A physical map of Brassica oleracea shows complexity of chromosomal changes following recursive paleopolyploidizations.
ABSTRACT Evolution of the Brassica species has been recursively affected by polyploidy events, and comparison to their relative, Arabidopsis thaliana, provides means to explore their genomic complexity.
A genome-wide physical map of a rapid-cycling strain of B. oleracea was constructed by integrating high-information-content fingerprinting (HICF) of Bacterial Artificial Chromosome (BAC) clones with hybridization to sequence-tagged probes. Using 2907 contigs of two or more BACs, we performed several lines of comparative genomic analysis. Interspecific DNA synteny is much better preserved in euchromatin than heterochromatin, showing the qualitative difference in evolution of these respective genomic domains. About 67% of contigs can be aligned to the Arabidopsis genome, with 96.5% corresponding to euchromatic regions, and 3.5% (shown to contain repetitive sequences) to pericentromeric regions. Overgo probe hybridization data showed that contigs aligned to Arabidopsis euchromatin contain ~80% of low-copy-number genes, while genes with high copy number are much more frequently associated with pericentromeric regions. We identified 39 interchromosomal breakpoints during the diversification of B. oleracea and Arabidopsis thaliana, a relatively high level of genomic change since their divergence. Comparison of the B. oleracea physical map with Arabidopsis and other available eudicot genomes showed appreciable 'shadowing' produced by more ancient polyploidies, resulting in a web of relatedness among contigs which increased genomic complexity.
A high-resolution genetically-anchored physical map sheds light on Brassica genome organization and advances positional cloning of specific genes, and may help to validate genome sequence assembly and alignment to chromosomes.All the physical mapping data is freely shared at a WebFPC site (http://lulu.pgml.uga.edu/fpc/WebAGCoL/brassica/WebFPC/; Temporarily password-protected: account: pgml; password: 123qwe123.
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ABSTRACT: BACKGROUND:Homoeologous sequences pose a particular challenge if bacterial artificial chromosome (BAC) contigs shall be established for specific regions of an allopolyploid genome. Single nucleotide polymorphisms (SNPs) differentiating between homoeologous genomes (intergenomic SNPs) may represent a suitable screening tool for such purposes, since they do not only identify homoeologous sequences but also differentiate between them.RESULTS:Sequence alignments between Brassica rapa (AA) and Brassica oleracea (CC) sequences mapping to corresponding regions on chromosomes A1 and C1, respectively were used to identify single nucleotide polymorphisms between the A and C genomes. A large fraction of these polymorphisms was also present in Brassica napus (AACC), an allopolyploid species that originated from hybridisation of A and C genome species. Intergenomic SNPs mapping throughout homoeologous chromosome segments spanning approximately one Mbp each were included in Illumina's GoldenGate(R) Genotyping Assay and used to screen multidimensional pools of a Brassica napus bacterial artificial chromosome library with tenfold genome coverage. Based on the results of 50 SNP assays, a BAC contig for the Brassica napus A subgenome was established that spanned the entire region of interest. The C subgenome region was represented in three BAC contigs.CONCLUSIONS:This proof-of-concept study shows that sequence resources of diploid progenitor genomes can be used to deduce intergenomic SNPs suitable for multiplex polymerase chain reaction (PCR)-based screening of multidimensional BAC pools of a polyploid organism. Owing to their high abundance and ease of identification, intergenomic SNPs represent a versatile tool to establish BAC contigs for homoeologous regions of a polyploid genome.BMC Genomics 01/2014; 15(1):560. · 4.40 Impact Factor
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ABSTRACT: Polyploidization has provided much genetic variation for plant adaptive evolution, but the mechanisms by which the molecular evolution of polyploid genomes establishes genetic architecture underlying species differentiation are unclear. Brassica is an ideal model to increase knowledge of polyploid evolution. Here we describe a draft genome sequence of Brassica oleracea, comparing it with that of its sister species B. rapa to reveal numerous chromosome rearrangements and asymmetrical gene loss in duplicated genomic blocks, asymmetrical amplification of transposable elements, differential gene co-retention for specific pathways and variation in gene expression, including alternative splicing, among a large number of paralogous and orthologous genes. Genes related to the production of anticancer phytochemicals and morphological variations illustrate consequences of genome duplication and gene divergence, imparting biochemical and morphological variation to B. oleracea. This study provides insights into Brassica genome evolution and will underpin research into the many important crops in this genus.Nature Communications 01/2014; 5. · 10.74 Impact Factor
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ABSTRACT: BACKGROUND:Brassica oleracea is a valuable vegetable species that has contributed to human health and nutrition for hundreds of years and comprises multiple distinct cultivar groups with diverse morphological and phytochemical attributes. In addition to this phenotypic wealth, B. oleracea offers unique insights into polyploid evolution, as it results from multiple ancestral polyploidy events and a final Brassiceae-specific triplication event. Further, B. oleracea represents one of the diploid genomes that formed the economically important allopolyploid oilseed, Brassica napus. A deeper understanding of B. oleracea genome architecture provides a foundation for crop improvement strategies throughout the Brassica genus.RESULTS:We generate an assembly representing 75% of the predicted B. oleracea genome using a hybrid Illumina/Roche 454 approach. Two dense genetic maps are generated to anchor almost 92% of the assembled scaffolds to nine pseudo-chromosomes. Over 50,000 genes are annotated and 40% of the genome predicted to be repetitive, thus contributing to the increased genome size of B. oleracea compared to its close relative B. rapa. A snapshot of both the leaf transcriptome and methylome allows comparisons to be made across the triplicated sub-genomes, which resulted from the most recent Brassiceae-specific polyploidy event.CONCLUSIONS:Differential expression of the triplicated syntelogs and cytosine methylation levels across the sub-genomes suggest residual marks of the genome dominance that led to the current genome architecture. Although cytosine methylation does not correlate with individual gene dominance, the independent methylation patterns of triplicated copies suggest epigenetic mechanisms play a role in the functional diversification of duplicate genes.Genome Biology 01/2014; 15(6):R77. · 10.30 Impact Factor
RESEARCH ARTICLEOpen Access
A physical map of Brassica oleracea shows
complexity of chromosomal changes following
Xiyin Wang1,4, Manuel J Torres1, Gary Pierce1, Cornelia Lemke1, Lisa K Nelson1, Bayram Yuksel1,9, John E Bowers1,
Barry Marler1, Yongli Xiao2, Lifeng Lin1, Ethan Epps1, Heidi Sarazen1, Carl Rogers1, Santhosh Karunakaran1,
Jennifer Ingles1, Emily Giattina1, Jeong-Hwan Mun5, Young-Joo Seol5, Beom-Seok Park5, Richard M Amasino6,
Carlos F Quiros7, Thomas C Osborn8, J Chris Pires3, Christopher Town2and Andrew H Paterson1*
Background: Evolution of the Brassica species has been recursively affected by polyploidy events, and comparison
to their relative, Arabidopsis thaliana, provides means to explore their genomic complexity.
