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4. GENETIC DIVERSITY...
4.2.2. DNA analysis
Total DNA was isolated from individual plants according to the modified CTAB procedure
(Annex 13.4). Five types of molecular markers were used in the studies. Sequences
of all primers used are given in Annex 13.5. Chloroplast DNA was analysed by restriction
digestion (DralI, EcoRI and HaeIII) of the non-coding region psbC-trnS in chloroplast genome
(Annex 13.6). Mitochondrial DNA was analysed by amplification polymorphism of intron between
B and C exons of nad1 gene (Annex 13.7). Total DNA was analysed by random
amplified polymorphic DNA - RAPD (Annex 13.8), intron splice junction polymorphism - ISJ
(Annex 13.9) and transposon based sequence specific amplification polymorphism - SSAP
(Annex 13.12). After initial screening of 30 RAPD (kit A from Operon and ten designed primers)
and 12 ISJ primers, in total nine RAPD primers (KP01-KP07, OPA02, OPA03) and four
ISJ primers (ISJ1-ISJ4) were chosen. The SSAP transposon specific primer was designed to
amplify the Lolium DNA transposon, Tpo1 belonging to the CACTA superfamily (Langdon et
al. 2003). Moreover two primers, LTR1 and LTR2 designed on the basis of consensus Ty1-
copia LTR sequences (RNAseH-LTR junction) were applied. The LTR1 was based on Lolium
multiflorum and Oryza sativa sequences while LTR2 on Saccharum officinarum and Triticum
aestivum sequences. These three primers were used in combinations with three AFLP primers,
two MseI specific primers (Mse-CT, Mse-ACA) and a single PstI specific primer (Pst-AT).
In the case of RAPD, ISJ and SSAP all bands that could be reliable read were treated as
single dominant loci and scored present (1) or absent (0) across all genotypes. Identity of the
majority of bands with the same mobility in L. multiflorum and L. perenne and their nuclear
inheritance were previously confirmed in genetic mapping studies (Chapter 6). Thus, RAPD,
ISJ and SSAP can be treated as nuclear dominant markers.
Data analysis
Allele frequency in each population, the average number of alleles over all loci (A), effective
number of alleles (N e
), percent of polymorphic loci (P), gene diversity (heterozygosity)
at each locus (h) and the average gene diversity in a population over all loci (H) were calculated
by the POPGENE version 1.32 (Yeh and Yang 1999) according to Nei (1987).
Population genetic statistics were also calculated for ecotypes, cultivars and for each
species that were treated as populations divided into a number of subpopulations. The following
statistics were calculated on a population basis: the mean number of alleles per locus
( ), the mean effective number of alleles ( ), average gene diversity at a locus ( ), the total
gene diversity (H T
) decomposed into the gene diversities within subpopulations (H S
) and
between subpopulations (D ST
), the coefficient of gene differentiation, G ST
that is identical to
Wright F ST
. The gene diversity (H T
, H S
, D ST
and G ST
) were first calculated for each locus separately
and then averaged. Moreover the fixation index in a group of populations (F IT
) and the
average in each population (F IS
) was calculated. The deviation of F IT
and F IS
from zero was
calculated by t-test. If the number of alleles is large, the distribution of parameters is approximately
normal, and the ordinary statistical tests may be used. Therefore, the statistical significance
of population genetic parameters corresponding to the different groups (ecotypes,
cultivars) or species was evaluated by one-way analysis of variance with LSD test using
STATISTICA 7.1 software. The overview of this statistics is presented in Annex 13.15.