Cardenas-Flores et al.

Clonal lineage / Origin / Year of supply
BEL / Glomeromycota in vitro Collection (GINCO), Mycothèque de l’Université catholique de Louvain (MUCL), Microbiology unit, Université catholique de Louvain, Belgium. / 2001
CAN / Biosystematics Research Centre, lodged by the AAC Eastern Cereal and Oilseed Research Centre, Ottawa, Canada. / 1992
ITA / Dipartamento di Biologia Vegetale, Universita di Torino, Torino, Italy. / 2005
SPA / Estación Experimental del Zaidín, CSIC, Grenade, Spain. / 1995
SWI / Institute of Botany, University of Basel, Switzerland. / 2005

Cárdenas-Flores, A., Draye, X., Bivort, C., Cranenbrouck, S. and S. Declerck*. Impact of multispores in vitro subcultivation of Glomus sp. MUCL 43194 (DAOM 197198) on vegetative compatibility and genetic diversity detected by AFLP. Mycorrhiza

* Place Croix du Sud, 3 box 6, 1348 Louvain-la-Neuve, BELGIUM. Tel. +32 10 47 46 44; Fax. +32 10 45 15 01; E-mail:

Contents

1)  Table S1 (page 1)

2)  Figure S2 (page 2)

Table S1. Extended affiliations of the laboratories where MUCL 43194 was subcultured and the year when it was supplied to them.

Figure S2

Figure S2. Glomeraceae group 1b clade extracted form the phylogenetic (rDNA) 18S-based tree of Glomerales. The global tree comprised the alignment used by Schwarzott et al. (2001) and sequences of MUCL 43194 and 41833 tested in this study (names in bold with accession numbers) using E. pisiformis (X58724) and M. polycephala (X89436) sequences as an out-group. Neighbour-joining values of 1000 bootstrap replicates (1800 characters, under Kimura 2 parameters) near branches. The bootstrap (from the 50% majority rule consensus tree) support of MUCL 43194 AFTOL is in bold. Bar = 0.001 substitutions per site.

Method: The replication reactions were conducted on 100 ng of the genomic DNA amplification product adding: 1x PCR buffer (20 mM Tris-HCl pH 8.4; 50 mM KCl; Sigma, Missouri, USA); 1.5 mM MgCl2; 0.2 mM of each rDNA primer; 200 mM each dNTP (Sigma, Missouri, USA) and 2.5 units of the Taq DNA polymerase (Sigma, USA)

The two primers used were: Geo A2 (5’-ACC TTG TTA CGA CTT TTA CTT CC-3’) and Geo 11 (5’-CCA GTA GTC ATA TGC TTC TCT C-3’) (Schuβler et al., 2001). The amplification was performed in a Mastercycler (Eppendorf, Germany) by taking the following steps: (i) denaturation at 94 °C for 5 min followed by (ii) 40 cycles at 94 °C for 30 s, 60 °C for 1 min, and 72 °C for 2 min and (iii) a final elongation step at 72 °C for 10 min. Each SSU PCR product was cloned using the Qiagen PCR cloning kit (Qiagen Gmbh, Germany) and sequenced using the kit GenomeLab™ Dye Terminator Cycle Sequencing (Beckman Coulter, USA). The sequences were obtained using eight different primers: NS1, NS2, NS3, NS4, NS5, NS6, NS7, NS8 (White et al. 1990). The sequences were analyzed with an automatic sequencer (CEQ™ 2000xl DNA Analysis System, Beckman Coulter, USA).

SSU-based phylogenetic analysis

The sequences were edited with Sequencher v.4.1.4 (Gene Code Corporation, USA), aligned with Clustal X 1.5 and corrected manually. To complete the analysis, the resulting SSU sequences were compared with the alignment (ALIGN_000124) used by Schwarzott et al. (2001) (sequences available on-line at http://www.amf-phylogeny.com) including sequences of Glomus spp: MUCL 41833: (AJ852534), MUCL 41835 (AJ852533), MUCL 43196 (AJ852532), MUCL 43194 (AJ852526) and MUCL 43194 AFTOL (AY635831), listed in the GeneBank database (http://www.ncbi.nlm.nih.gov).

The phylogenic trees were obtained after a neighbour joining analysis of 1800 characters under Kimura 2 parameters, run on PAUP v.4.0b10. The trees were evaluated by bootstrap analysis (100 replicates for parsimony and 1000 for neighbour joining) and the shortest tree used for evaluation and interpretation of data.

Schwarzott D, Walker C, Schüßler A (2001) Glomus the largest genus of the arbuscular mycorrhizal fungi (Glomales) is non-monophyletic. Mol. Phyl. Evol. 21: 190-197.

White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and direct 827 Q3 sequencing of fungal ribosomal RNA genes for phylogenetics. 828 In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR 829 protocols: a guide to methods and applications. Academic, New 830 York, pp 315–322

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