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6. DO ANY SPECIES BOUNDARIES....
were further assigned as B-SAP (Bacteria Specific Amplification Polymorphism). Sequences
of all primers used in the mapping studies are given in Annex 13.5 whereas amplification and
electrophoresis conditions for all marker types are described in Annexes 13.8-13.14.
Linkage analysis and software used
Linkage between enzymatic loci was analysed in four F 2
populations whereas the linkage
map was constructed for the BR3 x NZ15 interspecific population. To determine if two
loci are linked, the logarithm to the base of ten of the likehood odds ratio statistics was used
(LOD). This statistics informs about the conditional probability of an odd number of crossovers
between the markers. The LOD score was calculated for various recombination values
(Θ). The (Θ)value, which gives the highest LOD score, was taken as the best estimate of the
recombination fraction. A LOD score of 3 or greater for the recombination fraction between
two loci lower than 0.49 confirmed the linkage. A LOD score of lower values excluded linkage.
The programme MAPL98 (Ukai 2004), originally developed for rice genome mapping,
was used first. It grouped markers by connecting a marker A with another marker B which
had the smallest recombination value with the marker A. Starting with an arbitrary chosen
marker the connection was repeated until no partner was found and a chain of markers was
constructed. A marker at the end of the chain was judged as having no partner with a recombination
value lower than a threshold value. The threshold value was determined so that
probability of marker connection by error was 0.05. Multidimensional scaling allowing finding
the shortest way between markers was used for final marker ordering. Then map distances
were calculated using Kosambi’s mapping function, which allowed for modest interference
(Kosambi 1944). At the second stage of analysis, the programme Carthagene (Schiex et al.
2005) was employed. This software was comparable with the more widely used MAPMAKER
but it worked in WINDOWS XP environment and used WINDOWS interface. Additionally it
allowed comparing different maps. The map was drawn with help of MapChart (Voorrips
2002) and transformed into CorelDraw 12 (2004) format in order to prepare it for printing in
high resolution.
Due to huge amount of data, the map was built successively by separate mapping of
each marker, and combining the maps into a single linkage map using MAPL98. Final mapping
was done for each linkage group separately. Markers were coded as 1 for a female and
2 for a male type. Additionally, for codominant markers a heterozygous type was recognised
as 3. For some dominant markers segregating in F 2
, parental phenotypes were identical i.e.,
both had a band. In these cases F 1
and parental plants must have been heterozygotes for
null alleles. This was owing to the fact that L. multiflorum and L. perenne are allogamous
species and therefore parental plants were not completely homozygous lines. Consequently,
linkage phase was unknown in a certain part of data. In these cases the presence of a band
was arbitrary coded as 1 and the lack as 2. Then counterpart data in which “1” and “2” were
mutually converted were made and linkage analysis was done on the original and counterpart
data. The underlying assumption was that the markers with incorrect phase assignments
would not be linked to any of the markers where the correct phases were known. For a given
marker category, first all linked markers were checked altogether and then separately. This
set of data was selected that produced fewer groups, shorter map and fewer solitary mark-