Introduction to Freshwater Algae - Freshwater Algae: Identification, Enumeration and Use as Bioindicators - page 11

DNA sequence analysis, using a standard sequen-
cer or by applying the relatively new technique of
pyrosequencing (Ronaghi, 2001). Comparison to
known 16S and 18S rRNA gene sequences in a
standard database such as NCBI leads to tentative
species identification, normally requiring a match
of at least 90%.

Ultimately, species definition and identification in
both prokaryote and eukaryote algae may depend on
molecular analysis, with determination of unique and
defining DNA sequences followed by development
of species-specific nucleotide probes from these (see
next section).This approach would be particularly rel-
evant in the case of blue-green algae, but there are a
number of problems in relation to species-specificity
in this group (Castenholz, 1992).

The pattern of bands in the DGGE gel gives an
estimate of community complexity, while the inten-
sity of individual bands (derived from particular
species) provides a measure of species population
size. In addition to providing taxonomic information
where classical morphological characteristics do not
apply, molecular identification also has the advantage
that the whole of the microbial community (commu-
nal DNA sample) is being analysed in an objective
way. Non-photosynthetic bacteria, Archaea and pro-
tozoa are also identified in addition to prokaryote and
eukaryote algae (Droppo et al ., 2007; Galand et al .,
2008).
Potential limitations of molecular analysis are that
identification may be tentative (often only to genus
level) and taxonomic quantitation (relative numbers
of different algae) is difficult. The technique has
been particularly useful in relation to the biodiversity
of marine blue-green unicellular algae and is now
increasingly used with freshwater systems (Table
1.4). Molecular techniques can be used to probe par-
ticular enzymes (e.g. nitrogenase) as well as more
general taxon-specific gene sequences.

Polyploidy (multiple genomic copies per cell)
may occur, with variation between the multiple
genomes. As many as 10 multiple genome copies
have been observed in some blue-green algae.

Horizontal gene transfer means that some DNA
fragments are dispersed over a range of species.

DNA/RNA sequence analysis

Analysis of DNA sequences has been widely used
for the identification of both blue-green (16S rRNA
genes) and eukaryote algae (18S rRNA and chloro-
plast DNA). This technique has been used by Droppo
etal .(2007),forexample,todeterminethetaxonomic
composition of biofilms - identifying bacteria, blue-
green algae and some eukaryote unicellular algae.
The technique involves:

Collection of a sample of biomass from the entire
microbial community.

Enzyme probes: Nitrogenase ( nif ) genes
Molecular probes have been used to monitor genes
encoding both the large ( nif K) and small ( nif H) nitro-
genase subunits.
Stancheva et al . (2013) monitored nitrogenase
gene expression in benthic river algae by develop-
ing primers to the nif K gene, then using real-time
reverse transcriptase PCR. The technique was used
to validate the potential role of N 2 -ixing blue-green
algae as indicators of low ambient concentrations of
inorganic nitrogen.
Foster et al . (2007) used DNA quantitative PCR
(QPCR) technology to amplify and detect the pres-
ence of species-specific nif H genes in blue-green

Obtaining a DNA sample. This may involve extrac-
tion from a mixed environmental sample such as
biofilm or soil (Miller et al., 1999).

Polymerase chain reaction (PCR) amplification of
a specific nucleotide region - typically 16S or 18S
rRNA genes, about 1500 bp in length.

Separation of the amplified strands by denaturing
gradient gel electrophoresis (DGGE), or purifica-
tion of the PCR products using a rapid purification
kit.