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4. GENETIC DIVERSITY...
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4.2. MATERIAL AND METHODS
Twenty five populations of L. multiflorum and L. perenne representing cultivars and wild
ecotypes and two cultivars of L. hybridum were analysed by means of isoenzymes and from
twelve to twenty by different DNA markers. The list of populations and the methods of material
development are presented in Annex 13.1. On average 30 plants from each population
were used in isozyme analysis and 10 plants per a population in DNA analyses. For each
plant about 5-10 g of newly emerged leaves were harvested four weeks after the autumn cut
(i.e., in early October). Then they were packed in labelled bags, placed on ice, transported
to the laboratory and frozen in 30 o C for enzyme and DNA isolation. Since there was no loss
of enzyme activity in frozen plants in comparison with fresh ones, only frozen material was
used in further studies. The enzyme analysis and DNA isolation were carried 1-4 months
after the leaf harvesting.
4.2.1. Isoenzyme analysis
Isozymes were assayed by horizontal starch gel electrophoresis in Lithium borate/Triscitrate
buffer system. Polymorphism was measured at seven enzyme systems: esterases
(EST), fluorescent esterases (EST-flu), aspartate transaminase (AAT), cytosol aminopeptidase
(CAP), NAD-depended malate dehydrogenase (MDH), peroxidases (PER) and superoxide
dismutase (SOD). The methods, buffer recipes and staining procedures were taken
from Zielinski (1987) with own modifications. All procedures and enzyme systems used are
described in Annex 13.3.
Interpretation of zymograms
Due to the lack of knowledge about genetics of all studied isozymes but AAT, all loci
were identified by comparison with a model plant - Hordeum vulgare. The relatively high similarity
between genomes of Poaceae makes possible to use a single species as a model for
the analysis of the others. Therefore, with a broad knowledge about genetics and structure
of the majority of enzymes, relatively close similarity to Lolium and the same chromosome
number (2n=14), barley is a very suitable model in analysis of isozymes in ryegrasses. Later
on the manner of inheritance of all studied isozymes was confirmed in crossing experiments
(Chapter 6).
In the present work the standardised numbering system proposed by Kahler and Allard
(1970) for barley was adopted. Enzyme systems were referred to by upper case letters
according to standard names (e.g., CAP, EST etc.). Specific loci were noted using enzyme
system identification but with lower case letters except for the first one (e.g., Cap1, Est1).
For each locus the bands were labelled according to their distance in mm from the origin.
Subsequent bands were numbered for example, 20, 30, 35 etc. Homozygous genotypes
were thus, labelled 20/20 for example and heterozygous 20/30 for example. Specific alleles
were denoted after hyphen, Cap1-20, Est1-25. This system allows both for easy comparison
between own and literature data and numbering of new alleles. The similar system was also
adopted for AAT, GPI and ACP by Hayward et al. (1995).