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

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 enzyme-choosing 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.Terminal restriction fragment length polymorphism (TRFLP) profiling was originally developed as a means of genotyping mixed DNA samples (30) and is currently being employed in fungal community ecology studies (3, 5, 6, 7, 10, 13, 19, 22, 26, 27, 29, 33, 38), despite a number of technical and conceptual difficulties (11). Briefly, TRFLP profiling involves amplifying the DNA in pools of mixed genetic material with fluorescently labeled primers, digesting the products with restriction endonucleases, and sizing the labeled terminal fragments in a sequencer. The difference in the positions at which the different restriction enzymes cleave DNA is thought to provide enough variability for such DNA mixtures to be characterized and the contributing organisms to be identified.However, the technique is not without its problems. DNA extraction and PCR amplification biases burden most modern molecular techniques, including TRFLPs (18, 25). Additionally, concerns exist regarding the ability of the differences between primer-enzyme pairs (PEPs) to generate sufficiently different fragment sizes (2), the success of enzymatic cleavage (2), the dependency on the detection threshold of the sequencer (4), and the accuracy of DNA sizing (1). The choice of the primer pairs and restriction enzymes to be used has also been a matter of concern since the appearance of TRFLP profiling. Liu et al. (30) performed virtual digestion of all the bacterial RNA sequences in the Ribosomal Database Project database (release V) with 10 different enzymes and four primer pairs. This pioneering work showed the importance of avoiding enzymes with highly conserved target motifs, something that later became recognized as a major source of TRFLP bias (2, 14, 16, 32). Similar studies have been performed by Osborn et al. (36), Dunbar et al. (12), Engebretson and Moyer (15), and Cardinale et al. (8).The first virtual TRFLP analysis involving a database of fungal DNA sequences was performed by Edwards and Turco (14). This consisted of virtual digestion, by use of six restriction endonucleases, of 316 internally transcribed spacer (ITS) sequences belonging to a number of ectomycorrhizal genera. Avis et al. (2) found only small differences in the diversity of the TRFLPs produced in silico by three PEPs when using their own fungal ITS database, although these differences increased with sample number in iterative analysis. Recent advances using automated resources, such as REPK software (9), have allowed optimal enzyme selection for TRFLP profiling of previously defined communities of organisms. This software selects up to four restriction endonucleases capable of discriminating a desired number of sequence groups. However, this system relies on a priori information, which in real biological communities may not available.The aim of the present work was to improve selection of restriction enzymes for use in the TRFLP profiling of the ITS sequences of unknown fungal communities.