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6. DO ANY SPECIES BOUNDARIES....
that enzymes resolved by any protein electrophoresis are not a random sample but they are
pre-selected based on easy detection by ordinary staining procedure. Notwithstanding these
difficulties, enzymes are useful in the identification of linkage groups between many maps
providing that sufficient number of enzymatic loci is employed. With 16 enzymatic loci it is
possible to mark nearly all linkage groups. It is a pity that these rapid, reliable and cheap
markers are so rarely used for map alignment.
An important outcome from the current map is the identification of some enzymatic loci
for the first time. They include loci encoding aconitase hydratase, alcohol dehydrogenase,
cytosol aminopeptidase, isocitrate dehydrogenase, malate dehydrogenases, peroxidases
and shikimate dehydrogenase. In Lolium, similarly to other plants, two different isoenzymes
oxidize L-malate, oxaloacetate and some other 2-hydroxydicarboxylic acid. The first enzyme
(MDH) uses NAD as a cofactor while another NADP (MDHP). Two loci for both of them are
identified by means of starch gel electrophoresis. Due to similarity of their zymograms, it was
suspected that they may represent a single locus. Nevertheless, mapping studies demonstrated
that these two enzymes are indeed encoded by two different loci allocated to different
linkage groups, Mdh2 locus is mapped on LG3 whereas Mdhp2 locus on LG2.
Another spectacular achievement is that for the first time markers revealed by primers
complementary to bacterial sequences are mapped (B-SAP). In spite of transposons, this
is the most unique feature of the present map. Until now there has no such a map either in
Lolium or in the other plants. Two types of bacteria-specific markers are located, IS markers
revealed by primers complementary to IS6110 insertional element of M. tuberculosis and
katG markers based on KatG gene of the same bacterium. Their utility in generation of species
specific markers has been confirmed in several plant species (Zielinski and Polok 2005)
however, there has not been clear what kind of sequences they are. It has been suggested
that markers based on IS6110 can be related with transposon sequences whereas katG
markers with peroxidases due to the fact that KatG gene encodes catalase-peroxidase. Nevertheless,
this hypothesis remained to be corroborated in more direct genetic studies such
as this. A counterproposal that can easily be expressed is a contamination of plant material
by bacteria. In that case, most markers derived from bacterial sequences would segregate
in a non-Mendelian fashion and would not be linked with any plant sequence. Consequently,
they would be identified as solitary markers. Fortunately, the last theory is unlikely in the light
of the Mendelian inheritance of nearly 90% of B-SAP markers and their allocation to linkage
groups of L. multiflorum x L. perenne. Furthermore, their map position is not random, but
they are predominantly linked with enzymatic loci, including both loci responsible for plant
peroxidases.
The bacterial catalase-peroxidase belongs to the class I of peroxidases and as the
only member of this family possesses two functions - it can oxidaze (catalase activity) and
reduce hydrogen (peroxidase activity). Catalase-peroxidase of M. tuberculosis encoded
by KatG gene is one of the most studied bacterial peroxidases due to its role in acquiring
resistance to antibiotics. The gene is characterized by rather high variability and therefore,
a set of primers derived from KatG gene is commonly used for strain identification.
On the other hand, the plant peroxidases of class I originated from a common prokaryotic
ancestor but they lost their catalase activity. The detailed Zamocky’s (2004) studies
revealed that they possess three corresponding areas involved in catalytic mechanism.