Results: A genome-wide physical map of a rapid-cycling strain of B. oleracea was constructed by integrating high-
information-content fingerprinting (HICF) of Bacterial Artificial Chromosome (BAC) clones with hybridization to
sequence-tagged probes. Using 2907 contigs of two or more BACs, we performed several lines of comparative
genomic analysis. Interspecific DNA synteny is much better preserved in euchromatin than heterochromatin,
showing the qualitative difference in evolution of these respective genomic domains. About 67% of contigs can be
aligned to the Arabidopsis genome, with 96.5% corresponding to euchromatic regions, and 3.5% (shown to
contain repetitive sequences) to pericentromeric regions. Overgo probe hybridization data showed that contigs
aligned to Arabidopsis euchromatin contain ~80% of low-copy-number genes, while genes with high copy
number are much more frequently associated with pericentromeric regions. We identified 39 interchromosomal
breakpoints during the diversification of B. oleracea and Arabidopsis thaliana, a relatively high level of genomic
change since their divergence. Comparison of the B. oleracea physical map with Arabidopsis and other available
eudicot genomes showed appreciable ‘shadowing’ produced by more ancient polyploidies, resulting in a web of
relatedness among contigs which increased genomic complexity.
Conclusions: A high-resolution genetically-anchored physical map sheds light on Brassica genome organization
and advances positional cloning of specific genes, and may help to validate genome sequence assembly and
alignment to chromosomes.
All the physical mapping data is freely shared at a WebFPC site (http://lulu.pgml.uga.edu/fpc/WebAGCoL/brassica/
WebFPC/; Temporarily password-protected: account: pgml; password: 123qwe123.
Keywords: Comparative genomics, polyploidy, Arabidopsis thaliana
Flowering plants have extensively and often recursively
experienced polyploidization [1-4]. The resulting dupli-
cated regions, especially those produced recently, offer
the means to further study the contributions of
segmental and/or whole-genome duplication/triplication
to the evolution of a lineage, but add to genome com-
plexity. The high abundance of repetitive DNA
sequences in some flowering plants adds further to gen-
ome complexity. At present, many plant genomes have
been or are being sequenced. Draft genome sequences
can lack sufficient contiguity in many genomic regions
to support cross-species comparison of genome organi-
zation and structure, which is crucial to understanding
* Correspondence: email@example.com
1Plant Genome Mapping Laboratory, University of Georgia, Athens, GA
Full list of author information is available at the end of the article
Wang et al. BMC Genomics 2011, 12:470
© 2011 Wang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
plant evolution and speciation. In concert with sequence
assemblies, independent physical maps often facilitate
the correct ordering of DNA segments on chromosomes
and thus clarify the genome organization changes
revealed by multiple species comparisons [5,6].
Brassica is in the tribe Brassiceae, a well-defined clade
in the family Brassicaceae that also includes Arabidopsis
thaliana, the source of the first flowering plant genome
to be sequenced. Brassica and Arabidopsis are thought to
have shared common ancestry ~14-20 million years ago
[7-10]. The genus Brassica has great scientific and eco-
nomic importance . Crops of the genus Brassica are
widely used in the cuisine of many cultures and provide
much of world-wide edible vegetable oil supplies. Six
Brassica species are widely cultivated, including three
diploids: B. rapa (AA, 2n = 20), B. nigra (BB, 2n = 16)
and B. oleracea (CC, 2n = 18), and three amphidiploids
(allotetraploids): B. juncea (AABB, 2n = 36), B. napus
(AACC, 2n = 38) and B. carinata (BBCC, 2n = 34).
Study of B. oleracea offers particularly great promise
of new insights into morphological evolution that com-
plement and extend upon what is available in Arabidop-
sis [12-14]. In B. oleracea, morphological divergence has
been unusually rapid relative to reproductive isolation, i.
e., this single species has a stunning range of morpholo-
gies among genotypes that are readily intercrossed.
While domestication of most crops resulted in enhance-
ment of a single plant part for use by humans, such as
the seeds/grains of cereal crops, the fruits of some trees,
or the roots of some vegetable crops, the B. oleracea
crops are a striking exception. They include forms that
have been selected for enlarged vegetative meristems at
the apex (cabbages, B. oleracea subspecies capitata) or
in the leaf axils (Brussels sprouts, subsp. gemmifera),
forms with proliferation of floral meristems (broccoli,
subsp. italica) or even aborted floral meristems (cauli-
flower, subsp. botrytis), and forms with swollen bulbous
stems (kohlrabi, subsp. gongylodes), or orate leaf patterns
(kales, subsp. acephala). These morphologically diver-
gent genotypes (’morphotypes’) are freely intercrossing.
