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15. SUMMARY
At different stages of analyses, in total 45 populations representing ecotypes and
cultivars of L. multiflorum and L. perenne, two F 2
populations derived from interspecific
crosses between L. multiflorum and L. perenne (470 individuals), one L. multiflorum
F 2
population (205 individuals) and one L. perenne F 2
population (44 individuals) were
used. Moreover, the remaining representatives of the genus Lolium i.e., L. loliaceum,
L. persicum, L. remotum, L. temulentum, L. rigidum were used in phylogenetic studies in addition
to closely related F. pratensis, P. pratensis and representatives of the Triticeae tribe
(H. vulgare, T. aestivum, S. cereale, Triticale) and Aveneae tribe (A. sativa and A. strigosa).
The model species A. thaliana was used as outgroup.
A wide range of morphological, statistical and molecular methods was used to asses
genetic variation, construct genetic map, map QTLs and resolve phylogenetic relationships
within the genus. In total 23 quantitative, morphological traits including seedling characters,
vegetative and generative traits were analysed in addition to seedling root fluorescence.
Three types of random, genome scanning markers, RAPD, ISJ and AFLP were employed
at different stages of studies. Insertional polymorphism was analysed using SSAP. Furthermore
SSR markers and STS markers derived from Lolium and cereal sequences coupled
with restriction site polymorphism analysis were used in genetic mapping and phylogenetic
studies. Own marker system was elaborated based on primers derived from bacterial
sequences and used in mapping and phylogenetic studies (B-SAP). The analysis
of organelle DNA (cpDNA, mtDNA) was employed in genetic diversity and phylogenetic
studies whereas isozymes were used in genetic diversity analyses and genetic mapping.
In total 67 RAPD primers, 12 ISJ primers, 36 AFLP primer combinations, nine SSAP primer
combinations specific to the DNA transposon Tpo1, and two Ty1-copia retrotransposons
(Lolcopia1, Lolcopia2), seven SSR pair of primers, 12 STS sets of primers derived from
low copy genes or RFLP probes originated from L. perenne, L. temulentum, H. vulgare and
A. sativa, a pair of universal primers amplifying ITS1 region, a pair of primers specific to
L. perenne ITS region, a pair of primers amplifying spacer between tRNA-leu genes and
15 sets of B-SAP primers complementary to M. tuberculosis sequences were used. Among
B-SAP primers, apart from 12 sets derived from KatG gene encoding catalase-peroxidase,
primers complementary to insertion element IS6110, and hot regions of rpo and pol genes
were used. During studies a wide range of statistical methods and programmes from the
field of population genetics, phylogenetics, map construction and QTL mapping were employed.
The morphological analysis of taxonomic traits did not confirm the separation of
L. multiflorum and L. perenne into two species. They were similar in the majority of studied
characters with the overlapping range of variation. Both species had the same genetic
structure as indicated by a wealth of molecular markers including enzymes, chloroplast
and mitochondrial DNA, RAPD, ISJ. All markers analysed revealed nearly the same level
of polymorphism in L. multiflorum and L. perenne and the markers specific to a species
were hardly found. High molecular similarity strongly supported the classification of
L. multiflorum and L. perenne as a single species. To confirm these results, transposonbased
markers, highly effective in evolutionary studies were used for the first time. Three
different transposon sequences were applied including, DNA transposon from the CACTA
family, Tpo1 and two Ty1-copia retrotransposons, Lolcopia1 and Lolcopia2. The analy-