The Plant Journal (2006) 47, 977–986
doi: 10.1111/j.1365-313X.2006.02840.x
TECHNICAL ADVANCE
Advanced resources for plant genomics: a BAC library specific
for the short arm of wheat chromosome 1B
Jaroslav Janda1, Jan Šafář1, Marie Kubaláková1,2, Jan Bartoš1, Pavlı́na Kovářová1, Pavla Suchánková1, Stephanie Pateyron3,
Jarmila Čı́halı́ková1,2, Pierre Sourdille4, Hana Šimková1, Patricia Faivre-Rampant3, Eva Hřibová1, Michel Bernard4, Adam
Lukaszewski5, Jaroslav Doležel1,2,* and Boulos Chalhoub3
1
Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovská 6, CZ-77200 Olomouc,
Czech Republic,
2
Department of Cell Biology and Genetics, Palacký University, Šlechtitelů 11, CZ-78371 Olomouc, Czech Republic,
3
Organization and Evolution of Plant Genomes, Unité de Recherches en Génomique Végétale (INRA-URGV), 2 rue Gaston
Crémieux, CP 5708, F-91057 Évry Cedex, France,
4
Génétique Moléculaire des Céréales, UMR INRA-UBP, Domaine de Crouelle, 234 Avenue du Brézet, F-63039 Clermont-Ferrand
Cedex 2, France, and
5
Department of Botany and Plant Sciences, University of California, Riverside, CA 92521, USA
Received 1 November 2005; revised 2 May 2006; accepted 30 May 2006.
*For correspondence (fax þ 420 585 205 853; e-mail [email protected]).
Summary
Common wheat (Triticum aestivum L., 2n ¼ 6x ¼ 42) is a polyploid species possessing one of the largest
genomes among the cultivated crops (1C is approximately 17 000 Mb). The presence of three homoeologous
genomes (A, B and D), and the prevalence of repetitive DNA make sequencing the wheat genome a daunting
task. We have developed a novel ‘chromosome arm-based’ strategy for wheat genome sequencing to simplify
this task; this relies on sub-genomic libraries of large DNA inserts. In this paper, we used a di-telosomic line of
wheat to isolate six million copies of the short arm of chromosome 1B (1BS) by flow sorting. Chromosomal DNA
was partially digested with HindIII and used to construct an arm-specific BAC library. The library consists of
65 280 clones with an average insert size of 82 kb. Almost half of the library (45%) has inserts larger than 100 kb,
while 18% of the inserts range in size between 75 and 100 kb, and 37% are shorter than 75 kb. We estimated the
chromosome arm coverage to be 14.5-fold, giving a 99.9% probability of identifying a clone corresponding to any
sequence on the short arm of 1B. Each chromosome arm in wheat can be flow sorted from an appropriate
cytogenetic stock, and we envisage that the availability of chromosome arm-specific BAC resources in wheat
will greatly facilitate the development of ready-to-sequence physical maps and map-based gene cloning.
Keywords: wheat, genomics, physical mapping, BAC resources, chromosome sorting, flow cytometry.
Introduction
Common wheat (Triticum aestivum L.) is grown on a
larger acreage than any other crop plant. Because of its
socio-economic importance, a sustained increase in production must be ensured to keep pace with the growing
human population. It is expected that the breeding of
improved varieties will be accelerated by a better knowledge of the wheat genome. However, genomics in wheat
has been hampered by the size and the complexity of its
genome. Common wheat is an allohexaploid species
consisting of three closely related genomes (AABBDD,
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd
2n ¼ 6x ¼ 42), resulting in sequence redundancy (Gill
et al., 2004). The estimated size of the hexaploid wheat
genome is 17 billion bases (Bennett and Smith, 1991),
mainly consisting of repetitive DNA (approximately 80%,
Smith and Flavell, 1975), and is >100 times larger than the
genome of the model plant Arabidopsis thaliana. Each
individual wheat chromosome is larger than the recently
sequenced rice genome (Goff et al., 2002). These unique
features pose great challenges for gene discovery and
genome sequencing.
977
978 Jaroslav Janda et al.
Because of its large size and complexity, sequencing of
the wheat genome requires careful consideration and
development of appropriate strategies and technologies.
In addition to clone-by-clone sequencing and selective
sequencing of random bacterial artificial chromosome
(BAC) clones from gene-rich regions, shotgun sequencing
of ‘gene-enriched’ DNA obtained after genome filtration was
suggested (Gill et al., 2004). Regardless of which strategy is
chosen, a robust physical map will be required. This holds
true not only for the sequencing approaches based on largeinsert DNA clones but also for short sequences obtained
from gene-enriched DNA that will have to be ordered on the
genomic scaffold (Meyers et al., 2004). Beside its utility for
genome sequencing, the availability of a global physical
map would greatly accelerate positional gene cloning.
Physical maps are produced by ordering DNA clones from
large-insert (typically BAC) libraries on the basis of a clone
fingerprint pattern (Luo et al., 2003). Several genomic BAC
libraries have been constructed for bread wheat (Allouis
et al., 2003; Liu et al., 2000; Ma et al., 2000; Nilmalgoda
et al., 2003). To achieve the required genome coverage, one
to two million BAC clones have to be fingerprinted.