The plasticity of B. oleracea makes it a potential
model for the study of plant morphological evolution in
much the same manner that the dog (Canis spp.) is an
attractive model for mammalian evolution. While a few
genes like the homologs of Arabidopsis mutants such as
“CAULIFLOWER” are thought to play roles in some
Brassica morphologies [15-17], these morphologies are
under complex genetic control [18-21]. Some Brassica
QTLs map to locations that correspond to relevant Ara-
bidopsis mutants, suggesting positional candidates – but
many do not, suggesting the opportunity to identify
functions recalcitrant to mutation in Arabidopsis [22,23]
or that escaped detection due to small phenotypic
Due to their close phylogenetic relationship, Brassica-
Arabidopsis comparative genomics promises to identify
genetic determinants of a much broader spectrum of
variation than might be accessible using Arabidopsis
alone [12-14]. The close relationship of Brassica to Ara-
bidopsis motivated NSF-funded low-coverage (0.6×)
sequencing of B. oleracea (BO) genotype TO 1000 .
However, while the physiology and developmental biol-
ogy of Arabidopsis and Brassica are similar, the genomes
of Brassica species are much more complex than that of
A. thaliana [26-28]. The ‘diploid’ Brassica genomes are
3-5 times larger than that of Arabidopsis, ranging from
0.97 pg/2C (468 Mb/1C) for B. nigra to 1.37 pg/2C (662
Mb/1C) for B. oleracea, partially as a result of multiple
rounds of polyploidy during their ancestry [29,30]. One
round of ancient whole-genome triplication (gamma) in
an early eudicot ancestor and two whole-genome dupli-
cations (beta and alpha) occurred before the Arabidop-
sis-Brassica split [4,31,32]. Additional polyploidization(s)
occurred in the Brassica lineage after its divergence
from Arabidopsis, reflected by large duplicated segments
in the genetic maps of each of three diploids [B. rapa
(syn. rapa,), B. nigra and B. oleracea] [27,33-36]. The
corresponding duplicated structure of the B. rapa and
B. oleracea maps indicates that species divergence was
after polyploidization, resulting whole-genome triplica-
tion [29,37-39]. It was estimated that the genome tripli-
cation event and the initial diversification of the
Brassiceae must have occurred between 7.9 and 14.6
mya , which might be the hypothesized single and
major evolutionary event that have gave rise to the early
lineages . According to the analysis of the FLOWER-
ING Locus C region, it was further estimated that the
Brassica triplication occurred 13 to 17 mya, very soon
after the Arabidopsis and Brassica divergence at 17-18
Significant progress has been made in developing
genomic resources to expedite Brassica research [41-44].
A detailed genetic linkage map of B. rapa has been con-
structed containing 545 sequence-tagged loci distributed
on 10 linkage groups covering 1287 cM, with an average
interval of 2.4 cM between markers . Genetic linkage
maps were constructed for four B. oleracea populations,
with an average length of 863.6 cM and a total of 367
loci were detected in the constructed composite map
with an average interval between loci of 2.35 cM ,
which revealed at least 19 chromosomal rearrangements
differentiating B. oleracea and Arabidopsis. Linkage
maps of immortal mapping populations of rapid cycling,
self-compatible lines from B. rapa and B. oleracea were
recently developed, which included 224 and 279 mar-
kers, respectively . A genome-wide physical map of
the B. rapa genome was constructed by high-informa-
tion-content fingerprinting (HICF) , which facilitates
Wang et al. BMC Genomics 2011, 12:470
Page 2 of 15
improved physical map construction in both throughput
and quality by exploiting the fluorescence-labeled fin-
ger-printing approach. The map provided 242 anchored
contigs on 10 linkage groups to serve as seed points
from which to continue bidirectional chromosome
extension for genome sequencing. There are also efforts
to refine genetic linkage maps. Genome sequencing pro-
jects involving “A” and “C” genomes are on-going or
planned [47,48]. The Multinational Brassica Genome
Project (MBGP) and Brassica rapa Genome Sequencing
Project (BrGSP) are aiming to completely sequence the
genome of Brassica rapa inbred line ‘Chiifu” (http://
Here we report a physical map of a rapid-cycling
strain of B. oleracea (accession TO1434), integrating
high-information-content fingerprinting (HICF) of Bac-
terial Artificial Chromosome (BAC) clones with overgo
hybridization data from 2882 probes, including about
600 that have been genetically mapped. By integrating
the B. rapa physical map, we explored genome-wide
microsynteny between Arabidopsis and Brassica, and
found probable (peri)centromere-related contigs. Com-
parison of the B. oleracea map with Arabidopsis and
other available eudicot genomes showed appreciable
‘shadowing’ produced by more ancient polyploidies,
resulting in a web of relatedness among contigs which
increased genomic complexity, and interchromosomal
breakpoints during their diversification. This physical
map is of immediate value for gene isolation, and will
serve as a valuable genomic resource for Brassica “C”
genome sequencing, assembly of BAC sequences and
further comparative genomics between Brassica
BAC fingerprinting and physical map assembly
We fingerprinted a total of 73728 clones from 192 384-
well plates. Fingerprints containing less than 30 and
more than 200 bands were excluded from FPC analysis,
which used a dataset of 53048 clones.
FPC (Finger-Printed Contigs, v9.0)  was used to
construct BAC contigs. To produce an FPC-accessible
dataset (FPC does not accept color labels or fractional
sizes), the size of each fragment was multiplied by 10,
after which the decimal part was dropped. This resulted
in fragments with sizes ranging from 500 to < 6000
units. Secondly, the color labels were converted to non-
overlapping numeric ranges by adding offset values
6000, 12000, or 18000 to three of the four colors, which
eventually resulted in fragments ranging from 0 to
We designed and used overgo hybridization probes to
support contig construction. A total of 4226 probes
were designed by using Arabidopsis and Brassica
sequences, and they are often from conservative
domains (see Methods for details). After removing the
probes that hit > 50 BAC clones, a subset of 2882
probes were involved in the physical map assembly
Well-to-well contamination produces many problems
during assembly of HICF data. Therefore, before run-
ning FPC to construct the contigs, we removed the
likely contaminated BACs in the dataset by implement-
ing a de-contamination function in FPC. After an initial
round of contig construction (cutoff = 1e-50 and toler-
ance = 4 and best of 100 repetitive constructions), a
FPC program named DQer was run to eliminate possi-
ble questionable clones (Q-clones) for contigs > 15% Q-
clones. Multiple iterations of end-to-end and singleton-
to-contig merges were then adopted with successively
less and less stringent settings (Figure 1).
