Agriculture Reference
In-Depth Information
6X BAC library of a 17,000 Mb genome requires
1 million clones with an average insert size of
100 kb, and is not a small undertaking. Early
gene-isolation strategies in wheat therefore used
the smaller rice genome as a vehicle for map-based
cloning. Markers fl anking the gene that controlled
a trait of interest in wheat were used to initiate a
chromosome walk in rice and, in turn, markers
identifi ed in rice were mapped in wheat until two
rice markers were found that closely fl anked the
target gene in wheat. The underlying assumption
is that the rice sequence spanning the two fl anking
markers contains the rice homologue of the trait
that is being analyzed in wheat. Depending on the
physical size of the region, the rice sequence
should therefore yield one or multiple candidate
genes that can be functionally characterized. This
strategy was used by Moore and colleagues in
their early efforts to isolate the wheat
Ph1
gene,
which controls homologous pairing (Foote et al.,
1997). The pitfall of this approach is that, since
colinearity between rice and wheat is hardly ever
perfect, the target gene may have been subjected
to a rearrangement in either the wheat or the rice
lineage. This was the case for the
cdc2
genes,
which are hypothesized to underlie the
Ph1
phe-
notype in wheat but are absent in the orthologous
rice region (Griffi ths et al., 2006). A walk in rice
to isolate the barley gene
Rpg1
, a resistance gene
for stem rust caused by
P. graminis
f. sp.
tritici
,
also came to a halt with the absence of a
Rpg1
orthologue in rice (Han et al., 1999).
Chromosome walking in rice to isolate genes
in wheat became less laborious as the rice
genomic sequence became available, but remained
a hit-or-miss process. This was particularly
true for the isolation of genes that undergo rapid
reorganization such as disease resistance genes.
This prompted wheat researchers to start the
enormous endeavor of generating BAC libraries
for wheat. The fi rst libraries were generated for
the diploid wheats,
T. monococcum
and
Ae. taus-
chii
. The
T. monococcum
library, developed from
the genotype DV92, consisted of 276,000 clones
with an average insert size of 115 kb and covered
the genome at 5.6X (Lijavetzky et al., 1999). The
fi rst
Ae. tauschii
library, developed from genotype
Aus 18913, consisted of 144,000 clones with
an average insert size of 120 kb, representing
4.2X genome coverage (Moullet et al., 1999).
Several other
Ae. tauschii
BAC libraries, totaling
300,000 clones and representing 12.8X coverage,
have since been constructed of the acces-
sion AL8/78 (http://www.plantsciences.ucdavis.
edu/Dubcovsky/BAC-library/ITMIbac/
ITMIBAC.htm). In polyploid wheat, a 5X BAC
library was generated for the tetraploid
T. turgi-
dum
ssp.
durum
cv. Langdon (516,000 clones)
(Cenci et al., 2003) and a 9.3X library for the
hexaploid
T. aestivum
cv. Chinese Spring
(1,200,000 clones) (Allouis et al., 2003). A number
of other diploid, tetraploid, and hexaploid wheat
libraries representing fewer genome-equivalents
are also available (Liu et al., 2000; Nilmalgoda
et al., 2003; also see http://www.plantsciences.
ucdavis.edu/Dubcovsky/BAC-library/
ITMIbac/ITMIBAC.htm).
Positional cloning has focused on genes
that are located in the more gene-rich high-
recombinant regions of the wheat genome. To
date, four disease resistance genes, three vernal-
ization response genes, a domestication-related
gene, and a homologous pairing-control gene
have been isolated and validated (Faris et al.,
2003; Feuillet et al., 2003; Huang et al., 2003;
Yan et al., 2003, 2004, 2006; Yahiaoui et al.,
2004; Griffi ths et al., 2006; Simons et al., 2006;
Cloutier et al., 2007).
Disease resistance genes
The four disease resistance genes that have been
isolated to date include three conferring resis-
tance to leaf rust (caused by
P. triticina
Eriks.),
Lr1
,
Lr10
, and
Lr21
, and one conferring resis-
tance to powdery mildew [caused by
Blumeria
graminis
(DC) E.O. Speer f. sp.
tritici
],
Pm3b
.
Isolation of
Lr21
, the fi rst disease resistance gene
cloned in wheat, was facilitated by the high
genetic-to-physical distance ratio in the region
surrounding
Lr21
. Furthermore, a RFLP marker
was available at the start of the cloning experi-
ment that cosegregated with the resistance phe-
notype and which was, in fact, the resistance gene.
The strategy used in the isolation of
Lr10
was
much more typical and involved screening a large