Although it is possible to fingerprint such large numbers
of clones with the automated fingerprinting technique of
Luo et al. (2003), it is doubtful that biological constraints due
to polyploidy and current contig assembly technology
would allow contigs and physical maps to be built from
such a large number of fingerprints (Jan Dvořák, University
of California, Davis, CA, USA, personal communication).
One approach to simplify this task is to employ diploid
relatives of wheat whose genomes are smaller. Several BAC
libraries representing genomes of individual wheat progenitors have been constructed. Moullet et al. (1999) created a
BAC library from the D genome donor of wheat Aegilops
tauschii, and Lijavetzky et al. (1999) developed a BAC library
from T. monococcum, which is related to T. urartu, the
progenitor of the A genome. BAC libraries from T. urartu
and Ae. speltoides, the presumed progenitor of the B genome, were developed recently (Akhunov et al., 2005).
However, the hexaploid wheat has diverged compared with
its wild diploid progenitor species. Polyploidization events
cause important DNA loss and rearrangements (Akhunov
et al., 2003; Anderson et al., 2003; Chantret et al., 2005;
Dvorak et al., 2004; Kong et al., 2004; Wicker et al., 2003)
suggesting that the diploid species constitute poor surrogates for genomic studies in bread wheat.
We believe that a fundamental solution to the problem of
sequencing large genomes is to create BAC libraries specific
for small and defined parts thereof. In line with this, we
have developed procedures for purification of individual
chromosomes and chromosome arms by flow cytometry
(Kubaláková et al., 2002, 2005; Vrána et al., 2000) and a
protocol for preparation of intact DNA from sorted chromosomes that is suitable for cloning (Šimková et al., 2003).
These advances, and a highly efficient protocol for BAC
cloning (Chalhoub et al., 2004), have allowed the creation of
two sub-genomic BAC libraries from hexaploid wheat: a
composite library specific for chromosomes 1D, 4D and 6D
(Janda et al., 2004) and a library specific for chromosome 3B
(Šafář et al., 2004). Our ability to sort single chromosome
arms from polyploid wheat (Kubaláková et al., 2002, 2005)
offers the possibility of constructing BAC libraries from even
smaller parts of the genome. The size of individual wheat
chromosome arms ranges from 224 to 582 Mb, representing
only 1.3–3.4% of the whole genome. This is within the size
range of plant genomes that have already been sequenced
and/or will be sequenced in the near future.
Even with a reliable chromosome arm purification protocol, creation of a BAC library still represents a challenge
because of the minute amounts of DNA obtained from flowsorted chromosome arms. To the best of our knowledge, no
chromosome arm-specific BAC library has been constructed
for any organism. In this paper, we describe the construction
and characterization of a BAC library specific for the short
arm of chromosome 1B (1BS) of wheat. This chromosome
arm represents only 1.9% of the hexaploid wheat genome
and carries a large number of important genes, including
disease and pest resistance genes and genes for storage
proteins that directly affect grain quality (Erayman et al.,
2004; Sandhu et al., 2001). The Wheat Gene Catalog (http://
wheat.pw.usda.gov/ggpages/wgc/98/) is a good reference to
the gene content of chromosome arm 1BS. The ability to
create a BAC library from a particular chromosome arm
opens avenues for the development of a physical map of the
complex hexaploid wheat genome and its analysis in a
targeted and step-wise manner, working with one particular
chromosome arm at a time. In addition to simplifying the
physical mapping efforts by several orders of magnitude,
this chromosome-based approach has the potential for
division of labor during sequencing of the wheat genome
(Gill et al., 2004).
Results
The short arm of chromosome 1B (1BS) was purified from
a di-telosomic line of bread wheat cultivar ‘Pavon 76’. The
work involved preparation of liquid suspensions of intact
chromosomes from synchronized root tip meristems,
staining with a DNA-specific fluorescent dye 4¢,6-diamidino-2-phenylindole (DAPI), and analysis of the fluorescence
intensity of stained chromosomes using flow cytometry.
We obtained a reproducible distribution of relative fluorescence intensity (‘flow karyotype’), which was characterized by five peaks (Figure 1). We confirmed the
chromosome content of each peak by sorting particles
from each peak onto microscope slides and performing
fluorescence in situ hybridization (FISH) with probes for
GAA microsatellites and the Afa repeat. The two probes
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 47, 977–986
Chromosome arm-specific BAC libraries in wheat 979
Number of events
I
1BS
III
II
3B
Relative fluorescence intensity
Figure 1. Histogram of relative fluorescence intensity (‘flow karyotype’)
obtained after flow cytometric analysis of DAPI-stained suspensions of
chromosomes prepared from a 1BS di-telosomic line of wheat cultivar ‘Pavon
76’. The flow karyotype consists of three composite peaks (I, II, and III)
representing specific groups of chromosomes, and a peak representing
chromosome 3B. In addition, a peak representing the short arm of chromosome 1B (1BS) is clearly discriminated.
facilitated identification of any chromosome arm in
hexaploid wheat (Pedersen and Langridge, 1997). We
established that peaks I–III were composites of various
chromosomes and that the small peak to the right of peak
III represented chromosome 3B (Kubaláková et al., 2002;
Vrána et al., 2000). The peak on the far left was found to
represent the short arm of chromosome 1B.
