Genome Organization and Comparative Genomics - Wheat Science and Trade - page 326

extend the contig proximally, the most proximal
of the two BAC clones was sequenced at low
redundancy. Low-pass sequencing is considered
standard for identifying low-copy sequences that
can be mapped onto the genetic map and used for
contig extension. In wheat, BAC-end sequences,
the markers traditionally used in a chromosome
walk, have limited use due to the high-repeat
content of the genome. A low-copy marker
obtained by Stein et al. (2000) from the sequence
information identifi ed a new BAC clone that
extended the contig by 80 kb to form a 350-kb
contig that spanned Lr10 . Some 211 kb of T.
monococcum DV92 was shotgun-sequenced to 8X
redundancy and shown to carry fi ve putative
genes, including two resistance gene analogues,
rga1 and rga2 (Wicker et al., 2001).
A potential problem with map-based cloning in
a line that is not known to have the phenotype of
interest is that the gene may be absent from that
line. Haplotype analysis showed that this was not
the case for DV92. The identity of rga1 as Lr10
was demonstrated by the identifi cation of three
independent mutations in rga1 but not rga2 in
EMS mutants that had lost resistance to an aviru-
lent race of leaf rust carrying AvrLr10 . Transfor-
mation of the susceptible wheat cultivar Bobwhite
with the rga1 gene conferred resistance and con-
fi rmed that rga1 encoded Lr10 .

2007). These markers were subsequently used to
screen a BAC library of the hexaploid wheat cul-
tivar Glenlea (Nilmalgoda et al., 2003), which
contains the Lr1 resistance gene. A second round
of screening was carried out with markers devel-
oped from the positive BAC clones. All BAC
clones identifi ed during the two rounds of screen-
ing were fi ngerprinted and hybridized with
PSR567. Additional sequence information was
obtained from Glenlea for PSR567A, B, and D,
which were originally cloned from Ae. tauschii
and were non-polymorphic in the mapping popu-
lation. The markers used in the BAC screens,
together with the newly developed PSR567
markers, were used for genetic mapping in 400
F 1 -derived doubled haploid lines and in a recom-
binant inbred population, also of some 400 lines.
The Lr1 gene was shown to cosegregate with one
of the Xpsr567 fragments, designated RGA567-5.
Two overlapping BAC clones containing
RGA567-5 were shotgun sequenced. Four open
reading frames were identifi ed, three of which
were separated from Lr1 by recombination events.
Transformation of the susceptible cultivar Fielder,
which lacks Lr1 , with RGA567-5 confi rmed its
identity as Lr1 . In a second confi rmation experi-
ment, Lr1 activity in the resistant cultivar Thatch-
er Lr1 was down-regulated using VIGS, resulting
in virulence of the leaf rust AvrLr1 gene.

Lr1
The leaf rust resistance gene Lr1 present in
common wheat germplasm had been mapped
to the distal region of chromosome arm 5DL
(Feuillet et al., 1995). Saturation of this region
with SSR and RFLP markers from 5DL and
from orthologous barley and oat regions in two
fi ne-mapping populations, consisting of 2,826
and 832 F 2 plants, delimited the location of Lr1
to a 0.16-cM region. Lr1 was fl anked proximally
by Xpsr567 at 0.04 cM and distally by Xabc718 at
0.12 cM (Ling et al., 2003). Screening of Ae.
tauschii BAC libraries with PSR567 led to the
isolation of the partial resistance gene analogues
567A, 567B, and 567C (Ling et al., 2003) and
three additional low-copy markers (Cloutier et al.,

Pm3
At least 10 alleles have been identifi ed for the
powdery mildew resistance gene Pm3 . The gene
had been mapped to the distal region of wheat
chromosome arm 1AS, fl anked by the markers
WHS179 and BCD1434 (Hartl et al., 1993; Ma et
al., 1994). In an F 2 population of 1,340 plants,
WHS179 mapped 0.9 cM proximal to Pm3b . The
end clone of a BAC, identifi ed with BCD1434,
mapped 3.9 cM distal to Pm3b (Yahiaoui et al.,
2004). Existing wheat maps yielded a SSR marker,
PSP2999, that mapped 0.07 cM distal to Pm3b
and was used as the starting point for a chromo-
some walk. Walking was conducted simultane-
ously in T. monococcum accession DV92 and in T.
durum cultivar Langdon. Two cycles of low-pass