During the optimization of our processes, and later to
improve quality of some below-average batches of
BACs, we repeated fingerprinting of some 96-well ‘sub-
plates’, with 72 subplates (5184 BACs) duplicated, and
12 subplates (830 BACs) triplicated. For each BAC
Figure 1 FPC analytical pipeline used to assemble the Brassica oleracea physical map.
Wang et al. BMC Genomics 2011, 12:470
Page 3 of 15
repeated, the fingerprint having numbers of bands near-
est the global average (120) was used in assembly.
A total of 46,006 BACs were used in contig assembly,
yielding 2907 contigs each containing 2 or more BACs,
and 2323 singletons. An average contig contained 8.7
BACs and 3.2 overgo probes. Two contigs (ctg03293
and ctg02560) contain more than 1000 BACs, ~60% of
whose end sequences could be linked to Brassica repeti-
tive sequences determined by running BLAST. Five con-
tigs contained more than 100 BACs. Sixteen contigs
(ctg00857, ctg01639, ctg02159, ctg02194, ctg02197,
ctg02490, ctg02560, ctg02626, ctg02695, ctg02754,
ctg02830, ctg03202, ctg03304, ctg02470, ctg03571,
ctg04056) are RNA-related, and may help to decipher
the rRNA and tRNA genes in Brassica. Six contigs
(ctg01958, ctg02241, ctg02829, ctg03476, ctg03627,
ctg04065) are likely chloroplast-related, and five contigs
(ctg01690, ctg01958, ctg02241, ctg02960, ctg04062) are
likely mitochondrion-related, including two contigs that
are both chloroplast- and mitochondrion-related. These
contigs may be chimeric, involving both nuclear and
organelle DNA, or just nuclear DNA produced by lateral
gene transfer from organelle to nucleus as previously
discussed in Arabidopsis , and sorghum . DNA
similarity between BESs and organelle DNA can provide
some clue about the identity of potentially chimeric
contigs: BESs from chimeric contigs may have high
identity with organelle DNA, e.g., DNA similarity > 98%
over a long stretch, while laterally transferred DNA may
not. We infer that ctg04065 (265 BACs) may be a chi-
meric contig of chloroplast DNA (188 BACs) and
nuclear DNA (77 BACs). The DNA similarity of most
involved BESs against chloroplast DNA are often > 99%
in up to 800 bp, but some BESs have DNA similarity <
95%, perhaps reflecting a mix of extant chloroplast
DNA and laterally transferred ones. We also suggest
that ctg02241 and ctg04062 are chimeric mitochon-
drion-nuclear contigs inferred based on similar criteria.
The latter contains most mitochrondrial BACs (14 of 25
in the contig). BESs of other organelle-related contigs
have low similarity with extant organelle DNA, suggest-
ing their origins by lateral gene transfer.
Comparative genomic analysis
With the help of BAC end sequences and probe
sequences, both B. oleracea and B. rapa contigs were
mapped onto the Arabidopsis genome sequence (Figure
2). Neighboring hits < = 200 Kb from one another were
used to infer DNA synteny between B. oleracea and
Arabidopsis, and the longest syntenic region inferred is
more than 870 Kb, with most regions less than 400 Kb
(Figure 3A). For anchoring B. rapa contigs to
Figure 2 An example of a Brassica FPC contig linked to different Arabidopsis regions. The contig was displayed with 2 or 3 rows,
including assembled BAC clones, overgo probes, and merging information (if available) during contig assembly. Dashed lines between Brassica
BAC clones, probes and Arabidopsis genomic regions show interspecific chromosomal synteny.
Wang et al. BMC Genomics 2011, 12:470
Page 4 of 15
Arabidopsis, the extension parameter was reduced to
100 Kb because a higher density of BESs made it easier
to find cross-species synteny. A subset of 1990 B. olera-
cea and 1006 B. rapa contigs (68.5% and 70.4% of the
total of respective datasets) hit one or more Arabidopsis
regions. DNA sequence similarity revealed by the anchor
sequences peaked at 92% (Figure 3B), which supports a
14.5-20.4 million year divergence time between Arabi-
dopsis and Brassica [2,3,7-10]. Interestingly, the Blast E-
value showed a bi-modal distribution (Figure 3C), which
Figure 3 Characteristics of B. oleracea (Bo) contigs mapped onto the A. thaliana (At) genome. (A) Size of anchored regions based on
length of Arabidopsis sequences covered; Sequence similarity (B) and BLAST E-values (C) between anchored Bo and At sequences.
Wang et al. BMC Genomics 2011, 12:470
Page 5 of 15
may imply at least two different sets of anchored regions
in Arabidopsis, possibly reflecting the ancient duplica-
We found clear evidence of ancient duplication events
in the extant Brassica genomes. About 88% and 93% of
Arabidopsis genome sequences have been covered by
the anchored B. oleracea and B. rapa contigs, respec-
tively (Figure 4). At least 70% of regions have been cov-
ered to a depth > = 2, surely a result of multiple
homologous regions in Brassica (Figure 4). The peak is
around 3, covering nearly 20-25% of Arabidopsis gen-
ome sequences. There is a sharp decrease from coverage
3 to 4, supporting previous propositions of triplication
of at least portions of the Brassica ancestral genome
after its divergence from Arabidopsis. The 13% of the
Arabidopsis genome covered in depth 4, and total of
20% covered in depths > = 4, are shown below to be
partly explained by the ‘shadows’ of more ancient gen-
By checking Arabidopsis genomic regions known to
correspond to one another due to ancient duplication,
we revealed that 186 B. oleracea contigs (9.3% of all
anchored ones) were anchored to both members of a-
duplicated segment pairs and another 54 (2.7%) to b- or
g-duplicated regions. However, it is often possible to dis-
tinguish the orthologous regions from the outparalogous
regions (produced by ancient duplications before the
Arabidopsis-Brassica divergence). The inferred Arabi-
dopsis-Brassica orthologous regions always share
BLASTN E-values < 1e-30, while the outparalogous
regions share E-values ~ 1e-10. Excluding the identified
outparalogous regions from evaluation made the peak
Figure 4 A map of Brassica oleracea and Brassica rapa contigs anchored to Arabidopsis chromosomes. Chromosomes are arranged in
curved boxes, accompanied by gene densities (red), repetitive sequence densities (green), and distributions of overgo probes (blue ticks). The
external light-blue and green blocks show the distribution of syntenic Brassica oleracea and Brassica rapa contigs along Arabidopsis
chromosomes, respectively. Lines between chromosomes link syntenic genes in Arabidopsis, with colors distinguishing different duplicated
Wang et al. BMC Genomics 2011, 12:470
Page 6 of 15
around coverage depth 2 and 3 even more prominent
(Table 1), and the higher coverage-depth portion of Ara-
bidopsis became smaller.