Subsequently, we sorted six million copies of chromosome arm 1BS representing approximately 4 lg DNA in
batches of 105 and embedded them in 60 agarose plugs. The
sorting exercise took 27 working days, and approximately
Figure 2. Insert size analysis of 22 randomly
chosen clones from the TA-1BS BAC library (H1
sub-library).
BAC DNA was digested with NotI to release the
insert, separated by pulsed-field gel electrophoresis and stained with ethidium bromide. Lanes M
contain a k PFGE marker. The 7.5 kb band
represents the BAC vector.
M
7000 seeds were required to prepare 267 samples of chromosome suspensions. We repeatedly checked the purity of
the flow-sorted fractions by microscopic observation of
chromosomes sorted on a glass slide and subjected to FISH
with probes for GAA microsatellites and Afa repeats. On
average, the sorted fractions consisted of 89% chromosome
1BS. Contaminating particles were arms and chromatids of
other chromosomes without an apparent prevalence of
specific types. Pulsed-field gel electrophoresis showed that
the DNA of sorted chromosome arms was intact, indicating
its usefulness for BAC library construction (data not shown).
The DNA of sorted chromosomes was partially digested
with HindIII and used to prepare an 1BS-specific BAC library.
The complete 1BS-specific BAC library (TA-1BS) consists of
five sub-libraries created from five independent ligation
reactions using different size classes of DNA: H01, 50–75 kb;
H02, 75–100 kb; H1, 100–150 kb; H2, 150–200 kb; H3, 200–
250 kb. We estimated the average insert size of the library
after the analysis of 96 BAC clones selected randomly from
the five sub-libraries (Figure 2). The H01 sub-library has 9600
clones and an average insert size of 62 kb, and is similar to
the H02 sub-library with 23 808 clones and average insert
size 66 kb. Sub-library H1 is the largest, with 27 264 clones
and an insert size of 96 kb, while sub-library H2 is the
smallest sub-library, with only 384 clones and an average
insert size of 110 kb. Finally, the H3 sub-library has 4224
clones and its average insert size is 118 kb. The complete
1BS-specific BAC library comprises 65 280 clones ordered in
170 384-well plates with an average insert size of 82 kb.
Almost a half of the TA-1BS library (45%) has inserts larger
than 100 kb, while 18% of inserts are 75–100 kb, and 37% of
inserts are shorter than 75 kb (data not shown).
Taking into account the wheat genome size of
16 974 Mb 1C)1 (Bennett and Smith, 1991) and the relative
BAC clones
M
kb
–145.5
–97
–48.5
–7.5
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 47, 977–986
980 Jaroslav Janda et al.
chromosome lengths given by Gill et al. (1991), we estimate
the molecular size of the short arm of chromosome 1BS to
be 315 Mb. Thus, this chromosome arm represents only
1.9% of the hexaploid wheat genome. Given the number of
clones, the average insert size and the frequency of contaminating particles, this library represents 14.5 size equivalents of 1BS. Using the formula of Clarke and Carbon
(1976), we estimated the probability of recovering any DNA
sequence present on chromosome 1BS in the library to be
99.9%. On the other hand, the probability of finding any DNA
sequence from other chromosomes is as low as 4.5%.
To confirm the chromosome arm specificity and verify the
14.5-fold genome coverage, we screened the TA-1BS library
by PCR using a set of microsatellite (SSR) markers. To do this,
we divided the library into 170 pools; each pool representing
one 384-well plate. Then we screened the pools with nine SSR
markers specific for the short arm of chromosome 1B. The
number of pools that gave PCR products of the expected
lengths with each SSR marker ranged from 10 to 21 (Table 1).
Based on these results, we estimated the coverage of the TA1BS BAC library to be at least 15.2-fold.
Cytogenetic mapping is needed to support the development of physical contig maps by anchoring BAC contigs
onto chromosomes. In order to test the usefulness of our
new BAC library for cytogenetic mapping, we hybridized 75
randomly selected ‘low-copy’ BAC clones to wheat chromosomes by FISH. Only four of them (5.3%) localized exclusively on 1BS. Another BAC clone (4B9) gave a distinct signal
on the satellite of 1BS and on chromosome arms 5AS, 5BS,
1DS and 5DS. Five BAC clones gave FISH patterns similar to
that of repetitive DNA family pSc119 (Bedbrook et al., 1980),
while FISH with four BAC clones resulted in a distinct signal
on 1BS and dispersed signals on all wheat chromosomes.
Fifty-two BAC clones hybridized to all wheat chromosomes
along their length, indicating the presence of interspersed
repeated sequences. The remaining nine BAC clones did not
yield any hybridization signals.
