commercially, whereas stronger effects can be observed
when higher concentrations are used (Ratcliff et al. 2006).
Similarly, the effect of agricultural practices on soil bacterial
communities has been demonstrated with BIOLOG plates,
which has been confirmed by the T-RFLP fingerprint
method (Widmer et al. 2006).
8.4.2 Approaches Targeting Genotype
Diversity
8.4.2.1 Approaches Targeting Nucleic Acids (DNA)
DNA Reassociation
For this technique, DNA is extracted from a given biotope,
denatured, and allowed to renature. Originally it was used to
determine the size of a genome: At a given set of concentration and temperature, the rate of reassociation of denatured
DNA strands is a function of the complexity of the DNA. The
DNA of a single taxon will then reassociate very quickly,
whereas DNA retrieved from a biotope containing thousands
of taxa will take much more time to reassociate. Using this
technology, Torsvik et al. (1990) estimated that agricultural
soils typically contain thousands of taxa per gram of soil,
making it the most diverse habitat known (2002). Gans and
collaborators (2005) refined the approach and obtained even
higher values (around 10
6 bacterial taxa per 10 g of soil). This
technology is applicable to any habitat. However, its application is complex and it gives no indication of the taxa present,
which explains why it has been rarely used.
All the approaches described below target nucleic acids and
are based on PCR using DNA primers, which are short
sequences that anneal to conserved regions of genes. The
concept of “universal primers” is often used, but this concept
is to be taken with caution because the primers are designed
based on known taxa, for example, the symbiotic bacteria
Nanoarchaeum could not be amplified using these primers,
and it had to be characterized by Southern/cloning/sequencing
of its 16S rRNA gene. This permitted to show that there were
two mismatches in one of the so-called universal primers,
which prevented polymerization (Huber et al. 2002). Conversely, adequately targeting a narrow taxonomic group, e.g.,
Actinobacteria (Stach et al. 2003), poses less problems.
Linkage Disequilibrium Analysis Through
Polymorphism Association (on Isolates)
Two populations of microorganisms can be compared by the
tools of population genetics in order to quantify the frequency
of genetic exchange among populations. Bacteria belonging to
the same species may exchange (by conjugation or otherwise)
genes, which will cause genetic mixing. The frequency of
these exchanges is quantified by the study of linkage disequilibrium between two markers. This can be extended to quantify the genetic diversity among several isolates.
Denaturing Gradient Gel Electrophoresis (DGGE) and
Temperature Gradient Gel Electrophoresis (TGGE)
These techniques were developed for the study of human
polymorphism (Sheffield et al. 1989) to distinguish very
similar DNA or RNA sequences because they still have
different denaturation parameters that will result in
differences in gel migration. Indeed, the concentration of
denaturant (DGGE) or the temperature (TGGE) at which a
double-stranded sequence is separated into two singlestranded chains varies between very similar sequences. It is
also known that a sequence of double-stranded nucleic acid
being more compact will migrate much faster than a singlestranded sequence in an acrylamide gel. In these approaches,
a GC-rich clamp (GC-clamp) keeps the two strands together
in order to have a higher sensitivity of the method. This
technique was initially applied to microbial ecology by
Muyzer and Smalla (1998) in a series of works on different
biotopes that began in 1993. This revealed the existence of
hundreds of microbial taxa in the environments studied
(Muyzer et al. 1993). In January 2013, a Web of Science
search with DGGE or TGGE as keywords permitted to
retrieve 7,753 references, most of them corresponding to
microbial ecology papers. DGGE has been increasingly
used since the early 1990s, though no such increased use is
observed after 2010 (Fig. 8.1).
Single-Strand Conformational Polymorphism (SSCP)
While the principle of DGGE is based on the complementarity between the two strands of DNA, the one of SSCP is
based on sequence complementarity within strands. SSCP
differs from DGGE in that DNA is denatured and then
allowed to renature before (not during) gel migration. The
denatured DNA sequences, even closely related ones, have
very different migration rates. This technique was used, for
instance, to characterize the diversity of the archaeal community in 44 anaerobic digesters (Leclerc et al. 2004). A
Web of Science search in January 2013 with SSCP as keyword permitted to retrieve 2,081 references. This method has
been increasingly used between 1990 and 1998, whereas a
progressive decrease of its use is observed during the
1998–2012 period (Fig. 8.1).
Ribosomal Intergenic Spacer Analysis (RISA)
Gene order is highly conserved in ribosomal operons with a
primary transcript comprising the 16S (rrs) at the 5
0 end, the
23S (rrl) in the middle, and the 5S (rrf) at the 3
0 end in all
microbial species studied so far (Normand et al. 1996). The
only known exception is Wolbachia where rrs genes are
located away from the rrf and rrl genes (BensaadiMerchermek et al. 1995). By contrast, what varies greatly
from one bacterial species to another is the length of the
intergene between rrs and rrl genes, which led Fisher and
Triplett (1999) to use a pair of primers to amplify this region,
8 Biodiversity and Microbial Ecosystems Functioning
269
when higher concentrations are used (Ratcliff et al. 2006).
