Agriculture Reference
In-Depth Information
Until now, FISH has been used in wheat
mainly to determine the genomic distribution of
DNA sequences, to identify individual chromo-
somes, and to study structural changes in chro-
mosomes (Zhang et al., 2007). However, the
development of physical contig maps will need
cytogenetic mapping to ascertain the position of
some BAC contigs, to determine their orienta-
tion, and to estimate contig gaps. In other crops,
this has been done by FISH with BAC clones
(Cheng et al., 2002; Budiman et al., 2004). Unfor-
tunately, BAC FISH has been hampered in wheat
by the prevalence of repetitive DNA, which has
resulted in dispersed FISH signals (Zhang et al.,
2004b; Janda et al., 2006). Thus, although FISH
on fl ow-sorted chromosomes offers a powerful
approach for cytogenetic mapping, its use to
support the development of physical contig maps
in wheat is hampered by the lack of suitable
probes that localize to single loci.
correspond to gene sequences and/or by PCR
using microsatellites, cleaved amplifi ed polymor-
phic site (CAPS), or sequence tagged site (STS)
markers. To anchor the 3B physical contigs to
the genetic map, PCR screening was performed
with more than 2,000 molecular markers follow-
ing two approaches. The fi rst strategy was to use
markers (SSR, EST) that have been located
either on genetic or cytogenetic maps of chromo-
some 3B (Munkvold et al., 2004) to screen the
BAC library and anchor the corresponding FPC
contigs. The second approach comprised marker
development from the BAC contigs and place-
ment of the markers onto the genetic maps. To
do that, 19,400 BAC-end sequences were gener-
ated representing a cumulative length of nearly
11 Mb (1.1% of the chromosome length) distrib-
uted among the contigs of chromosome 3B (Paux
et al., 2006). The systematic identifi cation of
junctions between transposable elements allowed
the development of retrotransposon-based inser-
tion polymorphism (RBIP) markers (Flavell et
al., 1998) that consist of PCR amplicons span-
ning the junctions. This type of marker has
several advantages: (i) it is highly abundant since
more than 80% of the wheat genome consists of
transposable elements that are frequently nested
within one another; (ii) it is mostly unique in the
genome as there is a low chance that the same
insertion event occurred at another locus; (iii) it
is genome-specifi c since it originated from a spe-
cifi c chromosome sequence (Paux et al., 2006).
More than 700 ISBP markers have been devel-
oped across chromosome 3B and have been used
for anchoring the contigs on the genetic and
cytogenetic maps (Paux et al., 2008). Finally, a
minimum tiling path (MTP) has been established
for chromosome 3B by selecting clones with
minimal overlap. The MTP can now be used for
structural and functional studies on chromosome
3B.
It is very cost-effective to use PCR-based
methods to perform the anchoring of physical
maps to genetic maps. However, with large
genomes such as wheat, the number of PCRs
required to identify all BAC clones bearing a
single target marker sequence can become very
high. To reduce signifi cantly the number of reac-
Physical map of chromosome 3B—
a case study
The availability of a BAC library specifi c for
chromosome 3B ( ˇ afᡠet al., 2004) provided an
opportunity to test the feasibility of constructing
a physical map of the hexaploid wheat genome
using a chromosome-specifi c-based approach
(Gill et al., 2004). To establish a physical map
of chromosome 3B, high-information-content
(HIFC) fi ngerprints were generated from the
67,968 BAC clones of the 3B BAC library using
a modifi ed SNaPshot protocol (Luo et al., 2003)
and assembled into contigs using the FPC soft-
ware (Soderlund et al., 2000). Using a single ABI
3730 XL capillary sequencer (Applied Biosys-
tems, Foster City, California), it was possible to
fi ngerprint all BAC clones of the 3B BAC library
within 10 weeks. To date, the physical map con-
sists of about 1,000 contigs that cover nearly 80%
of the chromosome (Paux et al., 2008).
The most laborious task in the construction of
a physical map is the anchoring of the fi ngerprint
contigs to the genetic map. In most of the plant
genome physical mapping projects published so
far, anchoring has been performed through
hybridization with RFLP and overgo probes that
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