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9. PHYLOGENETIC RELATIONSHIPS...
was eluted from the gel, re-amplified and PCR products were digested. In total six restriction
enzymes were used, AluI, BamHI, EcoRI, HindIII, RsaI and TaqI. In a case of multiple bands
the polymorphism was assessed with respect to amplification products.
The utility of B-SAP for phylogenetic studies was checked using primers derived from
M. tuberculosis gene encoding catalase-peroxidase, KatG and composing of 4 801 bp.
Twelve pairs of katG primers were designed for every 280 bp fragments according to Zielinski
and Polok (2005). The strategy of primer designing and PCR conditions are described in
Annex 13.13.
The consensus tree was based on 2894 markers including RAPD, ISJ, AFLP, SSAP,
SSR markers in addition to B-SAP markers derived from KatG gene, IS6110, rpo and pol
from M. tuberculosis, markers derived from low-copy sequences of Lolium (LOLPISO5A,
LOLPISO1A, LOLOPIB, LTLHAB, ASNL, Trx, Gln2), H. vulgare (ASN, AS1, HS1, BCD450)
and A. sativa (CDO504, CDO1508).
The similarity was estimated on the basis of shared amplification products or restriction
fragments according to Nei and Li (1979). The UPGMA and squared Euclidean distance
were used for clustering.
Data from genetic mapping were compared with barley and oat maps. In particular, the
location of enzymatic sequences and the abundance of transposons were compared.
9.3. RESULTS AND DISCUSSION
9.3.1. Phylogenetic relationships between the genus Lolium and representativesof
“Core Pooids”
PCR-mediated gene sequencing for phylogenetic studies has increased explosively in
recent years. Sequence data provide high-resolution picture of molecular diversity but sacrifice
genetic information from many loci. Typically, sequence analysis involves a single gene
in one or two individuals per a species. In that sense the DNA sequence data permit recovery
of genetic information at less detailed level than some other kind of molecular markers. In
Lolium the phylogenetic relationships are better resolved using different categories of molecular
markers than analysing the LOLMTI mitochondrial sequence or the LOLPISO5A gene
encoding L. perenne pollen allergen. Even the analysis of ITS in rDNA gives worse resolution
than a wealth of molecular markers. A general view emerging from phylogenetic studies
based on one or a few sequences is that they usually support for the higher taxa level but the
support for the species level is very low. Furthermore, gene sequences are far more prone
to phylogenetic noise than molecular markers because a nucleotide has only four alternative
states. A practical income from analyses of DNA sequences is that a position of a given species
tends to change depending on a gene used.
To use as many markers as possible to interfere the phylogenetic relationships seems
to be the most fruitful approach, especially if many different marker categories are available
and the band identity is confirmed in mapping studies. The data from the genus Lolium
(Chapter 8) as well as the current tree of Pooideae subfamily demonstrate the high reliability
of such strategy. The majority of branches were resolved well and the species relationships