Chromosome sorting in
the Triticeae: progress and plans
for the near future
Jaroslav Doležel*, Pavla Suchánková, Pavlína
Kovářová and Marie Kubaláková
Laboratory of Molecular Cytogenetics and Cytometry,
Institute of Experimental Botany, Sokolovská 6, CZ-77200
*Corresponding Author: Phone: (+420) 585 205 852; E-mail:
dolezel@ueb.cas.cz
ABSTRACT
Although some species of the tribe Triticeae possess small genomes, the
genomes of barley (~5000 Mb/1C), rye (~8000 Mb/1C), durum wheat (~13,000 Mb/1C)
and bread wheat (~17,000 Mb/1C) are complex, consisting mainly of various
classes of repetitive DNA sequences. In addition, the recent evolution of wheat
involved two polyploidization events giving rise to allotetraploid genome of
durum wheat and allohexaploid genome of bread wheat. These features hamper
physical mapping and gene cloning. Purification of individual chromosomes by
flow cytometry can simplify these tasks by providing small and defined genome
fractions. Unfortunately, only one chromosome can be discriminated and sorted
in each of the four species due to small differences in DNA content. We have
demonstrated that this problem can be overcome by flow sorting from deletion
and alien chromosome addition lines. Thus, it is possible to purify any
chromosome arm of barley, durum wheat and bread wheat. In rye, each of the
seven chromosomes can be sorted. This advance provides a universal platform for
dissecting the nuclear genomes of wheat, barley and rye. Because the DNA of
sorted chromosomes is intact, they are suitable for a range of applications
including the construction of subgenomic DNA libraries and molecular
cytogenetic mapping. Currently the most attractive application concerns the
construction of BAC libraries. However, their preparation requires sorting of
many millions of chromosomes. We have optimized a protocol for sorting so that
enough chromosomes for the construction of a BAC library can be obtained in two
months. The utility of chromosome-specific BAC libraries for wheat genomics has
been exemplified by a progress in development of a physical contig map of wheat
chromosome 3B by the group of C. Feuillet (INRA, Clermont-Ferrand, France). We
are now exploring possibilities for the use of flow-sorted chromosomes for
high-resolution and high-throughput cytogenetic mapping to aid in assembling
physical contig maps. The selection of chromosomes for construction of the
first three BAC libraries from sorted chromosomes (Janda et al., Theor. Appl.
Genet. 109:1337, 2004; Šafář et al., Plant J. 39:960, 2004) was driven
primarily by a need to explore in a step-wise manner a feasibility of creating
subgenomic, chromosome-specific and ultimately chromosome arm-specific BAC libraries.
Our current target is to complete BAC resources for the homeologous group 3 of
wheat by sorting the arms of chromosomes 3A and 3D. A medium-term plan under
discussion includes the homeologous group 5. The availability of similar BAC
resources for barley and rye would not only accelerate gene cloning but open
avenues for comparative analysis of genome evolution at a level not attainable
until now. Facing this need, we have purified the short arm of rye chromosome
1R and we are planning to sort both arms of barley chromosome 3H. This work has been supported by the
Czech Science Foundation (grant awards 522/03/0354, 521/04/0607, 521/05/0257
and 521/05/H013).