DNA breakages distinguishing Brassica and Arabidopsis
To locate DNA breakages distinguishing the two species,
we divided Arabidopsis chromosomes into ‘bins’, which
were further linked to Brassica BESs to find multiple
associations of bins with different BESs. In total, we
found 39 synteny discontinuities between the two
lineages (Table 2), with 32 that imply interchromosomal
rearrangements, and 7 that imply intra-chromosomal
rearrangements. We identified tens of cases in which
paired BAC ends fell in different duplicated regions.
This added to the credibility of the analysis by showing
that the approach finds actual associations in that the
duplicated regions possibly share appreciable sequence
Heterochromatin vs. euchromatin
The chromosomal distribution of conserved Arabidop-
sis-Brassica synteny was striking, preserved almost uni-
versally in gene rich and repeat poor regions
presumably representing the Arabidopsis euchromatin,
and almost absent from the heterochromatin or pericen-
tromeric regions (Figure 4). About 14% of Arabidopsis
sequences were not covered by B. oleracea contigs,
occurring mainly in the pericentromeric regions (Figure
4). Among 1990 anchored B. oleracea contigs (excluding
the largest 5 contigs, suspected to be mosaics), 97%
(1920) could be aligned to the 104 Mb euchromatic
regions in Arabidopsis, involving 80% (2316) of
anchored probes, which may correspond to low-copy-
number genes in Brassica, and 91% (32415) of anchored
BACs. In contrast, only 6.7% of contigs, 20% of
anchored probes and 9% of anchored BACs aligned to
the 15 Mb heterochromatic regions. About 3% of B.
Table 1 Coverage depth of Brassica contigs anchored onto Arabidopsis genome sequence
Before removing ancient duplication
Coverage depth Covered length (Kb)
After removing ancient duplication
Covered length (Kb)FractionFraction
Wang et al. BMC Genomics 2011, 12:470
Page 7 of 15
oleracea contigs can be anchored to both euchromatic
and heterochromatic regions.
A total of 950 B. oleracea contigs that could not be
aligned to Arabidopsis were hypothesized to be peri-
centromere-related, based on four lines of evidence.
First, these contigs were gene-scarce, accounting for
33% of total contigs but less than 1% (25) of gene-
derived probes. Second, these 33% of contigs account
for only 16% of BACs, indicating that the underlying
BACs are relatively recalcitrant to assembly, consistent
with low DNA sequence complexity resulting from
high repetitive DNA content. Third, 46% of the BACs
were repeat-related based on their end-sequences (see
above), the same as those aligned to the Arabidopsis
heterochromatin and much higher than the 34% of
BACs aligned to the euchromatin (P-value = 0).
Fourth, we searched the BES against two Brassica-cen-
tromere-specific repeats (CentrBr1 and CentBr2, each
176 bp), and found that non-anchored contigs had a
similar abundance of centromeric elements (18%) as
known heterochromatin-aligned contigs (19%), and
much more than euchromatin-aligned contigs (10%).
Table 2 Identified breakpoints between Brassica oleracea and Arabidopsis thaliana
Bins (base pair)BAC# in Bin1Inconsistent Bins (base pair)BAC# in Bin2 BAC# in commonPaired BAC# between bins
Wang et al. BMC Genomics 2011, 12:470
Page 8 of 15
Accordingly, many of the non-anchored contigs may
Ribosomal-RNA-related contigs correspond mainly to
two pericentromeric regions on Arabidopsis chromo-
somes 2 and 3, showing possible expansion of their
related orthologous copies in Brassica. The regions cov-
ered to the greatest depths are not RNA-related but
possibly related to other repeats like transposons.
Evolution of centromeric repeats
Identified first in B. rapa , we found thousands of
CentBr1 and CentBr2 repeat sequences in the BESs
from both B. oleracea and B. rapa, which permitted us
to perform a comparative analysis of their evolution. We
hereafter refer to them as CentB1 and CentB2, since
they are not confined to B. rapa. A subset of 791 and
563 B. oleracea BESs, or 2% of the total, are CentB1-
and CentB2-related, respectively. Many B. rapa BESs
(20%) were also related to these elements, and showed
unbalanced relatedness to the two repeat classes, with
17156 and 1132 BESs related to two classes, respectively.
About 50% of the BACs in both species were related to
the same repeat class at both ends, while only a small
fraction (~0.5%) were related to different elements at
each end, suggesting a relatively separate distribution
and expansion of the two element families in the Bras-
From the BESs we retrieved 2894 and 62222
sequences of the two centromeric repeat classes and
randomly selected 100 B. oleracea sequences and 200 B.
rapa sequences for phylogenetic analysis (Figure 5). As
expected, the CentB2 repeats grouped together, forming
a subtree in which repeats from each species form two
subgroups, each clustered with repeats from the other
species. This illustrates the separate divergence and
expansion of family members in each species. The
CentB1 repeats from the two species are much more
interleaved with one another, though forming many
clusters and showing separate expansion. This phyloge-
netic distribution suggests a clear origination and initial
divergence of these repeat families in a rapa-oleracea
common ancestor. Possible cross-species gene transfer
cannot be ruled out due to the existence of many sub-
groups containing genes from both species.
