28
communities because TRF size for one primer enzyme combination could be similar but not for the other combinations. T-RFLP is extensively used in the studies of
structural attributes of the microbial communities under an agricultural system
(Malghani et al. 2016; Liu et al. 2016a, b) and also for the restoration of contaminated sites and bioenergy feedstock production (Mossa et al. 2017).
3.5 Automated Version of RISA (Ribosomal Intergenic
Spacer Analysis (ARISA)
The automated version of ribosomal intergenic spacer analysis (RISA), ARISA, is a
useful tool for exploring microbial diversity (Lee et al. 2012). It is a sensitive and
highly reproducible technique in comparison to RISA and exploits the length heterogeneity of the internal transcribed spacer (ITS) that lies between 16S and 23S
rRNA sequences of the ribosomal RNA operon. PCR amplification of the ITS
sequence is done with 5′-fluorescently labelled primer. After being labeled, PCR
products are analysed by an automated DNA analyser, and the results show polymorphism in the PCR product length. The length heterogeneity of each PCR product represents an operational taxonomic unit (OTU). Cloning and sequencing of the
ARISA-PCR products and their further BLAST analysis reveals the identity of the
microbes. Another approach for rapid phylogenetic characterization of microbial
communities could be the direct phylogenetic analysis of the T-RFLP products
through genetic libraries (Brown and Fuhrman 2005; Collins et al. 2006). ARISA
offers more phylogenetic resolution as it targets the 16S-23S ITS rather than the 16S
rRNA region targeted in T-RFLP studies (Hewson and Fuhrman 2004). Some
microorganisms share ITSs of equal length and their diversity would not be shown
by ARISA. On the other hand, some microorganisms may have multiple copies of
rRNA operons having ITS of variable regions. Thus, a single microorganism may be
overrepresented as different microorganisms in a community profile (Collins et al.
2006). Because ARISA is a PCR-dependent technique, it may face some artefacts of
PCR-based amplification, but it offers better resolution of bacterial communities at
species and strain levels (Kovacs et al. 2010a, b). The technique is economic and
relevant for microbial community structure analysis under conservation agriculture
practices (Likar et al. 2017; Butterly et al. 2016; Bertani et al. 2016) and for contaminated land phytoremediation studies (Fernandes et al. 2017).
3.6 Single-Strand Conformation Polymorphism (SSCP)
Single-strand conformation polymorphism (SSCP) is a screening technique for
identifying different genomic variants in a microbial population from environmental samples (Fischer and Lerman 1979). The technique can detect sequence
3 Methods for Exploring Soil Microbial Diversity
communities because TRF size for one primer enzyme combination could be similar but not for the other combinations. T-RFLP is extensively used in the studies of
structural attributes of the microbial communities under an agricultural system
(Malghani et al. 2016; Liu et al. 2016a, b) and also for the restoration of contaminated sites and bioenergy feedstock production (Mossa et al. 2017).
3.5 Automated Version of RISA (Ribosomal Intergenic
Spacer Analysis (ARISA)
The automated version of ribosomal intergenic spacer analysis (RISA), ARISA, is a
useful tool for exploring microbial diversity (Lee et al. 2012). It is a sensitive and
highly reproducible technique in comparison to RISA and exploits the length heterogeneity of the internal transcribed spacer (ITS) that lies between 16S and 23S
rRNA sequences of the ribosomal RNA operon. PCR amplification of the ITS
sequence is done with 5′-fluorescently labelled primer. After being labeled, PCR
products are analysed by an automated DNA analyser, and the results show polymorphism in the PCR product length. The length heterogeneity of each PCR product represents an operational taxonomic unit (OTU). Cloning and sequencing of the
ARISA-PCR products and their further BLAST analysis reveals the identity of the
microbes. Another approach for rapid phylogenetic characterization of microbial
communities could be the direct phylogenetic analysis of the T-RFLP products
through genetic libraries (Brown and Fuhrman 2005; Collins et al. 2006). ARISA
offers more phylogenetic resolution as it targets the 16S-23S ITS rather than the 16S
rRNA region targeted in T-RFLP studies (Hewson and Fuhrman 2004). Some
microorganisms share ITSs of equal length and their diversity would not be shown
by ARISA. On the other hand, some microorganisms may have multiple copies of
rRNA operons having ITS of variable regions. Thus, a single microorganism may be
overrepresented as different microorganisms in a community profile (Collins et al.
2006). Because ARISA is a PCR-dependent technique, it may face some artefacts of
PCR-based amplification, but it offers better resolution of bacterial communities at
species and strain levels (Kovacs et al. 2010a, b). The technique is economic and
relevant for microbial community structure analysis under conservation agriculture
practices (Likar et al. 2017; Butterly et al. 2016; Bertani et al. 2016) and for contaminated land phytoremediation studies (Fernandes et al. 2017).
3.6 Single-Strand Conformation Polymorphism (SSCP)
Single-strand conformation polymorphism (SSCP) is a screening technique for
identifying different genomic variants in a microbial population from environmental samples (Fischer and Lerman 1979). The technique can detect sequence
3 Methods for Exploring Soil Microbial Diversity