Table 1 Results of TA-1BS library screening on pools of 384-well
plates with 1BS-specific SSR markers
SSR marker
Marker code
Reference
Number of positive pools
gwm 18
gwm 264
gwm 413
gwm 550
gpw 2067
gpw 3122
gpw 4069
gpw 7059
gpw 7070
Average
a
a
a
a
b
b
b
b
b
21
14
17
12
10
13
14
21
15
15.2
a
Röder et al. (1998).
b
Sourdille and Bernard (unpublished data).
With the aim of overcoming the difficulties observed with
BAC-FISH, we explored a possibility of using ‘low-copy’
subclones for FISH. Out of the 75 BAC clones tested, eight
BAC clones with different FISH patterns were chosen for
subcloning (Table 2). This strategy provided shotgun subclones that hybridized exclusively to unique loci on 1BS.
FISH with subclones selected from BAC clones 77E3, 79P7
and 81P18, which hybridized to a single locus on 1BS and
multiple loci on all chromosomes, resulted in discrete
signals on 1BS only. To some measure, the subcloning
strategy was also successful with BAC clones that hybridized
evenly over the whole chromosome complement. We succeeded in selecting ‘low-copy’ subclones hybridizing exclusively to specific loci on 1BS from two such BAC clones
(78M16 and 81L2). However, subclones obtained from two
other BAC clones (80E19, 80M16) gave only dispersed
hybridization signals. Finally, FISH with two subclones
(4B9-A9 and 4B9-F6) resulted in the same hybridization
pattern as FISH with the complete BAC 4B9. Representative
examples of FISH with BAC clones and subclones are shown
in Figure 3.
Selected ‘low-copy’ subclones that gave a range of FISH
patterns were completely sequenced (Table 3). DNA sequences were assembled and edited using BioEdit software
(http://www.mbio.ncsu.edu/BioEdit/bioedit.html), and further analyzed using Tandem Repeats Finder (TRF; Benson,
1999) and JDotter (Brodie et al., 2003). All sequences were
successfully assembled, except subclones 4B9-A9 and
78M16-G18, for which TRF and JDotter showed the presence
of duplicated and tandem organized regions. In case of 4B9A9, this result could provide an explanation for the FISH
pattern obtained (Figure 3). A homology search was performed against the sequences deposited in GenBank, and
against the Triticeae Repeat Sequence Database (TREP;
http://wheat.pw.usda.gov/ITMI/Repeats/index.shtml). Most
of the subclones were found to carry various parts of
repetitive elements, the most frequent being various parts of
retrotransposons (Table 3). No homology was found for
subclones 81P18-B18 and 77E3-E15.
The cytogenetically mapped subclones were then used to
test the feasibility of developing chromosome sequence
tags. Based on the nucleotide sequences of BAC subclones,
we designed primers for PCR and tested their specificity on
flow-sorted chromosome arm 1BS, genomic DNA of ‘Pavon
76’, and di-telosomic line 1BL of ‘Pavon 76’. The primers
designed for 81P18-B18 and 77E3-F15 subclones were 1BSspecific and gave one PCR product of about 1000 bp
(Figure 4).
Discussion
In this paper, we report an important step forward in
developing a new generation of genomic resources for
bread wheat. We demonstrate that it is possible to create a
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 47, 977–986
Chromosome arm-specific BAC libraries in wheat 981
Table 2 Patterns of hybridization to wheat chromosomes observed after FISH with probes for putative ‘low-copy’ BAC clones selected from the
1BS-specific BAC library, and for their ‘low-copy’ shotgun subclones
BAC clones
BAC subclones
BAC
address
Insert
size (kb)
Hybridization pattern
Subclone
number
Insert
size (bp)
4B9
75
Single locus on 1BS, 5AS, 5BS, 1DS and 5DS
A9
2700
F6
2500
E15
F15
O23
A1
C22
G18
B1
2300
2700
2500
4900
1500
1900
2500
Single locus on 1BS, 5AS,
5BS, 1DS and 5DS
Single locus on 1BS, 5AS,
5BS, 1DS and 5DS
Single locus on 1BS
Single locus on 1BS
Dispersed on all chromosomes
No signal
No signal
Single locus on 1BS
Single locus on 1BS
B1
N3
O22
A1
C3
E17
N3
B14
B18
C3
2800
3200
1200
3000
2000
2900
2900
1000
2500
3500
Dispersed on all chromosomes
Dispersed on all chromosomes
No signal
Dispersed on all chromosomes
No signal
No signal
Single locus on 1BS
No signal
Single locus on 1BS
Single locus on 1BS
77E3
55
Single locus on 1BS and weakly
dispersed on all chromosomes
78M16
70
Dispersed on all chromosomes
79P7
45
80E19
80M16
50
60
Single locus on 1BS and weakly
dispersed on all chromosomes
Dispersed on all chromosomes
Dispersed on all chromosomes
81L2
35
Dispersed on all chromosomes
81P18
60
Single locus on 1BS and weakly
dispersed on all chromosomes
Hybridization pattern
Figure 3. Fluorescence in situ hybridization (FISH) on the short arm of wheat chromosome 1B (1BS) using three BAC clones that give three different types of
hybridization pattern, and three subclones derived from them.