Recursive polyploidizations and subsequent changes
Brassica provides an attractive system in which to study
polyploidy and its consequences, having been affected
by recursive polyploidizations including g triplication in
a common ancestor of most if not all rosids, b (< 70
mya) and a (< 32 mya) duplications in the Brassicales
after divergence from papaya, triplication (< 20 mya) in
the Brassica oleracea and B. rapa common ancestor,
and very recent duplications to form B. juncea (AABB),
B. napus (AACC), and B. carinata (BBCC). These pro-
vide good opportunities to study the relationship
between speciation and genome doubling/tripling.
Genome macro-structural changes during lineage evo-
lution can be enormous, but the types and rates of
change differ widely among lineages. For example, the
chromosome numbers of tetraploid Brassica species are
the sum of the chromosome numbers of their parental
diploids, showing no significant chromosomal changes
after genome doubling. In contrast, there have been
about 7 chromosomal fissions, fusions and merges in
the A. thaliana lineage since its divergence from A. lyr-
ata, the latter still showing near-perfect collinearity with
a member of a different genus, Capsella rubella [53-57].
Genomic resources in preparation for an outgroup,
Sisymbrium irio, may soon make it possible to deduce
the levels and patterns of change in the diploid Brassicas
since their divergence.
Gene losses after the Brassica triplication event have
been very extensive. One chromosome segment from
the rosid common ancestor would be represented in 36
copies through sequential episodes of two whole-gen-
ome triplications and two whole-genome duplications (3
× 2 × 2 × 3) in the B. oleracea (or B. rapa, or B. nigra)
genomes if all doubled/tripled copies had been pre-
served, with such a genome containing more than
400000 genes. The angiosperm genomes sequenced to
date are estimated to have about 25000 to 46000 pro-
tein-coding genes, with the largest set of predicted gene
models from soybean (46430) . All these genomes
have been affected by 1 to 3 whole-genome duplications
like Brassica. Therefore, the Brassica genomes must
have preserved only a small fraction of duplicated genes,
as reported previously . The physical map reveals a
clear impact of these recursive duplications on genome
complexity, with a web of syntenic patterns among
paleo-duplicated regions upon which the relatively
recent triplication is superimposed, making the genome
complicated to decipher.
Comparative analysis of B. napus and A. thaliana, has
been proposed to define 24 genomic blocks in the
ancestral Brassica karyotype (n = 8) . These blocks
were used to delineate the genome of B. rapa with each
block in 1-3 copies, revealing ~44 major rearrangements
during the evolution of B. rapa from the ancestral kar-
yotype. Our present analysis likewise suggests 39 syn-
teny discontinuities between B. oleracea and A. thaliana
genome sequences. Since the genomic structure of Ara-
bidopsis has been affected only by several major rearran-
gements , we predict that many of these synteny
discontinuities occurred during the evolution of B. oler-
acea and its close ancestors, perhaps mostly during a
period of genomic instability shortly after the lineage-
Wang et al. BMC Genomics 2011, 12:470
Page 9 of 15
specific whole-genome triplication. A similar analysis
was performed by mapping B. rapa BAC clones onto
the A. thaliana genome, inferring 19 inter-chromosomal
Synteny preservation and recombination
Most B. oleracea and B. rapa contigs and BACs, includ-
ing the majority of low-copy DNA hybridization probes,
could be anchored to the Arabidopsis euchromatin.
Br54 1Br120 1
Br179 1To59 1
Br191 1To48 1
Figure 5 Phylogeny of centromeric repeats in B. oleracea and B. rapa. B. oleracea repeat ids start with “To” and B. rapa repeat ids start with
“Br”. CentBr1 repeats end with “1”. CentBr2 repeats (ending with “2”) are denoted with red branches.
Wang et al. BMC Genomics 2011, 12:470
Page 10 of 15
Despite this synteny preserved between Brassica and
Arabidopsis euchromatin after 15-20 mya of divergent
evolution, pericentromeric regions tacitly assumed to be
heterochromatic appear substantially rearranged, as few
contigs can be anchored. Repetitive and centromeric
probes are enriched in the few contigs anchored here as
well as many non-anchored contigs, suggesting that the
latter belong here too. Not only is cross-species synteny
better preserved in euchromatin, but paralogous DNA
synteny produced by whole-genome duplications also
remains more evident (Figure 2). Indeed, the depth of
coverage of the Arabidopsis genome by Brassica BAC
contigs increases with distance from the Arabidopsis
pericentromeric space. An attractive future study would
be to compare on a nucleotide-for-nucleotide basis the
entire centromeric regions of Arabidopsis and Brassica
chromosomes, perhaps revealing small islands that are
preserved by selection acting on key functions
The Arabidopsis-Brassica comparison provides further
support for a model of genome evolution that has arisen
from comparison of the monocots rice and sorghum
 and is also supported by analysis of the soybean
sequence . Specifically, synteny preservation is high
and repetitive DNA abundance is low in genomic
regions where recombination is relatively frequent. In
sorghum, very recent LTR retroelement insertions are
approximately evenly distributed across the entire gen-
ome, while older insertions are largely in the hetero-
chromatin . Considering these data in view of
Muller’s ratchet , one would predict most rearrange-
ments to be slightly deleterious, in that gene arrange-
ment appears to be much more strongly preserved in
recombinogenic than non-recombinogenic regions such
as pericentromeric space .
