Genome Organization and Comparative Genomics - Wheat Science and Trade - page 325

mapping population for recombination events
in the region of interest, using a combination of
marker development, contiging, and genetic
mapping to narrow down the target region,
sequencing of the target region and, fi nally, con-
fi rming the identity of the candidate genes by
mutation analysis and/or transformation. The
isolation procedures used for Lr21 , Lr10 , Lr1 ,
and Pm3b are described later and provide exam-
ples of how pitfalls, such as the lack of polymor-
phism for a marker expected to be closely linked
to the target gene and the absence of a BAC
library of the resistant cultivar, can be managed.
The four studies also demonstrate the different
techniques that can be used to confi rm the iden-
tity of the resistance gene candidates. Validation
methods include resistance expressed in suscep-
tible cultivars after stable or transient transforma-
tion with the candidate resistance gene, breakdown
of resistance in lines that have undergone virus-
induced gene silencing (VIGS) of the resistance
gene after regions of the candidate gene were
introduced as double-stranded RNA, and com-
parative analysis of the sequence of the candidate
gene in several independent mutants that have
lost the resistance following irradiation or treat-
ment with a mutagen such as ethylmethylsulfo-
nate (EMS).

KSUD14 sequence cosegregated with Lr21 . The
KSUD14 sequence was used to isolate a cosmid
clone from an Lr21 -containing Ae. tauschii acces-
sion. The 43-kb insert contained seven predicted
genes, one of which was a nucleotide-binding site
(NBS)-leucine-rich repeat (LRR) type gene with
homology to KSUD14. Transformation experi-
ments confi rmed that KSU14 was, in fact, the
Lr21 gene (Huang et al., 2003).

Lr10
The leaf rust resistance gene Lr10 originated
from common wheat and is located in the distal
region of chromosome arm 1AS. A screen of near-
isogenic lines carrying different leaf rust resis-
tance genes in a Thatcher background with a
probe that encoded a serine-threonine protein
kinase was the fi rst step that ultimately led to the
isolation of the Lr10 gene. The serine-threonine
protein kinase was a member of a multigene
family, but a unique fragment was identifi ed in
the Thatcher line carrying Lr10 . This fragment,
designated Lrk10 , cosegregated with Lr10 in a
population of 128 F 2 plants developed from the
cross between the resistant near-isogenic line
Thatcher Lr10 and the susceptible cultivar Frisal
(Feuillet et al., 1997). The Lrk10 gene was sub-
sequently shown in a larger population to be
located 0.8 cM distal to Lr10 and was used,
together with the marker cMWG645 which is
located 1.2 cM proximal to Lr21 , to identify
recombination events in the 2-cM region span-
ning Lr10 (Stein et al., 2000). Ninety-six recom-
bination events were found in 3,120 F 2 plants.
The use of closely linked fl anking markers, identi-
fi ed in a population of a few hundred gametes, to
screen a population of a few thousand gametes for
individuals that carry a recombination event in
the region of interest is standard procedure in
positional cloning and limits fi ne-mapping efforts
to informative plants only.
A marker that cosegregated with Lr10 in the
fi ne-mapping population was used to screen the
T. monococcum DV92 BAC library, and two over-
lapping BAC clones totaling 270 kb were identi-
fi ed. One end of the contig mapped distally of
Lr10 , but no proximal marker was identifi ed. To

Lr21
Gene Lr21 originated from Ae. tauschii and was
fi rst incorporated in the wheat cultivar Thatcher
in the 1970s (Rowland and Kerber 1974). It was
later shown to be allelic to Lr40 , another Ae. taus-
chii gene employed in enhancing the resistance of
bread wheat to leaf rust (Huang and Gill 2001).
Gene Lr21 was an ideal candidate for map-based
cloning. The gene mapped to the distal region of
chromosome arm 1DS, which was gene-rich and
highly recombinogenic. Furthermore, the intro-
gressed Ae. tauschii fragment that carried Lr21
provided increased polymorphism levels which
facilitated mapping. Linkage analysis had already
shown that the RFLP marker XksuD14 was
closely linked with Lr21 , and fi ne mapping in a
population of 520 F 2 plants placed XksuD14
0.1 cM distal to Lr21 . The 3′ and 5′ ends of the