TAXONOMY, PHYLOGENY AND EVOLUTION - Cyanobacteria - page 21

gene restriction patterns leading to the establishment of 'ribotyping' as potential taxonomic tool for
the enumeration of bacterial diversity. The molecular genetic basis of ribotyping has been elucidated
(Bouchet et al ., 2008). This is considered to be a rapid method for comparisons of rDNAs (Laguerre
et al ., 1994; Moyer et al ., 1994). It involves digestion of rDNAs (obtained by PCR amplifi cation by
using universal primers) with restriction enzymes that have 4-bp recognition sites. The restriction
digests are analysed by agarose gels. For community analysis, the large number of fragments can be
resolved by using polyacrylamide gels to produce community specifi c pattern (Martínez-Murcia et
al ., 1995; Massol-Deya et al ., 1995). The disadvantage with this method is that it is of limited use for
detecting specifi c phylogenetic groups or for estimating species richness or evenness.
A variation of the above method is to subject the whole genomic DNA for restriction analysis
instead of a single gene as in ARDRA. This is termed as amplifi ed fragment length polymorphisms
(AFLPs). Naturally occurring DNA polymorphisms are basically due to point mutations or
rearrangements (i.e. insertions and deletions) in the DNA. AFLP analysis is based on selective
amplifi cation of DNA restriction fragments (Vos et al ., 1995). Restriction enzyme digestion of
genomic DNA is follwed by PCR amplifi cation and the products are subjected to separation by
electrophoresis. This enables us to detect the presence or absence of such polymorphisms by the
presence or absence of banding patterns. The variation in banding patterns may directly refl ect
the genetic relationships between the bacterial strains examined. So these banding patterns in
otherwords represent genomic fi ngerprints allowing numerical analysis for characterization and
identifi cation. Genome-wide variations in the strains can be scanned by AFLP. Thus it will greatly
help in the resolution of taxonomic problems which are otherwise problematic and answers for
these are diffi cult to obtain through morphological features or by other molecular methods. The
broad taxonomic applicability of this technique has been tested in case of bacteria (Huys et al ., 1996)
and fungi (Majer et al ., 1996). The advantage of AFLP over other techniques is that multiple bands
are derived from all over the genome. This prevents over interpretation or misinterpretation due
to point mutations or single-locus recombination, which may affect other genotypic characteristics.
The main disadvantage of AFLP markers is that alleles are not easily recognized (Majer et al ., 1998).
Due to the inherent advantages of its utility, reproducibility and effi ciency, the AFLP technique has
been used to investigate cyanobacterial populations as well (Janssen et al ., 1996).
RAPD fi ngerprinting in conjunction with PCR has been used to identify microorganisms to
the strain level (Welsh and McClelland, 1990). The sequences frequently used are short tandemly
repeated repetitive sequences (STRR). These are of the nature of heptanucleotide sequences that
are present nearly at the rate of 100 copies per genome of Calothrix sp. strain PCC 7601. These can
be used for special identifi cation in the taxonomy of heterocystous cyanobacteria. Three types of
the STRR sequences identifi ed are STRR1 [CCCCA(A/G)T], STRR2 [TT(G/T)GTCA] and STRR3
[CAACAGT]. Of these, the former two are present 100 copies each per Calothrix genome (Mazel et
al ., 1990). These can be used as oligonucleotide probes (Rouhiainen et al ., 1995) or as primers for the
generation of PCR-amplifi ed DNA profi les (Rasmussen and Svenning, 1998) for the identifi cation
of toxic cyanobacteria in cultures as well as from fi eld samples. Besides the STRR sequences,
the repetitive extragenic palindrome sequences (REP) and Enterobacterial Repetitive Intergenic
consensus sequences (ERIC) (Sharples and Lloyd, 1990; Hulton et al ., 1991; Wilson and Sharp, 2006)
have been used for the identifi cation of bacteria. DNA polymorphisms in phycocyanin locus were
used to generate RAPD fi ngerprinting profi les to differentiate members of the genera Anabaena and
Microcystis (Neilan et al ., 1995). Symbiotic and free-living cyanobacterial cultures were identifi ed by
PCR fi ngerprinting (Weiwen et al ., 2002) and genotypes of Microcystis were discriminated by RAPD
fi ngerprinting technique (Nishihara et al ., 1997).