BAC 81L2 (left) and its subclone 81L2-A1 hybridize to the entire length of 1BS except the NOR region. BAC 4B9 (centre) and its subclone 4B9-A9 hybridize to a single
locus on 1BS. In contrast, BAC 81P18 hybridizes strongly to a single locus and weakly along the entire length of 1BS, while its subclone 81P18-B18 maps to a single
locus on 1BS. The probes were labeled by digoxigenin and the sites of probe hybridization were detected using anti-digoxigenin–FITC (yellow). The chromosomes
were counterstained by DAPI (shown in red pseudocolor). Three representative examples of hybridizations on 1BS are given.
BAC library from a specific chromosome arm. The TA-1BS
library is specific for the short arm of chromosome 1B, and is
the third sub-genomic BAC library produced from individual
plant chromosomes, after the composite BAC library specific
for wheat chromosomes 1D, 4D and 6D (Janda et al., 2004)
and a BAC library from wheat chromosome 3B (Šafář et al.,
2004). In addition to creating a unique resource for wheat
genomics, the current work confirms that our procedure for
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 47, 977–986
982 Jaroslav Janda et al.
Table 3 DNA sequences obtained from ‘low-copy’ BAC subclones
Homology with known DNA sequences
GenBank
TREP
Subclone
address
GenBank
Sequence accession
length (bp) number
DNA sequence
GenBank
accession
number
4B9-A9
1334*
DX572372 Gypsy-like retrotransposon
AY494981 8e)113
77E3-E15
77E3-F15
4079
2787
77E3-O23
2549
78M16-G18m13r 590*
78M16-G18r
718*
78M16-G18m13f 642*
81L2-A1
2376
DX572368
DX572367 Putative non-LTR
retroelement
reverse transcriptase
DX572366 Transposon protein, CACTA
DX572369 Germin-like protein 4
DX572370 Germin-like protein 4
DX572371
DX572365
81P18-B18
81P18-C3
DX572364
DX572363
2321
2513
Smallest
sum
probability DNA sequence
Retrotransposon,
gypsy
(Laura – 719C13)
TREP
Smallest
accession sum
number
probability
TREP1238 9e)31
NP922330 3e)28
ABA98219 3e)10
AAT67049 4e)14
AAT67049 4e)14
Transposon, CACTA TREP746
2e)22
Retrotransposon,
LTR, gypsy
TREP821
e)157
Unclassified
TREP1218 6e)20
n
vo
Pa
BL
t1
D
C
BA
1B
M
S
*Subclone could not be completely sequenced and/or assembled.
bp
500–
Figure 4. Agarose gel electrophoresis of PCR products obtained with primers
(5¢-TCTCCTCCCATGTAAGCTCTG-3¢ and 5¢-CTAGAAGGATGCCGAGGATG-3¢)
derived from BAC subclone 81P18-B18.
The reaction was performed on flow-sorted short arm of chromosome 1B
(1BS), BAC clone 81P18 (BAC), genomic DNA prepared from a line of wheat
cultivar ‘Pavon 76’ di-telosomic for 1BL (Dt1BL) and genomic DNA of ‘Pavon
76’. The absence of a product on Dt1BL and its presence on 1BS indicate
specificity of the primers for the short arm of 1B. Lane M,100 bp DNA Ladder
Plus marker (Fermentas, Vilnius, Lithuana).
the construction of large-insert DNA libraries from DNA of
flow-sorted chromosomes is reproducible, reliable and efficient. This advance, together with the availability of a complete series of telosomic lines in wheat (Sears, 1954) and the
possibility of purifying wheat telosomes by flow cytometry
(Kubaláková et al., 2002), make it possible to create a BAC
library from every chromosome arm of wheat.
The TA-1BS library consists of five sub-libraries differing
in the number of clones and average insert sizes. These
features offer considerable flexibility in the use of the library.
Sub-libraries H1, H2 and H3, with an average insert size of
about 100 kb and tenfold 1BS coverage, could be used
preferentially for contig assembly and chromosome walking. Sub-libraries H01 and H02, which contain clones with
smaller inserts of around 65 kb, represent useful resources
for FISH mapping. We have confirmed the representative
coverage of the short arm of chromosome 1B by library
screening with SSR markers that map to 1BS. The 15.2-fold
1BS coverage, as determined by screening with SSR markers, suggests that our estimate of clone representation using
average insert size (14.5-fold) is accurate.
Compared with other genomic and sub-genomic BAC
resources for hexaploid wheat, the TA-1BS library has a
comparable average insert size and the highest genome
coverage. Liu et al. (2000) reported on the construction of a
TAC (transformation-competent artificial chromosome) library with an average insert size of 54 kb and 3.07-fold
genome coverage. A genomic BAC library prepared by
Nilmalgoda et al. (2003) comprises 6.5 · 105 clones, an
average insert size of 79 kb, and 3.1-fold genome coverage.
