Ecological Archives E091-166-A1

Natasha Teutsch Hausmann and Christine V. Hawkes. 2010. Order of plant host establishment alters the composition of arbuscular mycorrhizal communities. Ecology 91:2333–2343.

Appendix A. Molecular methods for AMF characterization.

Cloning and sequencing

We generated an experiment-wide clone library using pooled amplicons from all root samples in this experiment as well as those in a complimentary study run at the same time using the same plant species and soil (Hausmann and Hawkes 2009). Fresh PCR products from all samples were pooled, gel purified (MinElute Gel Purification kit, Qiagen), cloned into the pCR 2.1-TOPO vector (Invitrogen), and transformed following manufacturer’s protocols. We sequenced 450 clones with the correct insert size (for details see Hausmann and Hawkes 2009). Sequences were auto-aligned and hand-edited to remove ambiguous bases (SequencherTM 4.2.2, Gene Code Corporation, Ann Arbor, Michigan, USA). We separated AMF sequences from non-target sequences using NCBI BLAST (Altschul et al. 1997), resulting in assignment of 234 clones to AMF and 79 to non-AMF fungi (primarily ascomycetes); unresolved (60) and unmatched (77) sequences were discarded. AMF sequences, their closest BLAST matches, and additional AMF sequences from a representative set of Glomeromycota (Schϋβler et al. 2001) were automatically aligned with ClustalX v.1.83.1 (Jeanmougin et al. 1998) and hand-edited using SeAl v2.0a11 (Rambaut 1996). We narrowly defined 24 AMF phylotypes in the clone sequence library using Bayesian phylogenetic analyses.

T-RFLP

T-RFLP analysis of clones was used to generate fragment length profiles of known sequences to identify T-RFLP profiles of experimental samples (Hausmann and Hawkes 2009). Polymorphic fragments were created by digesting clones with four enzymes HinFI (G^ANTC), MboI (^GATC), TaqI (T^CGA) (Fermentas, Hanover, Maryland), and Hsp92II (CATG^) (Promega, Madison, Wisconsin), excluding three uninformative fragments (AM1-HinFI, AM1-Hsp92II, AM1-MboI). Fragment lengths were analyzed and verified, with 99% matching expected lengths within five bp; clones without matches were discarded. The 24 AMF phylotypes were collapsed into 15 AMF OTUs based on clone fragment similarity using the TRAMPR package (Fitzjohn and Dickie 2007). OTUs were named sequentially according to genus based on phylogenetic analyses (Hausmann and Hawkes 2009). We followed the same procedure with non-AMF sequences and verified that no T-RFLP profiles were shared between AMF and non-AMF, effectively removing non-AMF sequences from the analysis. For the experimental samples, we pooled three replicate PCR reactions, gel purified the products, and digested them as described for the clones. We used the TRAMPR package in R to match sample T-RFLP profiles to clone T-RFLP profiles. An OTU was considered present if all five identifying fragments occurred within 1.5 bp of the known fragment length, supporting high confidence (P < 10-4) (Dickie and FitzJohn 2007). For details on the phylogenetics and enzyme profiles for these OTUs, as well as a discussion of our approach, see Hausmann and Hawkes (2009).

LITERATURE CITED

Altschul, S. F., L. S. Thomas, A. S. Alejandro, Z. Jingui, M. Webb, and J. L. David. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25:3389–3402.

Dickie, I. A. and R. G. FitzJohn. 2007. Using terminal restriction fragment length polymorphism (T-RFLP) to identify mycorrhizal fungi: a methods review. Mycorrhiza 17:259–270.

Fitzjohn, R. G. and I. A. Dickie. 2007. TRAMPR: an R package for analysis and matching of terminal-restriction fragment length polymorphism (TRFLP) profiles. Molecular Ecology Notes 7:583–587.

Hausmann, N. T. and C. V. Hawkes. 2009. Plant neighborhood control of arbuscular mycorrhizal community composition. New Phytologist 183:1188–1200.

Jeanmougin, F., J. D. Thompson, M. Gouy, D. G. Higgins, and T. J. Gibson. 1998. Multiple sequence alignment with Clustal X. Trends in Biochemical Science 23:403–405.

Rambaut, A. 1996. Se-Al: Sequence Alignment Editor. http://evolve.zoo.ox.ac.uk/.
Schϋβler, A., H. Gehrig, D. Schwarzott, and C. Walker. 2001. Analysis of partial Glomus SSU rRNA gene sequences: implications for primer design and phylogeny. Mycological Research 105:5–15.


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