Genome Organization and Comparative Genomics - Wheat Science and Trade - page 335

the second library can have a lower number of
clones with shorter inserts. This makes BAC
library construction less demanding and faster.
Recently, ˇ imková and colleagues (H. ˇ imková,
pers. comm.) created an ordered BAC library
from 1 × 10 6 3B chromosomes of cultivar Hope
in only 4 weeks, including chromosome sorting.
This specifi c BAC resource is being used in map-
based cloning of the Sr2 gene (W. Spielmeyer,
pers. comm.) after a contig of 1 Mb had been
established on the Chinese Spring 3B reference
physical map (C. Feuillet, unpublished data).
However, this strategy is only applicable to chro-
mosome 3B, and a group of 1D, 4D, and 6D
chromosomes that can be sorted from any hexa-
ploid wheat. To sort other chromosomes, teloso-
mic stocks will have to be developed from specifi c
genotypes. This may require less than 2 years (A.
Lukaszewski, pers. comm.).
In addition to facilitating the construction of
physical maps and map-based cloning, chromo-
some (arm)-specifi c BAC libraries aid in the
development of genome-specifi c markers for
marker-assisted selection. This is highly relevant
for polyploid species such as wheat in which the
presence of homoeologous sequences hampers the
effi cient development of genome-specifi c markers.
It has been shown recently that BAC-end
sequences from chromosome-specifi c BAC librar-
ies can be used very effi ciently to develop a large
number of genome-specifi c markers for genetic
and physical mapping in wheat (Paux et al., 2006)
by using the junctions between repetitive ele-
ments (Devos et al., 2005b). The main advantage
of these insertion site based polymorphism (ISBP)
markers or repeat junction markers (RJMs) is that
they are very specifi c, polymorphic, and distrib-
uted homogeneously along the chromosomes in
contrast to EST or SSR markers that can show
some bias in genomic distribution.

chromosomes deserve attention. The fi rst one is
the development of DNA markers from particu-
lar chromosomal regions for which BAC libraries
are not yet available. One option is to create a
short-insert DNA library from a small number
of fl ow-sorted chromosomes, enrich the library
for particular microsatellite motifs, and use mic-
rosatellite-containing DNA clones to develop
SSR markers (Po ˇ árková et al., 2002). Although
not yet explored in wheat, the results that were
obtained in rye demonstrate the potential of this
approach in species with complex genomes
(Kofl er et al., 2007). Another unexplored option
is to isolate markers from chromosomal DNA
using the DArT technology (Wenzl et al., 2004).
Chromosome specifi city of candidate markers
can be ascertained prior to genetic mapping by
PCR or hybridization on DNA from NT lines.
An alternative is to perform PCR on a small
number (100-500) of chromosomes that can be
sorted in a short time (Po ˇ árková et al., 2002). A
more powerful approach involves the use of
DNA amplifi ed from fl ow-sorted chromosomes
as probes on DNA arrays for mass parallel
mapping of DNA sequences ( ˇ imková et al.,
2008).
A marriage of fl ow and molecular cytogenetics
has resulted in a powerful tool for cytogenetic
mapping. Traditionally this has been done by
FISH on mitotic metaphase spreads from root-tip
meristems (Jiang and Gill 2006). The number of
metaphases that can be analyzed limits the
throughput, and the sensitivity and specifi city of
FISH are often compromised by the presence of
cell wall and cytoplasmic debris. As fl ow-sorted
chromosomes are free of cellular remnants and
can be sorted onto a microscopic slide in large
numbers, they are ideal targets for FISH and
PRINS. This facilitates physical mapping of
DNA sequences (Kubaláková et al., 2002, 2005)
and detection of rare structural variants (Kuba-
láková et al., 2003). A further advantage of using
fl ow-sorted chromosomes is that they can be
stretched longitudinally to improve the spatial
resolution of cytogenetic mapping up to 100-fold
compared with mitotic metaphase chromosomes
(Valárik et al., 2004).

Chromosome-based approach offers
more than subgenomic BAC libraries
Development of subgenomic BAC libraries has
been the most attractive use of fl ow cytogenetics
in wheat. However, other applications of sorted