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 47, 977–986
Chromosome arm-specific BAC libraries in wheat 983
Another published BAC library from hexaploid wheat was
created by Allouis et al. (2003). The library consists of
1.2 · 106 clones, with an average insert size of 130 kb, and
represents 9.3 haploid genome equivalents.
A single but serious disadvantage of genomic libraries
from hexaploid wheat is their large size, which makes their
screening laborious and expensive. Maintenance and handling of genomic libraries with approximately 106 BAC clones
remains a non-trivial task (Allouis et al., 2003). However,
sub-genomic BAC libraries offer a solution to this problem.
Our composite BAC library specific for chromosomes 1D, 4D
and 6D, which consists of only 87 168 clones with an average
insert size of 85 kb, represents at least 3.4-fold clone
coverage (Janda et al., 2004), while the BAC library specific
for chromosome 3B, which comprises only 67 968 clones
with an insert size of 103 kb, represents at least 6.2 equivalents of the chromosome (Šafář et al., 2004). The TA-1BS
library described in this paper comprises 65 280 clones with
an average insert size of 82 kb, and represents 14.5 size
equivalents of the short arm of chromosome 1B.
In addition to chromosome coverage, specificity is an
important quality parameter for chromosome- and chromosome arm-specific BAC libraries. To ensure that our library is
specific for chromosome 1BS, we have periodically checked
the flow-sorted fractions for contamination. The average
content of other chromosome arms and chromatids was
11% as determined by FISH using probes for the GAA
microsatellite and the Afa repeat. This FISH approach offers
unambiguous identification of all chromosome arms in
hexaploid wheat (Kubaláková et al., 2002). Our previous
studies (Janda et al., 2004; Šafář et al., 2004) demonstrated
that FISH analysis of sorted chromosome fractions provided
the most detailed and reliable data on the purity in sorted
fractions and the extent of contamination by other chromosomes. Hence, we did not consider it useful to check the
TA-1BS library for contamination using PCR with markers
specific for other wheat chromosomes.
DNA libraries cloned in a BAC vector are a favorable
genomic resource for use in map-based cloning. The
availability of a BAC library specific for the short arm of
1B, which represents only 1.9% of the bread wheat genome,
should accelerate cloning of important genes in wheat.
Preliminary results obtained with our BAC library specific for
chromosome 3B (Šafář et al., 2004) confirm these expectations. The small size and the specificity of the library greatly
facilitated map-based cloning of major QTL for Fusarium
head blight resistance (Liu et al., 2005) and a durable rustresistance gene (Kota et al., 2006).
Whole-genome physical maps provide a basis for genome
sequencing and will be needed whatever the sequencing
method employed (Meyers et al., 2004). However, the
genome of hexaploid wheat is so complex that the development of a whole-genome physical map seems out of
reach of the current technology (Jan Dvořák, University of
California, Davis, USA, personal communication). Chromosome- and chromosome arm-specific BAC libraries provide
a solution to this problem. As they represent only a small
fraction of the whole genome, sufficient coverage of a
particular chromosome can be achieved with about 50 000
BAC clones. Using a suitable high-throughput method, a
library of this size can be fingerprinted in a few months (Luo
et al., 2003). Furthermore, the reduced complexity should
greatly simplify the assembly of BAC contigs. Recent results
on the production of a physical contig map of wheat chromosome 3B (Paux et al., 2006) fully support these assumptions. The ability to create a BAC library from a particular
chromosome arm opens avenues for the development of a
physical map of the complex hexaploid wheat genome and
its analysis in a targeted and step-wise manner, working
with a particular chromosome arm at a time. In addition to
simplifying the physical mapping efforts by at least two
orders of magnitude, the chromosome-based approach has
the potential for a division of labor (Gill et al., 2004).
The development of physical contig maps requires support
from cytogenetic mapping to confirm the precise physical
positions of centromeres, to determine the distances from
the actual chromosome termini of markers near the termini of
linkage maps, to resolve the order of contigs and clones, and
to estimate the numbers and sizes of gaps (Harper and Cande,
2000). Our results demonstrate that mapping complete BAC
clones to specific loci in wheat may be difficult due to the
presence of repetitive DNA sequences that hybridize
throughout the genome. This conclusion is supported by
the observations of Zhang et al. (2004). In this paper, we show
that shotgun subcloning and the use of ‘low-copy’ subclones
may overcome this problem. Our success in the localization
of clones shorter than 2 kb was made possible by performing
FISH on flow-sorted chromosomes that are free of cell wall
and cytoplasmic remnants. Because thousands of chromosomes can be sorted on one slide, FISH on sorted chromosomes offers higher throughput, higher sensitivity and
higher resolution when compared to mitotic metaphase
spreads (Valárik et al., 2004). Hence, flow-based cytogenetic
mapping could play an important role in the development of a
sequence-ready physical map of bread wheat.
