsowski BM, Zaitsoff PD, Öpik M, Hart MM (2014) Where the wild things are: looking for uncultured Glomeromycota. New Phytol 204:171–179CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Soil Microbiology and Symbiotic SystemsEstación Experimental del Zaidín, CSICGranadaSpain
  2. 2.Department of Animal Biology, Plant Biology and EcologyUniversidad de JaénJaénSpain

Personalised recommendations