Similarly, the effect of agricultural practices on soil bacterial
communities has been demonstrated with BIOLOG plates,
which has been confirmed by the T-RFLP fingerprint
method (Widmer et al. 2006).
8.4.2 Approaches Targeting Genotype
Diversity
8.4.2.1 Approaches Targeting Nucleic Acids (DNA)
DNA Reassociation
For this technique, DNA is extracted from a given biotope,
denatured, and allowed to renature. Originally it was used to
determine the size of a genome: At a given set of concentration and temperature, the rate of reassociation of denatured
DNA strands is a function of the complexity of the DNA. The
DNA of a single taxon will then reassociate very quickly,
whereas DNA retrieved from a biotope containing thousands
of taxa will take much more time to reassociate. Using this
technology, Torsvik et al. (1990) estimated that agricultural
soils typically contain thousands of taxa per gram of soil,
making it the most diverse habitat known (2002). Gans and
collaborators (2005) refined the approach and obtained even
higher values (around 10
6 bacterial taxa per 10 g of soil). This
technology is applicable to any habitat. However, its application is complex and it gives no indication of the taxa present,
which explains why it has been rarely used.
All the approaches described below target nucleic acids and
are based on PCR using DNA primers, which are short
sequences that anneal to conserved regions of genes. The
concept of “universal primers” is often used, but this concept
is to be taken with caution because the primers are designed
based on known taxa, for example, the symbiotic bacteria
Nanoarchaeum could not be amplified using these primers,
and it had to be characterized by Southern/cloning/sequencing
of its 16S rRNA gene. This permitted to show that there were
two mismatches in one of the so-called universal primers,
which prevented polymerization (Huber et al. 2002). Conversely, adequately targeting a narrow taxonomic group, e.g.,
Actinobacteria (Stach et al. 2003), poses less problems.
Linkage Disequilibrium Analysis Through
Polymorphism Association (on Isolates)
Two populations of microorganisms can be compared by the
tools of population genetics in order to quantify the frequency
of genetic exchange among populations. Bacteria belonging to
the same species may exchange (by conjugation or otherwise)
genes, which will cause genetic mixing. The frequency of
these exchanges is quantified by the study of linkage disequilibrium between two markers. This can be extended to quantify the genetic diversity among several isolates.
Denaturing Gradient Gel Electrophoresis (DGGE) and
Temperature Gradient Gel Electrophoresis (TGGE)
These techniques were developed for the study of human
polymorphism (Sheffield et al. 1989) to distinguish very
similar DNA or RNA sequences because they still have
different denaturation parameters that will result in
differences in gel migration. Indeed, the concentration of
denaturant (DGGE) or the temperature (TGGE) at which a
double-stranded sequence is separated into two singlestranded chains varies between very similar sequences. It is
also known that a sequence of double-stranded nucleic acid
being more compact will migrate much faster than a singlestranded sequence in an acrylamide gel. In these approaches,
a GC-rich clamp (GC-clamp) keeps the two strands together
in order to have a higher sensitivity of the method. This
technique was initially applied to microbial ecology by
Muyzer and Smalla (1998) in a series of works on different
biotopes that began in 1993. This revealed the existence of
hundreds of microbial taxa in the environments studied
(Muyzer et al. 1993). In January 2013, a Web of Science
search with DGGE or TGGE as keywords permitted to
retrieve 7,753 references, most of them corresponding to
microbial ecology papers. DGGE has been increasingly
used since the early 1990s, though no such increased use is
observed after 2010 (Fig. 8.1).
Single-Strand Conformational Polymorphism (SSCP)
While the principle of DGGE is based on the complementarity between the two strands of DNA, the one of SSCP is
based on sequence complementarity within strands. SSCP
differs from DGGE in that DNA is denatured and then
allowed to renature before (not during) gel migration. The
denatured DNA sequences, even closely related ones, have
very different migration rates. This technique was used, for
instance, to characterize the diversity of the archaeal community in 44 anaerobic digesters (Leclerc et al. 2004). A
Web of Science search in January 2013 with SSCP as keyword permitted to retrieve 2,081 references. This method has
been increasingly used between 1990 and 1998, whereas a
progressive decrease of its use is observed during the
1998–2012 period (Fig. 8.1).
Ribosomal Intergenic Spacer Analysis (RISA)
Gene order is highly conserved in ribosomal operons with a
primary transcript comprising the 16S (rrs) at the 5
0 end, the
23S (rrl) in the middle, and the 5S (rrf) at the 3
0 end in all
microbial species studied so far (Normand et al. 1996). The
only known exception is Wolbachia where rrs genes are
located away from the rrf and rrl genes (BensaadiMerchermek et al. 1995). By contrast, what varies greatly
from one bacterial species to another is the length of the
intergene between rrs and rrl genes, which led Fisher and
Triplett (1999) to use a pair of primers to amplify this region,
8 Biodiversity and Microbial Ecosystems Functioning
269