In conclusion, this paper demonstrates that high-quality
sub-genomic BAC resources representing only few per cent
of the whole genome can be prepared for large-genome
species. While our two previous studies utilized flow-sorted
chromosomes to construct sub-genomic BAC libraries (Janda et al., 2004; Šafář et al., 2004), this study marks a
fundamental advance and reports on the creation of a
large-insert library specific for a particular chromosome
arm. We envisage that the use of chromosome arm-specific
BAC resources will accelerate the development of sequenceready physical contig maps and gene cloning, as well as
comparative analyses aiming at revealing the genome
changes accompanying the evolution of polyploid wheat.
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 47, 977–986
984 Jaroslav Janda et al.
Experimental procedures
Estimation of the insert size of BAC clones
Plant material
A di-telosomic line of hexaploid wheat Triticum aestivum L. cv. ‘Pavon 76’ (2n ¼ 40 þ 2t1BS) carrying the short arm of chromosome 1B
(1BS) in the form of a telosome was used. Seeds were germinated in
the dark at 25 0.5C on moistened filter paper for 3 days to obtain a
root length around 2–3 cm. Approximately 7000 seeds were germinated in batches of 25–30 for the preparation of 267 chromosome
suspensions which were further used for flow sorting.
Preparation of chromosome suspensions and flowcytometric sorting
Cell-cycle synchronization and the preparation of suspensions of
intact chromosomes were performed according to the method
described by Vrána et al. (2000). Briefly, each of the 267 samples of
chromosome suspension was prepared by mechanical homogenization of 25 formaldehyde-fixed meristem root tips in 1 ml icecold isolation buffer (Šimková et al., 2003). Chromosomes in suspension were stained with 2 lg ml)1 DAPI (4¢,6-diamidino-2-phenylindole) and analyzed using a FACSVantage flow cytometer
(Becton Dickinson, San José, CA, USA) equipped with a UV laser set
to multi-line UV and running at 300 mW output power. In order to
sort the chromosome 1BS, the sort window was set on a dot plot of
fluorescence pulse area versus fluorescence pulse width. The
chromosome arm was sorted in aliquots of 1 · 105 into 160 ll of
1.5x isolation buffer. The purity of sorted fractions was checked
regularly by sorting 2000 particles into a 15 ll drop of PRINS buffer
(Kubaláková et al., 2001) supplemented with 5% sucrose on a
microscope slide. After air-drying, sorted chromosomes were
identified using fluorescence in situ hybridization (FISH) with
probes for the GAA microsatellite and Afa repeat, which give
chromosome-specific fluorescent labeling patterns (Kubaláková
et al., 2002).
BAC library construction
Preparation of high-molecular-weight DNA and BAC library construction were performed as described previously (Chalhoub et al.,
2004; Janda et al., 2004; Šafář et al., 2004). Briefly, each batch of 105
flow-sorted 1BS chromosomes was pelleted (200 g), and the chromosomes were embedded in 15 ll of low-melting-point agarose
(0.8% w/v). In total, 60 agarose mini-plugs representing 6 · 106
sorted chromosomes were prepared and used for DNA cloning.
Chromosomal DNA was partially digested with HindIII and sizeselected by pulsed-field gel electrophoresis (PFGE) in 0.25x TBE
buffer at 6 V cm)1, with a 1–40 sec switch time ramp, angle 120, for
12 h, and with a 2.5–5.5 sec switch time ramp, angle 120, for 6 h at
14C. Five regions of the gel, corresponding to approximately 50–
75 kb (H01 fraction), 75–100 kb (H02 fraction), 100–150 kb (H1 fraction), 150–200 kb (H2 fraction) and 200–250 kb (H3 fraction) were
excised, the high-molecular-weight DNA was electro-eluted and
ligated into a dephosphorylated vector pIndigoBAC5 (Caltech, Pasadena, CA, USA), which was prepared according to the method
described by Chalhoub et al. (2004). Ligation mixtures were incubated at 16C, and, after de-salting, transformed into Escherichia
coli ElectroMAX DH10B competent cells (Gibco BRL, Gaithersburg,
MD, USA) by electroporation. The library was ordered in 384-well
microtitre plates filled with freezing medium (Woo et al., 1994). The
plates were incubated at 37C overnight, and stored at )80C after
replication.
Ninety-six BAC clones were randomly selected from all five ligations (H01–H3), and incubated overnight at 37C in 1.5 ml of 2YT
medium (Sambrook and Russel, 2001) containing 12.5 lg ml)1
chloramphenicol. BAC DNA was extracted by a standard alkaline
lysis method and digested with NotI restriction endonuclease
(0.25 U/20 ll). PFGE was used to separate DNA fragments in 1%
agarose gel with 0.5x TBE buffer at 6 V cm)1, with a 1–40 sec switch
time ramp, angle 120, for 14 h at 14C. The insert sizes were estimated after comparison with a lambda size standard run in the
same gel.
