Selection of Enzymes for Terminal Restriction Fragment Length Polymorphism Analysis of Fungal Internally Transcribed Spacer Sequences

Abstract

Terminal restriction fragment length polymorphism (TRFLP) profiling of the internally transcribed spacer (ITS) ribosomal DNA of unknown fungal communities is currently unsupported by a broad-range enzymechoosing rationale. An in silico study of terminal fragment size distribution was therefore performed following virtual digestion (by use of a set of commercially available 135 type IIP restriction endonucleases) of all published fungal ITS sequences putatively annealing to primers ITS1 and ITS4. Different diversity measurements were used to rank primer-enzyme pairs according to the richness and evenness that they showed. Top-performing pairs were hierarchically clustered to test for data dependency. The enzyme set composed of MaeII, BfaI, and BstNI returned much better results than randomly chosen enzyme sets in computer simulations and is therefore recommended for in vitro TRFLP profiling of fungal ITSs.

Consequences of minimum soil tillage on abiotic soil properties and composition of microbial communities in a shallow Cambisol originated from fluvioglacial deposits

“…Only at the sites, where a strong SOC accumulation might be expected in response to shifts in tillage management, archaea might also be affected, as a result of the autotrophic lifestyle of ammonia oxidizers, which has been recently shown by Souza et al (2013) using a metagenomic approach. However, as the sensitivity of the T-RFLP approach depends on the variability of the amplified region of the marker gene as well as on the used restriction enzyme (Liu et al 1997;Alvarado and Manjon 2009), it cannot be excluded that the sensitivity of the used fingerprinting approach was insufficient to identify shifts in the archaeal phylum. Here, sequencing approaches of 16S rRNA gene amplicons might be of use to confirm the observed response pattern in the future.…”

“…only peaks with an intensity of >50 fluorescence units and a size of >50 bases were selected for further analysis to avoid problems with primer dimers). In addition, the number of TRFs in the fingerprints depends on the selected primer pairs and restriction enzymes (Liu et al 1997;Alvarado and Manjon 2009). In many cases, Buniversal primers^for one phylum are not really universal when the bias of PCR has been considered.…”

Impact of postfire logging on soil bacterial and fungal communities and soil biogeochemistry in a mixed-conifer forest in central Oregon

“…Restriction enzyme HinfI was selected over HaeIII for T-RFLP community analysis after comparison trials on replicate samples revealed its ability to identify the greatest amount of variation. Both HinfI and HaeIII are widely used in fungal T-RFLP profiling (Avis et al 2006;Dickie and FitzJohn 2007;Alvarado and Manjon 2009). GeneMapper software 4.0 (Applied Biosystems Inc., Foster City, CA) was used to determine fragment fluorescence, OTUs were binned to 1 bp in width, and a binary analysis of presence or absence was performed following the methods of Rinehart (2004).…”

Investigation of fungal root colonizers of the invasive plant Vincetoxicum rossicum and co-occurring local native plants in a field and woodland area in Southern Ontario

“…Amplicons were digested following the manufacturer's instructions using restriction enzymes EcoRII and FspBI for 2 hours at 37°C (Fermentas Canada Inc., Burlington, Ontario) (isoschizomers for MaeII and BstNI respectively. Alvarado and Manjón (2009) performed an in silico study of terminal fragment size distributions to test primer-enzyme pairs, and recommended these enzymes for T-RFLP using ITS1F-ITS1 primers based on their effectiveness in producing differential T-RFLP cuts reflective of fungal diversity. Digests contained 15 μl of PCR product, 2U each EcoRII and FspBI, 2 μl Tango™ 1X buffer, and 2.6 μl sterile water.…”