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9. PHYLOGENETIC RELATIONSHIPS...
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current data exemplify how the nature of evolutionary changes can be traced using molecular
appraisals. The documented examples of genome rearrangements responsible for the transition
from wild to domesticated forms are also more and more frequent and involved such
crops as maize, rice, sorghum and many others. However, all they document past events.
The genus Lolium and especially L. perenne is unusual due to the fact that it is in a half way, it
is diversifying both through the adaptation to more southern environments and domestication
processes, but still pending the birth of species boundaries. It is a kind of a model for tracing
“the evolution in action” through the use of vast molecular techniques available.
The results presented in the preceding chapters shed some light on the evolutionary
history of the genus and such as they are dealing with the past. Another challenge is to use
this knowledge to understand the nature of undergoing evolutionary processes driving by intensive
breeding and changing environment. It would have a tremendous effect on predicting
the potential influence of intergeneric hybrid cultivars or transgenic plants and finally help in
protecting biodiversity. Close relationships of the genus Lolium with cereals or more generally
with “Core Pooids” (Aveneae, Bromeae, Poeae, Triticeae) is an additional advantage
because evolutionary considerations can draw extensively on the knowledge and methods
developed for more studied species. Therefore, the present part intended to summarize the
knowledge about the position of the genus Lolium within the Poaceae family and its relationships
with cereals based on own results, with the aim of using it as a foundation for further
research on the mechanisms of evolution in plants.
9.2. MATERIAL AND METHODS
In addition to seven species from the genus Lolium, described previously (Chapter 8,
Annex 13.1) a set of representatives of cereals’ species was used. It involved members of
“Core Poids”, H. vulgare, T. aestivum, S. cereale (Triticeae) and artificial allopolyploid derived
from these two species - Triticale, and two oat species, A. sativa and A. strigosa (Aveneae).
Moreover, A. thaliana was used as outgroup.
Phylogenetic trees were constructed on the basis of cereal sequence tagged sites (STS)
previously used in mapping studies and selected from Taylor et al. (2001). Shortly, primers
were derived from genes encoding asparagine synthetase (AS1), L-asparaginase (ASN),
and HS1 protein of H. vulgare. In addition primers complementary to RFLP probes from
H. vulgare (BCD450) and A. sativa (CDO504 and CDO1508) were used. PCR conditions
were optimised during mapping studies so that to amplify a single band in L. perenne ssp.
multiflorum and L. perenne ssp. perenne, and these conditions were used for species comparisons.
Optimised PCR conditions are given in Annex 13.14. Primer sequences are given
in Annex 13.5. A single band was amplified by primers derived from H. vulgare asparagine
synthetase (AS1) and A. sativa RFLP probe CDO1508. Primers derived from H. vulgare
RFLP probe BCD450 and A. sativa probe CD0504 revealed two strong reproducible bands
whereas for ASN and HS1 only multi-band pattern was observed. In these cases the optimisation
was done to receive reproducible amplification. At the next step DNA of all species was
amplified at PCR conditions specific to L. perenne. If a single band was obtained, PCR products
were subjected to restriction digestion. If two bands were obtained, the strongest band