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4. GENETIC DIVERSITY...
deed concentrated on the whole genus and only single samples of L. multiflorum and
L. perenne were assessed.
The examples cited above indicate that direct comparisons of Italian and perennial
ryegrasses have been avoided. Instead, considerable attention has been devoted to
comparison of L. perenne with L. rigidum (Balfourier et al. 1998) or even with members
of the other grass genera (Warren et al. 1998; Kolliker et al. 1999). Restriction analysis
of chloroplast genome has been used in order to explain the present distribution area of
natural populations of L. perenne and L. rigidum (Balfourier et al. 2000). It is so strange
that L. rigidum has hardly economic importance otherwise being a rare and threaten weed.
L. multiflorum, on the other hand, is one of most important temperate forage species
that continues to cause problems during breeding due to high morphological similarity to
L. perenne. One likely explanation for the relative lack of data regarding L. multiflorum is that
the results have been too confusing and disagreed with the present taxonomic classification.
The aim of the present study was therefore, to review the differentiation of L. multiflorum
and L. perenne by means of several molecular marker systems including those not used previously.
To determine how much of the genetic variation has resulted from human mediated
gene flow and what remains of natural pattern of variation, both cultivars and wild ecotypes
were included in the analysis. To maximize effectiveness by combining high resolution with
broad coverage of individuals, isoenzymes with several DNA methods were applied. It is still
expensive and time-consuming to obtain DNA information from multiple Mendelian nuclear
loci among numerous individuals. Therefore, isozyme electrophoresis was applied at the
beginning. It remains one of the best techniques available for studies of population structure
also because of codominance. Restriction and PCR analysis of chloroplast and mitochondrial
DNA is valuable method for estimation of migration routes and up to our knowledge no
comparison between cpDNA and mtDNA of L. multiflorum and L. perenne has been made.
Among random DNA markers, RAPDs were chosen due to simplicity and efficiency in species
determination. So called semi-specific markers are similar to RAPDs in that they employ
a single primer to amplify DNA. However, they are based on the consensus sequences for
the intron splice junctions (ISJ). The junctions to exons are highly conserved sequences.
Introns are generally subjected to weak selective pressure and therefore, they are usually
highly variable in sequence length. These properties would appear to make the ISJs ideal
targets for the identification of polymorphism in PCR products (Weining and Landridge 1991).
This type of markers has never been used in ryegrasses but proved to be highly effective
in discrimination of pea and barley mutants (K. Polok, unpublished data). Moreover, transposon-based
profiling (SSAP) was optimized and for the first time applied for estimation of
genetic diversity in L. multiflorum and L. perenne. Polymorphism resulted from transposon
insertion is thought to be higher than revealed by AFLP and especially useful for intra-specific
comparisons (Syed et al. 2005). Transposons are able not only to increase significantly the
host genome size but can also mutate host genes. Therefore, they are likely to be a major
contributor to the generation of genetic diversity in plants (Kumar et al. 1997). To gain an
overall picture of this diversity, both DNA and retrotransposon based SSAP was used.