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13. SUPPLEMENTARY MATERIALS
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individually to pots. After about two months, multitillered plants were transplanted to the field
in Tomaszkowo and used for crossing experiments. In total 50 plants from five populations of
L. multiflorum and five populations of L. perenne were used.
Each F 1
hybrid was derived from a cross between a single female parent and a single
male parent. About ten different crosses were made in 2000. The female parent was chosen
to recognize hybrids from accidental self-pollination. Inflorescences of a female plant were
emasculated, and pollinated with pollen of a single male plant. All pollinated spikes were
isolated with a paper bag. When matured, F 1
seeds were collected, and germinated at 24 o C,
checked for fluorescence, and F 1
plants were transplanted into a greenhouse during the
winter 2000/2001. In total eight F 1
hybrids derived from interspecific crosses (from four different
combinations), five hybrids of L. multiflorum genotypes (from two combinations) and four
hybrids of L. perenne genotypes (from two combinations) were grown.
Each F 2
population was derived from self-pollination of a single F 1
hybrid plant so as to
ensure 3:1 segregation of dominant markers or 1:2:1 in a case of codominant ones. To prevent
from out-pollination and force to self-pollination, each F 1
plant was growing in a different
box with cereals or Poa. Additionally each F 1
plant was isolated with a paper bag. Seeds
were harvested from individual plants. In total from 50 to 400 seeds of F 2
were collected
per a single F 1
hybrid. Among interspecific crosses two biggest populations were chosen for
further studies. Among intraspecific crosses a population per a species was selected on the
basis of the number of individuals. Seeds of these populations were germinated in the laboratory
in the autumn 2001 and transplanted into the field in the spring 2002.
The methodology of F 2
seed germination, the fluorescence test, plant growing and cultivation
were nearly the same as for L. multiflorum and L. perenne populations. The modifications
were related with the recovery of plants with faint fluorescence (+/-) and a greater
number of replications. Each F 1
and F 2
plant and parental plants were divided into 27 ramets.
In total three blocks located within Tomaszkowo were set up. Each block consisted of three
replications and each replication consisted of three ramets as replicates of each genotype.
Hence, in each block a single genotype was represented by nine ramets. Quantitative characters
were analysed during two seasons (2002 and 2003) and during two crops (July, September)
to assess genotype x environment interaction.