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8. MOLECULAR PHYLOGENY OF THE GENUS LOLIUM
outgroups. The seed sources of all species and the methodology of plant growing are listed
in Annex 13.1. The identity of species from gene banks was checked using taxonomic keys.
Bulks from 20 to 30 plants were analysed per a species.
Total DNA was isolated from individual plants according to the modified CTAB procedure
(Annex 13.4). Several types of molecular markers were used in the studies. The analysis of organelle
DNA included: restriction site polymorphism of chloroplast DNA (only HaeIII was used
because only this enzyme revealed polymorphism in multiflorum and perenne) of the noncoding
region psbC-trnS in chloroplast genome (Annex 13.6), amplification polymorphism of
intron between B and C exons of nad1 gene (Annex 13.7), restriction polymorphism of the mitochondrial
gene, LOLMTI originated from L. perenne ssp. multiflorum (Annex 13.7). Nuclear
DNA was analysed by restriction site polymorphism of rDNA internal transcribed spacer using
universal primers complementary to the conservative sequences near 18S and 5.8S rDNA
borders as well as by primers complementary to L. perenne ssp. perenne spacer, LOLITS.
Amplification polymorphism of intergenic spacer between two nuclear tRNA-Leu genes was
included in the studies. Two sequences derived from Lol p I pollen allergen, LOLPISO5A,
LOLPISO1A and a sequence derived from Lol p Ib pollen allergen, LOLLOPIB were analysed
with respect to amplification polymorphism. A restriction map was made for LOLPISO5A using
enzymes previously selected on the basis of restriction maps of sequences deposited in
NCBI. The maps were created using NEB cutter program provided by New England Biolabs.
Furthermore, the polymorphism of several low copy sequences was analysed. They included
L. temulentum nuclear gene encoding chlorophyll binding protein type II (LTLHAB) and L. perenne
genes encoding L-asparaginase (ASNL), thioredoxin (Trx) and glutamine synthetase
(Gln2). Methodology of the analysis of all mentioned above sequences is given in Annex
13.14 while primer sequences in Annex 13.5. All random, genome scanning markers, RAPD,
ISJ, AFLP and transposon-based SSAP markers that have been previously mapped on linkage
map of multiflorum x perenne were included in phylogenetic analyses. In total 12 RAPD
primers, 12 ISJ primers, six AFLP primers, two combinations of primers specific to the DNA
transposon, Tpo1 and two combinations specific to the Lolcopia2 retrotransposon (Annex
13.08-13.12) were employed.
Genetic similarity was calculated based on the number of shared bands according to
Nei and Li (1979) with further modifications introduced by Clark and Lanigan (1993) for RAPD
data and Innan et al. (1999) for AFLP data. The resultant matrices were used in the cluster
analysis. Two clustering methods were applied, the UPGMA that assumes equal evolutionary
rates and the neighbour-joining (N-J) that allows for unequal rates. However, received
trees were nearly identical, therefore only the results of UPGMA were presented owing to the
possibility of using STATISTICA 7.1. Where necessary the principal component analysis was
employed. The mean number of nucleotide substitutions for restriction sites was calculated
according to Nei and Li (1979).