Screening the library with 1BS-specific probes
After growing overnight in 2YT medium supplemented with chloramphenicol (12.5 lg ml)1) in 384-well plates, BAC clones from
each of the 384-well plates were pooled, pelleted and resuspended
in 4 ml TE buffer (10 mM Tris, 1 mM EDTA). Bacterial suspensions
were lysed at 95C for 30 min, pelleted at 3000 g for 60 min, and the
supernatant was diluted 25-fold using deionized water for PCR
reactions. The reaction mix (10 ll) consisted of 2 ll template DNA,
1x PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 1.25 lM primers and
0.5 U AmpliTaq DNA polymerase (Roche, Mannheim, Germany).
The PCR reaction was performed as follows: nine cycles consisting
of 5 min at 94C, 12 sec at 65C and 18 sec at 72C, and 35 cycles of
12 sec at 94C, 12 sec at 55C and 18 sec at 72C, and a final
extension at 72C for 7 min. The presence of PCR products was
checked by electrophoresis on 4% MetaPhor agarose gel (FMC
Bioproducts, Philadelphia, PA, USA).
Selection of ‘low-copy’ BAC clones
A total of 6144 BAC clones from the H01 sub-library (sixteen 384well plates) were spotted onto two 8 · 12 cm Hybond Nþ filters (AP
Biotech, Piscataway, NJ, USA). The filters were hybridized with
wheat genomic DNA labeled with alkaline phosphatase using AlkPhos Direct (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) to reveal clones potentially containing a low amount of
repetitive DNA sequences. Then the filters were screened with a
probe for BAC vector to avoid the selection of non-growing clones.
Seventy-five ‘low-copy’ BAC clones showing no or weak hybridization signals were tested as BAC-FISH, and eight of them were
selected for subcloning. In addition, they were localized on flowsorted wheat chromosomes using FISH as described previously
(Šafář et al., 2004). No blocking DNA was used in the hybridization
mix for FISH.
Subcloning of ‘low-copy’ BAC clones
DNA of ‘low-copy’ BAC clones was isolated using the Large-Construct Kit (Qiagen, Valencia, CA, USA), and physically fragmented
using a HydroShear DNA shearing device (GeneMachines, San
Carlos, CA, USA). Fragments of 3–5 kb were ligated into pCR-XLTOPO vector (Invitrogen Life Technologies, Carlsbad, CA, USA).
Ligation mixtures were transformed into One-Shot TOP10 E. coli
(Invitrogen). A total of 384 subclones from each of the eight BAC
clones were ordered in 384-well plates filled with freezing medium
(Woo et al., 1994), incubated at 37C overnight, and stored at )80C.
All 3072 BAC subclones were spotted onto one 8 · 12 cm
Hybond Nþ filter and screened with labeled genomic DNA as described above. One to three subclones from each BAC showing a
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 47, 977–986
Chromosome arm-specific BAC libraries in wheat 985
weak signal after hybridization with labeled genomic DNA were
randomly chosen for mapping using FISH.
Fluorescence in situ hybridization
Preparation of digoxigenin- or biotin-labeled probes and FISH on
flow-sorted chromosomes were performed as described by Šafář
et al. (2004). The sites of probe hybridization were detected using
anti-digoxigenin–fluorescein isothiocyanate and streptavidin-Cy3,
and the chromosomes were counterstained with DAPI or propidium
iodide. The preparations were evaluated using a Olympus BX60
microscope (Olympus, Tokyo, Japan) with a CCD camera interfaced
to a PC running ISIS software (Metasystems, Altlussheim, Germany).
Sequence analysis of ‘low-copy’ BAC subclones
Six BAC subclones that were successfully localized by FISH to single
loci on the short arm of chromosome 1B and two that gave
hybridization patterns dispersed on all chromosomes were randomly chosen for sequencing. The sequencing was performed by
Agowa (Berlin, Germany). The sequenced parts of the subclones
were assembled in the Chromas software (Griffith University,
Southport, Qld, Australia), edited using BioEdit (http://www.
mbio.ncsu.edu/BioEdit/bioedit.html), and the assembled sequences
were deposited in GenBank (GSS database). They were then searched for similarities to known sequences in GenBank using BLASTN
(Altschul et al., 1997), and tested for the presence of repetitive units
using JDotter (Brodie et al., 2003) and Tandem Repeat Finder
(Benson, 1999). Finally, the sequences of subclones were used to
design primers for internal sequence amplification to verify their
specificity for 1BS. PCR was performed as described by Vrána et al.
(2000) on flow-sorted chromosome arm 1BS, BAC DNA, genomic
DNA of a di-telosomic line of cv. ‘Pavon 76’ carrying the long arm of
1B (Dt1BL), and genomic DNA of cv. ‘Pavon 76’.
Acknowledgements
We are grateful to our colleagues Arnaud Bellec, Michaela Libosvárová, Radka Tušková and Jitka Weiserová for excellent technical assistance. This work was supported by research grants from the
Czech Science Foundation (grant awards 521/04/0607, 521/05/P257
and 521/05/H013), the Ministry of Education, Youth and Sports of the
Czech Republic (grant award LC06004), research grants from the
French Ministry of Research and Agriculture, and by a collaborative
project ‘Barrande’ (2003-035-2) between the Ministry of Education,
Youth and Sports of the Czech Republic and the French Ministry of
Foreign Affairs and Education. J.J. acknowledges the receipt of a
Marie Curie PhD student Fellowship at URGV-INRA.
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Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 47, 977–986