Agriculture Reference
In-Depth Information
been created from the short arms of chromo-
somes 3A, 3D, and 7D and from the long arms
of 3A and 3D. Table 15.2 provides information
about chromosome BAC libraries constructed in
wheat so far.
to 30 times more clones (1,000 to >2,600 384-well
plates) (Allouis et al., 2003; Nilmalgoda et al.,
2003; Ling and Chen 2005; Ratnayaka et al.,
2005). Finally, the possibility of approaching the
hexaploid wheat genome one chromosome at a
time allows the establishment of a strategy for
physical mapping that is based on international
collaborations in which individual laboratories
develop physical maps of specifi c chromosomes
and chromosome arms for a reasonable cost and
labor investment, with progress in each labora-
tory independent of the others.
An apparent limitation of the chromosome
BAC libraries is the insert size, which is at the
lower range of published genomic BAC libraries
(Allouis et al., 2003; Nilmalgoda et al., 2003; Ling
and Chen, 2005; Ratnayaka et al., 2005). Although
the chromosome BAC libraries contain clones
with inserts up to 200 kb, the presence of clones
with inserts shorter than 50 kb compromises the
average insert size (J.
ˇ
afᡠand H.
ˇ
imková, pers.
comm.). Due to the small amount of starting
DNA, the original protocol of chromosome BAC
library construction involved only one DNA size-
selection step (
ˇ
afᡠet al., 2004). Recently,
ˇ
afáˇ
and
ˇ
imková (J.
ˇ
afᡠand H.
ˇ
imková, pers.
comm.) improved the protocol so that it is possi-
ble to make two size-selection steps even with
small amounts of starting DNA. BAC libraries
made using this protocol have average insert sizes
of about 110 kb (Table 15.2).
While the use of the aneuploid lines from
Chinese Spring is appropriate for the construc-
tion of a hexaploid wheat reference physical map,
the development of physical maps at target loci
from genotypes that carry specifi c traits of inter-
est such as disease resistance will be needed in
some cases. One option is to generate nongridded
genomic BAC libraries from desired genotypes
that are suitable for PCR screening (Ma et al.,
2000; Isidore et al., 2005). These libraries do not
need to have a high genome coverage and will
generally be used only for a few screening steps
to identify clones of interest. Another option is to
generate chromosome-specifi c BAC libraries. If a
physical contig spanning a region of interest is
available from the Chinese Spring reference map,
Advantages of subgenomic
BAC resources
The most obvious advantage of chromosome-
based BAC libraries is their specifi city. By apply-
ing these libraries for physical mapping and
positional cloning, most of the diffi culties due to
homoeology are avoided (see below). The level of
contamination of the libraries with other chromo-
somes depends on the accuracy of sorting.
Although it is possible to sort wheat chromosomes
that are 97% pure (Kubaláková et al., 2002), typi-
cally, purities in large-scale chromosome sorting
experiments have varied from 88% to 91% (Janda
et al., 2004, 2006;
ˇ
afᡠet al., 2004). The identity
of the sorted particles is confi rmed by fl uores-
cence
in situ
hybridization (FISH) or primed
in
situ
DNA labeling (PRINS) using probes and
primers that provide chromosome-specifi c fl uo-
rescent labeling (Vrána et al., 2000;
ˇ
afᡠet al.,
2004), as well as by screening of the BAC libraries
with chromosome-specifi c markers (Janda et al.,
2004, 2006;
ˇ
afᡠet al., 2004). The results show
that chromosome-specifi c BAC libraries contain
about 10% contaminating clones. However, the
fi rst physical mapping and positional cloning
experiments conducted with the chromosome-
specifi c BAC libraries (see below) indicated that
this low level of contamination has no effect on
the accuracy of the results and therefore does not
compromise the value of the chromosome-specifi c
BAC libraries.
In addition to simplifying the physical mapping
by targeting single chromosomes, the use of chro-
mosome BAC libraries offers important logistical
advantages. With the number of clones ranging
from 4×10
4
to 1×10
5
(ordered in 104 to 260
384-well plates), the libraries occupy limited
freezer space, and maintenance (replication,
pooling) and screening are easier and cheaper
than for genomic BAC libraries that comprise 10
