Modern techniques for studying ECM communities are based on molecular identification or profiling. Advances in molecular methods have facilitated the accurate identification and analysis of fungal communities, and in combination with morphological techniques, provide a powerful means of studying ECM communities. Common molecular techniques that are used to identify ECM fungi and study ECM communities are summarised in Table 1.1 (see Anderson & Cairney 2004 for review).
Molecular methods for ECM identification and community profiling are based on ribosomal DNA (rDNA). The Internal Transcribed Spacer (ITS) is a non-coding region of the rDNA that separates the small-subunit (SSU) and the large-subunit (LSU) and has been widely applied to the study of fungi, especially ECM fungi. The ITS region is highly conserved within species but exhibits enough variation between species allowing taxonomic resolution often to the species level (Gardes, White et al. 1991; Kåren, Hogberg et al. 1997; Taylor & Bruns 1999; Anderson & Cairney 2004; Nilsson, Ryberg et al. 2009; Ryberg, Kristiansson et al. 2009).
DNA can be extracted and amplified using fungal specific primers from any fungal structure such as sporocarps, mycorrhizal root tips, or mycelium. Two universal primers ITS1 and ITS4 were developed by White, Bruns et al. (1990) to amplify the ITS spacer of the rDNA using Polymerase Chain Reaction (PCR). These primers were first applied the study of mycorrhiza by Gardes, White et al. (1991). The primers ITS1-F and ITS4B were developed by Gardes and Bruns (1993) for specific amplification of fungal and Basidiomycete DNA. Also the reverse primer, ITS4A was designed for increased specificity for Ascomycete DNA (Larena, Salazar et al. 1999). These primers have been subsequently used extensively for fungal community studies (Erland, Henrion et al. 1994; Kåren, Hogberg et al. 1997; Farmer & Sylvia 1998; Chambers, Sawyer et al. 1999; Jonsson, Dahlberg et al. 1999; Viaud, Pasquier et al. 2000; Burke, Martin et al. 2005).
Many molecular techniques result in the identification of operational taxonomic units (OTUs), which are groups of fungi that have similar molecular profiles or sequences. OTUs are often used as a proxy for a single species, but differ from a species unit, as their morphological or sexual compatibilities are generally not considered. Both the molecular and morphological variation of an organism needs to be considered to determine the appropriate limits to each OTU.
1.0 General introduction
18
PCR-RFLP and T-RFLP are techniques that are useful for assessing community shifts and tracking presence and absence in samples (Horton & Bruns 2001; O'Brien, Parrent et al. 2005). The advantages of using these techniques are that each sample, either root tip or sporocarp, can be profiled, and profiles can then be grouped on similarity, removing the need for taxonomic identification of each individual sample. These techniques can be time consuming as most research has indicated that two restriction enzymes are needed to adequately differentiate fungal taxa, but in some cases, three or more may be required (Kåren, Hogberg et al. 1997; Glen, Tommerup et al. 2001b). Also, these techniques are limited in their ability to provide taxonomic affiliation or enumerate species richness of communities unless a large reference database is available, or they are combined with DNA sequencing (Horton & Bruns 2001; O'Brien, Parrent et al. 2005). Using PCR-RFLP or T-RFLP, in combination with DNA sequencing, does allow grouping of like profiles, which is
beneficial as it minimises the number of sequences (and therefore minimises time invested and expense) that need to be obtained for taxonomic identification.
T-RFLP is advantageous over PCR-RFLP as it allows automated sizing with great precision of restriction fragments and a high output of data, as well as multiple species identification from a single sample but also has many disadvantages including the formation of pseudo- restriction fragments (Egert & Freidrich 2003; see Avis, Dickie et al. 2006; and Dickie & FitzJohn 2007 for review).
Cloning of PCR amplicons to isolate individual DNA fragments from environmental samples has been used for assessing soil fungal diversity (Viaud, Pasquier et al. 2000; Chen & Cairney 2002; O'Brien, Parrent et al. 2005; Porter, Skillman et al. 2008). This method allows mixed samples, such as soil, or multiple mycorrhizal root tips to be processed together. The main limitation to this method is determining the number of clones for PCR-RFLP and sequencing so that common and rare sequences can be determined and diversity is fully sampled (Anderson & Cairney 2004). In highly diverse communities a large number of clones may need to be screened. For example O'Brien, Parrent et al. (2005) sampled 100 soil cores and sequenced all 863 clones that were recovered from these cores resulting in 412 fungi, with no saturation in the species-effort curve.
