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suspensions of intact chromosomes by mechanical
homogenization of formaldehyde-fi xed root tips.
Major advantages of the protocol include the
ease with which seedlings can be handled, the
karyological stability and high degree of cell cycle
synchrony in root tips, and the mechanical stabil-
ity of isolated chromosomes. The protocol, includ-
ing its modifi cation in which the fi xation step
was omitted (Lee et al., 1997; Gill et al., 1999),
stimulated the recent progress in fl ow cytogenetics
of wheat (Vrána et al., 2000; Kubaláková et al.,
2002).
The availability of suspensions of intact chro-
mosomes facilitated the analysis of wheat chro-
mosomes using fl ow cytometry (Lee et al., 1997;
Gill et al., 1999; Vrána et al., 2000). The chromo-
some content of individual peaks on the wheat
fl ow karyotype was determined by Vrána et al.
(2000), who found that only chromosome 3B
formed a discrete peak and could be sorted as a
single chromosome. The other chromosomes
formed composite peaks I, II, and III (Fig. 15.3a).
Thus, with the exception of chromosome 3B,
fl ow-sorting techniques at that time could not be
used to dissect the wheat genome into individual
chromosomes. To overcome this obstacle,
Kubaláková et al. (2002) screened a large set of
wheat cultivars and landraces to detect structural
chromosome changes and chromosome polymor-
phisms that would allow additional chromosomes
to be separated, but the observed alterations in
fl ow karyotypes were small and did not provide a
reliable basis for sorting other chromosomes.
Kubaláková et al. (2002) then suggested that
most wheat chromosome arms could be sorted
from lines carrying telocentric chromosomes, or
telosomes. Telosomes originate by centric fi ssion
of a chromosome and, after synthesis of new telo-
meres, they can be maintained as stable cytoge-
netic stocks. A complete set of telosome lines has
been developed in Chinese Spring (Sears 1954).
Chinese Spring has been chosen as a model geno-
type for wheat cytogenetics and genomics (Gill
et al., 2004) and will be used by the International
Wheat Genome Sequencing Consortium
(IWGSC, http://www.wheatgenome.org) for the
construction of the hexaploid wheat physical map.
Due to their relatively small size (DNA content),
Fig. 15.3 Histograms of relative fl uorescence intensities
(fl ow karyotypes) obtained after fl ow cytometric analysis of
DAPI-stained suspensions of mitotic chromosomes of hexa-
ploid wheat. (a) The fl ow karyotype—Chinese Spring—con-
sists of the chromosome 3B peak, the small composite peak
I containing chromosomes 1D, 4D, and 6D, and the two
large composite peaks II and III containing the remaining 17
chromosomes. (b) Flow karyotype obtained after the analysis
of ditelosomic line Dt1BS of cultivar Pavon. The peak repre-
senting the short arm of chromosome 1B is well discrimi-
nated, which facilitates its sorting. (c) The analysis of a Chinese
Spring line carrying an isochromosome for the long arm of
chromosome 5B (iso5BL) results in a fl ow karyotype with an
additional peak to the right of the peak of chromosome 3B.
(d) The fl ow karyotype of the double ditelosomic 3D line
(dDt3D) of Chinese Spring is characterized by additional
peaks representing the short arm telosome 3DS and the long
arm telosome 3DL, which facilitates their simultaneous
sorting.
telosomes form extra peaks on the left side of fl ow
karyotypes and do not overlap with peaks of other
chromosomes (Fig. 15.3b). The only exceptions
are the long arms of chromosomes 3B and 5B that
overlap with peak I, and the long arms of 4A, 1B,
and 7B, whose peaks are too close to peak I to be
separated reliably. These arms, however, can be
sorted from stocks carrying them as isochromo-
somes, that is, chromosomes with two identical
arms that originate by centric fusion of two iden-
tical telosomes (Fig. 15.3c).
These advances made it possible to sort
chromosomes at high speeds, typically 5 to 30
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