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6. DO ANY SPECIES BOUNDARIES...
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ers. Then the procedure was repeated for all solitary markers. Conversion was also repeated
each time when a new marker category was introduced onto the map. To produce a suitable
robust genetic map the final order of markers was transformed into the Carthagene format
using Excel (2003) transposition. Linkage groups were again determined using 2-points estimation
and a minimum LOD ratio of 6.0 and a maximum recombination rate of 0.2. Optimal
order was estimated using the annealing command and verified using validation methods
as implemented in Carthagene. Once the map was constructed, marker coverage was compared
with that of existing maps.
6.2.3. Numbering of linkage groups
The linkage groups were numbered in accordance with the corresponding linkage
groups of the first molecular marker based map of perennial ryegrass published by Hayward
et al. (1998) owing to the number of enzymatic loci. Consequently, Est4 and Aat2 determined
linkage group 1 (LG1), Hk1 - LG2, Gtdh2 - LG5. Alignment of LG3, LG4, LG6 and LG7 was
problematic due to limited number of common markers detecting a single locus and therefore
these groups were numbered arbitrary, yet taking into account the overall length of a given
linkage group. This numerical order was chosen because it was partially in agreement with
the chromosome numbers as allocated by Lewis et al. (1980) with primary trisomics. Alignment
with the ILGI L. perenne map was troublesome due to inconsistency in linkage group
numbering by different laboratories and different marker naming. For example Bert et al.
(1999) used alphabetical order for their AFLP map constructed in the p150/112 population.
The first SSR map of this population constructed by Jones et al. (2002a) employed numerical
order. Surprisingly, the same authors using also the p150/112 population numbered AFLP
linkage groups on the basis of conserved synteny with Triticeae (Jones et al. 2002b). Consequently,
the numerical order in their AFLP map was in agreement neither with the previously
published SSR map nor with the AFLP map of Bert et al. (1999). What is more, Jones
et al. (2002b) described that MseI/EcoRI primer combinations were used in AFLP analyses,
whereas names of AFLP loci on their map suggesting that they used Tru9I/EcoRI combinations.
Similar problem with the alignment of the ILGI linkage map was experienced by Muylle
et al. (2005) due to the detection of multiple loci by the respective SSR or RFLP probes.
6.2.4. Marker naming on linkage map
Due to large number of markers on the linkage map, and to keep the map readable,
some abbreviations had to be introduced (Table 6.2). In general, enzymatic loci were indicated
according to the names of their enzymes as indicated in Annex 13.3. DNA loci were
indicated by marker type and product number counting from the fastest band. AFLP loci were
named according to restriction sites flanked by adapters (MseI, EcoRI) and three-base extensions
of selective primers. Numerical nomenclature as it is on the ILGI map and some others
was avoided to improve readability without necessity to look at the standard list for AFLP
primer nomenclature. The same idea was employed for transposon-based primers.