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9. PHYLOGENETIC RELATIONSHIPS...
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and phenotype (Vision 2005). Unfortunately, the majority of comparative studies employ low
copy sequences whereas the rapid evolution is associated with intergenic sequences, often
retrotransposons. Consequently, treating the chromosome as simply linear order of genes is
a huge simplification.
Despite a number of success stories the comparative mapping is not as straightforward
as previously thought. The analysis of comparative maps in Poaceae shows that the probability
of two adjacent markers being syntenic can be as low as 50% (Gaut 2002). Typically,
comparative maps have been constructed using heterologous RFLP probes from the closely
related species (Gale and Devos 1998). The attempts to use this approach in Lolium have
been undertaken by Jones et al. (2002b). It has been possible to establish colinear regions
with Triticeae, but the problems have arisen in alignment of this map with the others, including
the SSR map also constructed by these authors. The limitations of heterologous RFLP
probes in different taxonomic families are that the 70-80% of sequence identity at the DNA
level is demanded for such probes to cross-hybridize (Vision 2005).
The emerging approach likely to overcome the barrier of high DNA identity takes advantages
from large expressed sequence tags (EST) or sequence specific tags (STS), which
dozens are deposited in GenBank. The primers can be designed to conservative regions
and then used to amplify a sequence in a species of interests. If it is possible to obtain
a reproducible amplification pattern, preferentially a single band, and if it is possible to find
polymorphism between parents of a cross then such ESTs or STSs can be mapped. Finally,
putative homologs can be searched using appreciate software available elsewhere.
Therefore, these mapped ESTs, STSs or any other sequences serve as anchor probes. The
high level of sequence conservation within genes should offer the opportunity of comparing
conserved genome organisation between species. Recently, more than 1 000 EST loci have
been mapped in barley and they provide new anchor points for map comparisons (Stein et
al. 2007). However, although the idea is simple, it is not easy to apply as it has been demonstrated
in the mapping studies in Lolium (Chapter 6) and also experienced in P. sativum
(K. Polok; unpublished data).
The STS sequences were selected from Taylor at al. (2001) based on the reported results
showing restriction polymorphism in L. perenne ecotypes. Unfortunately, all STS proved
to be useless in mapping studies due to no polymorphism between parents and the lack of
segregation in F 2
. Furthermore some problems were observed with the amplification of a specific
band although it was possible to obtain reproducible multi-band profiles. The example
includes ASN locus encoding L-asparaginase in barley. As reported by Taylor et al. (2001)
comparisons between H. vulgare and L. perenne sequences reveal conservation in gene
structure and overall similarity is 89%. The set of primers derived from asparagine synthetase
(AS1) gene was able to amplify a single band comparable to that of H. vulgare but it was not
polymorphic in spite of digestions with several different enzymes. Some difficulties were also
encountered with RFLP probes from oat and barley. For example, cDNA clone BCD450 from
barley was optimised to amplify two strong bands but they were twofold smaller than a band
of Taylor et al. (2001). The bands were identical not only in parents of the mapping population
but also in all studied species, thus suggesting the amplification of primers instead of a band.
Eventually, lower annealing temperature resulted in multi-band pattern. In contrast to Taylor
et al. (2001) results, reproducible amplification was received with oat probes CDO504 and