The extensive duplicated regions in Brassica gen-
omes provide much opportunity for illegitimate
recombination, which could lead to reciprocal (cross-
ing-over) or nonreciprocal (gene conversion) DNA
information transfer, or homeologous nonreciprocal
transposition . Illegitimate recombination is often
deleterious, incurring DNA mutations, deletions, and
inversions. Gene conversion can be explained as a
“copy and paste” process, which removes the informa-
tion of one DNA segment but doubles the effect of its
homologous segment, leading to changes in expression
dosage. Illegitimate recombination has a much greater
chance to occur between relatively young duplicated
blocks , or to recur between ancient blocks that
are kept very similar by its recurrence [6,62,63]. Dif-
ferent lines of cytological evidence show that
exchanges can occur between homeologous chromo-
somes of both resynthesized and natural B. napus
[64-66]. Though the Brassica triplication event may
have occurred as much as 18 mya , evidence from
rice-sorghum comparison supports illegitimate recom-
bination between 70 million-year-old duplicated
regions. Indeed, intragenomic study of rice shows that
70-my old duplicated regions have interacted as
recently as the past 400,000 years . Therefore,
another important future study, when the required
data are available, will be to investigate the impact of
illegitimate recombination on the evolution of Brassica
genes, genomes, and species.
Toward sequencing Brassica oleracea
Recursive polyploidizations may complicate assembly
of Brassica genome sequences, especially if they are
accompanied by frequent illegitimate recombination
events that render ‘islands’ of paralogous DNA
sequence (such as genes) homogeneous. Based on our
findings herein and those in previous publications,
there are many duplicated blocks, making Brassica
genomes very complex to decipher. Though the fre-
quency of homeologous recombination per generation
is very low , its cumulative effect over many gen-
erations may be high. Gene conversion or homeolo-
gous DNA translocation could keep two homeologous
DNA segments very similar, misleading efforts to
reconstruct the evolutionary history of genes or geno-
The physical map described herein, genetically
anchored and rich in landmarks such as BAC end
sequences and hybridization data to genetically-mapped
markers, provides a valuable adjunct to efforts in pro-
gress to sequence the rapid-cycling genotype from
which the BACs were made. Moreover, efforts are also
in progress to investigate the genomic basis of the
remarkable morphological diversity among cultivated
forms that distinguishes B. oleracea from any other
plant species we are aware of. The BACs provide an
excellent bridge between the resolution that might be
accomplished by QTL fine mapping , and the identi-
fication of determinant genes.
Based on the physical map of B. oleracea, we have
done a very preliminary comparative genomics analysis
with several eudicot plants. The future availability of
whole-genome sequences from Brassica species will
further expand scope for comparative analysis and shed
light on both genome-level and single-gene-level
changes that have contributed to the evolutionary trajec-
tory of Brassica.
A genetically-anchored, sequence-rich physical map for
B. oleracea sheds light on genome evolution of Brassica-
ceae species, and provides a valuable resource toward
the assembly of genome sequences, especially using
recent short-read technologies.
Wang et al. BMC Genomics 2011, 12:470
Page 11 of 15
BAC library BOTO1, constructed from the TO1434 line,
was prepared from partial HindIII digest of Brassica
oleracea genomic DNA. The library includes a total of
87168 clones, of which 73728 were gridded and finger-
printed and used in overgo hybridization. The expected
BAC size is ~100 Kb. Clones having < 30 or > 250
bands were removed from further analysis, which
resulted in a total of 61871 clones.
Probe design and hybridization
A total of 4226 B. oleracea overgo probes were hybri-
dized to the BOTO1 BAC clone library. Overgo probes,
40 bp each, were designed from Arabidopsis gene
sequences, with 603 probes [BOVG0001-BOVG0602,
and BOVG1153] designed from markers on genetic
map, 490 probes [BOVG0603-BOVG1152] from Brassica
genomic sequences matching a-singleton genes (defined
in Bowers et al., 2003), 576 probes [BOVG1154-
BOVG1729] from Brassica genomic sequences matching
a-duplicated genes, and the remainder from an assort-
ment of other Arabidopsis genes. For probe design,
source sequences were searched with BLASTN (at most
4 mismatched sites are allowed and at least 31 bp in
length of hit region) against all known plant sequences
to find conserved domains, and compared to known
plant repeats to screen out possible repetitive sequences.
The selected sequences were then chopped into 40 bp
segments and screened for GC content of between 40%
Probes were labeled using P-32 and applied to macro-
arrays of 18,432 BACs per membrane following methods
described previously . Briefly, multiplex experiments
were done by applying 576 probes at a time, in pools of
24 probes per bottle, by rows, columns and diagonals of
a 24 × 24 array of probes. Films were manually scored,
and scores digitized using text-recognition software
(ABBYY FINEREADER). Data were deconvoluted and
stored in our locally developed MS Access database sys-
tem “BACMan”. The hybridization data were involved
to construct BAC contigs while running FPC.
The high-information-content fingerprinting (HICF)
method was adopted, together with a commercially
available SNAPshot labeling kit. Plasmids were digested
with EcoRI, BamHI, XbaI, XhoI and HhaI. The ends of
restriction fragments were differentially labeled using
fluorochrome tagged ddNTPs after the first four enzyme
cuts, and the last enzyme further reduced fragment size
and produced a blunt end. The fingerprints were gener-
ated by an ABI sequencer and size files were generated
by GeneMapper Software v4.0 after processing the
chromatograms. Only the fragments from 50 to < 600
bp were preserved for further analysis, those beyond this
range being considered unreliable.
Well-to-well contamination causes major problems in
assembly. We screened possibly contaminated wells
before assembly using a de-contamination function
implemented in FPC v9.0. A clone was inferred to have
been contaminated if it had a statistically significant
number of overlapping bands (e.g. cutoff 1e-50) with
any of its neighboring clones within a 7 × 7 square of
wells. In total, 5477 clones were inferred at a cutoff 1e-
50, and tolerance 4 to have been potentially contami-
nated, and were excluded from assembly. Well-to-well
contamination also contributed much to forming an
unexpectedly large contig.
