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8. MOLECULAR PHYLOGENY OF THE GENUS LOLIUM
any Lolium representatives. None restriction site was common for all Lolium species. Either
a site was observed only in some of all analysed species or it was common to Lolium and
Festuca.
The restriction map of L. loliaceum mitochondrial gene confirmed its close affinity to
the group of out-pollinated taxa. It was the most alike L. perenne ssp. multiflorum that was
reflected by only 0.039 nucleotide substitutions between both species in comparison with
twofold more substitutions between L. loliaceum and perenne (Table 8.1). Additional reason
why L. loliaceum was related with multiflorum the most closely was the unique AluI site at 350
bp that was probably a sign of past introgression from F. pratensis to multiflorum and next to
L. loliaceum. The lack of this site in perenne and L. rigidum seemed to confirm the evidence
of introgression. Similar conclusion could be drawn from the lower number of nucleotide
substitutions differentiating both L. loliaceum and multiflorum from F. pratensis in comparison
with L. rigidum. The intermediate position of L. perenne ssp. perenne between L. rigidum and
multiflorum was apparent from the distribution of TaqI sites. Two sites (at 1600 bp and 1820
bp) were present in perenne, the first was shared with L. rigidum whereas the second with
multiflorum. It was also exemplified by the lowest mean number of nucleotide substitutions
between perenne and L. rigidum from one side and perenne and multiflorum from the other.
8.3.2. Diversity of spacers in genes encoding 18S-26S rRNA and leucine tRNA
The amplification of ITS1 region using common conservative primers resulted in the
single PCR product of the same size in all Lolium species in addition to F. pratensis and
P. pratensis. The ITS region of all nine species had the same restriction sites with respect
to EcoRV (data not shown) and TaqI (Figure 8.4), demonstrating inability to differentiate not
only Lolium species but also the closely related taxa, F. pratensis and more surprisingly,
P. pratensis. Eventually, minor differences were revealed by the digestion with HaeIII (Figure
8.4). In that case all autogamous species but L. loliaceum were clearly distinct from allogamous
ones. F. pratensis could easily be recognised, however the fingerprint of P. pratensis
was very alike Loliums. The better resolution was obtained when primers complementary to
the ITS region derived from L. perenne ssp. perenne - LOLITS were used for amplification.
The difficulties with obtaining a single PCR product in L. persicum proved its distinctiveness
from the other Lolium species. For the remaining taxa a specific PCR band was obtained
allowing further restriction digestions. Similarly to ITS1, LOLITS was not digested by EcoRV
and only minor differences could be observed in TaqI patterns. Nevertheless, the diges-