DNA sequencing consists of a series of biochemical reactions, fluorescently labels the nucleotides, and their order is determined by a DNA sequencer. Sequences from different samples can be aligned and checked for similarity, enabling groups of like sequences to be
1.0 General introduction
19
determined (e.g. species or operational taxonomic units) or searched for matches to known DNA sequences within a database.
Next-generation DNA sequencers (454- or Pyro-sequencing) that allow high quality sequence output based on the "sequencing by synthesis" principle are likely to supersede current instruments in the near future and will allow large-scale sequencing of complete fungal communities (Mardis 2008; Buee, Reich et al. 2009; Nilsson, Ryberg et al. 2009; Öpik, Metsis et al. 2009).Like all methodologies, 454-sequencing poses challenges in that the limited read lengths may be problematic for identification (lengths of individual reads of DNA sequence are in the neighbourhood of 300-500 nucleotides, shorter than the 800-1000 obtainable with chain termination methods). The composition depicted through this new style of sequencing and traditional sequencing may not be comparable (Nilsson, Ryberg et al. 2009). An alternative technique to sequencing, which also has the capacity for high throughput, is microarray analysis (Courty, Buée et al. 2010). In the past this technique has been used for genome-wide transcription profiling but is now being applied to the
identification of ectomycorrhizal communities (Reich, Kohler et al. 2009).
For this research, amplification of the ITS region by PCR of root tips and sporocarps using the fungal specific primers ITS1 and ITS4 was utilised in combination with DNA sequencing. PCR, soil cloning, PCR-RFLP and DNA sequencing were all used in combination for
1.0 General introduction
20
Table 1.1 Summary of common molecular techniques used for the identification and characterisation of ectomycorrhizal species and communities.
Molecular technique Description of process
DNA sequencing DNA extractions followed by PCR, then the order of nucleotides is determined by a DNA sequencer (Gardes, White et al. 1991; Stendell, Horton et al. 1999; Tedersoo, Jairus et al. 2008). The key principle of most methods of DNA sequencing (e.g. Sanger sequencing) is chain termination with dideoxynucleotide triphosphates (ddNTPs). 454- or Pyro-sequencing with next generation sequencers differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with
dideoxynucleotides (Buee, Reich et al. 2009; Nilsson, Ryberg et al. 2009; Jumpponen, Jones et al. 2010; Tedersoo, Nilsson et al. 2010).
Restriction fragment length polymorphism (RFLP)
DNA amplification, followed by restriction digestion with endonucleases that cleave the DNA at certain restriction sites. Digested DNA, containing restriction fragments is then
electrophoresed on high resolution agarose or acrylamide gel to determine RFLP patterns and restriction fragment sizes. The RFLP profiles from unknown material are then matched to profiles obtained from identified sporocarps or cultures (Erland, Henrion et al. 1994; Kåren, Hogberg et al. 1997; Glen, Tommerup et al. 2001a).
Terminal restriction fragment length polymorphism (T- RFLP)
Similar to PCR-RFLP but uses a fluorescent label unique to each PCR primer for detection of the terminal fragments of restriction- digested PCR products. These terminal restriction fragments contain the labelled primer and extend to the restriction site for the enzyme used (Liu, Marsh et al. 1997; Edel-Hermann, Dreumont et al. 2004; Burke, Martin et al. 2005).
1.0 General introduction
21 Molecular technique Description of process
Soil cloning DNA is extracted from an environmental sample, amplified and then cloned using bacterial phage cells. These cells are then cultured after which DNA is extracted, and amplified. RFLP or sequencing can then be used to identify species or community patterns (Viaud, Pasquier et al. 2000; Chen & Cairney 2002; O'Brien, Parrent et al. 2005; Porter, Skillman et al. 2008).