BAC end sequencing and analysis
A subset of BACs were end-sequenced using methods
described previously , yielding 85317 BAC end
sequences (BESs) http://www.ncbi.nlm.nih.gov/. By
searching against the TIGR Brassica Repeat Database
and our extended Brassica repeats database, especially
two Brassica-centromere-specific repetitive sequences
, ‘repeat-related’ and ‘centromere-repeat-related’
BAC end sequences were identified.
Inferring RNA-, chloroplast- and mitochondrion-related
The eudicot RNA gene sequences, Arabidopsis thaliana
complete chloroplast genome sequence (AP000423.1),
and Brassica napus complete mitochondrion genome
sequence (AP006444.1), were downloaded from Gen-
Bank, against which B. oleracea BAC end sequences
were searched at E-value < 1e-5. If more than 20% of
BAC end sequences of a contig hit these specific
sequences, it was inferred to be RNA-, chloroplast- and/
Comparative analysis of Brassica rapa and B. oleracea
The previously published B. rapa contigs  were
involved in the present analysis by anchoring them to
the Arabidopsis genome sequence using 100,666 BAC
end sequences http://www.ncbi.nlm.nih.gov/.
Mapping onto Arabidopsis genomes
Contigs were anchored to Arabidopsis  genome
sequence by performing BLASTN search with BAC end
sequences and probe sequences against the genome
sequence (E-value < 1e-10 for Arabidopsis and E-value <
1e-5 for other eudicots). BAC end sequences and probe
sequences having more than 50 hits were not used in syn-
teny analysis. Syntenic regions were identified by linking
neighboring hits < = 200 Kb on Arabidopsis genome
Wang et al. BMC Genomics 2011, 12:470
Page 12 of 15
sequences (Figure 6A). We checked whether a contig can
be linked to Arabidopsis duplicated regions. A Brassica
FPC contig may be linked to multiple duplicated regions
for recursive whole genome replication events, including
a, b, and g . If all replicated copies have been preserved,
a contig may be linked to one a-orthologous region, one
a-paralogous region, two b-paralogous regions and eight
g-paralogous regions (Figure 6B). However, wide-spread
DNA losses following replication events often lead to a
degenerate pattern of correspondence. One contig may be
related to multiple Arabidopsis regions, and it is often pos-
sible distinguish orthology from paralogy if sequence simi-
larity is considered (Figure 6C). To find possible
chromosomal breakpoints distinguishing Brassica from
Arabidopsis, we searched for paired B. oleracea BESs that
hit different Arabidopsis regions (Figure 6D). The proce-
dure is similar to the one used in B. rapa and Arabidopsis
comparison . To perform the search, Arabidopsis chro-
mosomal sequences were divided into bins of selectable
sizes of 500 Kb or 1 Mb. Each bin was linked to BESs by
BLASTN at E_value < 1e-30 (a parameter used previously
), and was then systematically compared to every other
bin to check for multiple associating (i.e. with 3 or more)
pairs of BESs. Different bin sizes made little difference to
the results, indicating the stability of the approach.
We acknowledge financial support from the US Department of Agriculture
Initiative for Future Agricultural and Food Systems (00-52100-9685), and the
National Science Foundation Plant Genome Comparative Sequencing
Program (IOS 0638418), Genes and Genome Systems Program (MCB
1021718), and Advances in Biological Informatics Program (DBI 0849896).
1Plant Genome Mapping Laboratory, University of Georgia, Athens, GA
30602, USA.2The Institute for Genomic Research, 9712 Medical Center Drive,
Rockville, Maryland, 20850, USA.3Division of Biological Sciences, Life Sciences
Center, University of Missouri, Columbia, MO 65211, USA.4Center for
Genomics and Computational Biology, College of Life Sciences, and College
of Sciences, Hebei United University, Tangshan, Hebei 063000, China.
5Genomics Division, National Academy of Agricultural Science, Rural
Development Administration, 150 Suin-ro, Gwonseon-gu, Suwon 441-707,
Korea.6Department of Biochemistry, University of Wisconsin, Madison WI,
53706, USA.7Department of Plant Sciences, University of California, Davis CA,
95616, USA.8Department of Horticulture, University of Wisconsin, Madison
WI, 53706, USA (current address Monsanto, St Louis MO.9The Scientific and
Technical Council of Turkey, Genetic Engineering and Biotechnology
Institute, P.O. Box 21, 41470 Gebze, Kocaeli.
AHP and XW conceived the research. GP, CL, LN, BY, JEB, LL, EE, HS, CR, SK,
JI, EG, CFQ, and RMA performed the experiments. XW, MT, JEB, JM, YS, BP,
Figure 6 Comparative mapping of Brassica FPC contigs onto the Arabidopsis genome. In subfigures (cartoons, not based on real data) A,
C and D, Brassica contigs are displayed with assembled BAC clones (depicted by overlapping lines), and interspecific chromosomal synteny is
shown in dashed lines. A). Interspecific chromosomal synteny inference. B). A Brassica contig (shown with a hexagon shape) is expected to be
linked to multiple homologous regions in Arabidopsis (shown with circles), at most one ortholog, one a-paralog, two b-paralogs, and eight g-
paralogs. DNA losses may have removed some of them (shown with dashed-lined circles). C). A Brassica contig is linked to Arabidopsis
duplicated regions. Unbalanced synteny often permits one to distinguish between orthology and paralogy, or reveals differential gene losses
among paralogous regions. D). Inference of synteny discontinuity is shown for a Brassica contig against two Arabidopsis regions, which may
indicate a chromosomal breakpoint during the diversification of the two species.
Wang et al. BMC Genomics 2011, 12:470
Page 13 of 15
and YX performed data analysis. BM constructed the online service. AHP,
TCO, JCP, and CT led the research. XW and AHP drafted the manuscript. All
authors read and approved the manuscript.
Received: 6 May 2011 Accepted: 28 September 2011
Published: 28 September 2011
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Cite this article as: Wang et al.: A physical map of Brassica oleracea
shows complexity of chromosomal changes following recursive
paleopolyploidizations. BMC Genomics 2011 12:470.
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