Sequence and structure of Brassica rapa chromosome A3
Mun et al. Genome Biology
Sequence and structure of Brassica rapa chromosome A3
Jeong-Hwan Mun 0
Soo-Jin Kwon 0
Young-Joo Seol 0
Jin A Kim 0
Mina Jin 0
Jung Sun Kim 0
Myung-Ho Lim 0
Soo-In Lee 0
Joon Ki Hong 0
Tae-Ho Park 0
Sang-Choon Lee 0
Beom-Jin Kim 0
Mi-Suk Seo 0
Seunghoon Baek 0
Min-Jee Lee 0
Ja Young Shin 0
Jang-Ho Hahn 0
Jee Young Park
Su Ryun Choi
Yong Pyo Lim
Andrew G Sharpe
Isobel AP Parkin
Beom-Seok Park 0
0 Department of Agricultural Biotechnology, National Academy of Agricultural Science, Rural Development Administration , 150 Suin-ro, Gwonseon-gu, Suwon 441-707 , Korea
Background: The species Brassica rapa includes important vegetable and oil crops. It also serves as an excellent model system to study polyploidy-related genome evolution because of its paleohexaploid ancestry and its close evolutionary relationships with Arabidopsis thaliana and other Brassica species with larger genomes. Therefore, its genome sequence will be used to accelerate both basic research on genome evolution and applied research across the cultivated Brassica species. Results: We have determined and analyzed the sequence of B. rapa chromosome A3. We obtained 31.9 Mb of sequences, organized into nine contigs, which incorporated 348 overlapping BAC clones. Annotation revealed 7,058 protein-coding genes, with an average gene density of 4.6 kb per gene. Analysis of chromosome collinearity with the A. thaliana genome identified conserved synteny blocks encompassing the whole of the B. rapa chromosome A3 and sections of four A. thaliana chromosomes. The frequency of tandem duplication of genes differed between the conserved genome segments in B. rapa and A. thaliana, indicating differential rates of occurrence/retention of such duplicate copies of genes. Analysis of 'ancestral karyotype' genome building blocks enabled the development of a hypothetical model for the derivation of the B. rapa chromosome A3. Conclusions: We report the near-complete chromosome sequence from a dicotyledonous crop species. This provides an example of the complexity of genome evolution following polyploidy. The high degree of contiguity afforded by the clone-by-clone approach provides a benchmark for the performance of whole genome shotgun approaches presently being applied in B. rapa and other species with complex genomes.
The Brassicaceae family includes approximately 3,700
species in 338 genera. The species, which include the widely
studied Arabidopsis thaliana, have diverse characteristics
and many are of agronomic importance as vegetables,
condiments, fodder, and oil crops . Economically, Brassica
species contribute to approximately 10% of the worlds
vegetable crop produce and approximately 12% of the
worldwide edible oil supplies . The tribe Brassiceae,
which is one of 25 tribes in the Brassicaceae, consists of
approximately 240 species and contains the genus
Brassica. The cultivated Brassica species are B. rapa (which
contains the Brassica A genome) and B. oleracea (C
genome), which are grown mostly as vegetable cole crops,
B. nigra (B genome) as a source of mustard condiment,
and oil crops, mainly B. napus (a recently formed
allotetraploid containing both A and C genomes), B. juncea (A and
B genomes), and B. carinata (B and C genomes) as
sources of canola oil. These genome relationships between
the three diploid species and their pairwise allopolyploid
derivative species have long been known, and are
described by Us triangle .
B. rapa is a major vegetable or oil crop in Asia and
Europe, and has recently become a widely used model for
the study of polyploid genome structure and evolution
because it has the smallest genome (529 Mb) of the
Brassica genus and, like all members of the tribe Brassiceae,
has evolved from a hexaploid ancestor [4-6]. Our previous
comparative genomic study revealed conserved linkage
arrangements and collinear chromosome segments
between B. rapa and A. thaliana, which diverged from a
common ancestor approximately 13 to 17 million years
ago. The B. rapa genome contains triplicated
homoeologous counterparts of the corresponding segments of the
A. thaliana genome due to triplication of the entire
genome (whole genome triplication), which occurred
approximately 11 to 12 million years ago . Furthermore, studies
in B. napus, which was generated in the last 10,000 years,
have demonstrated that overall genome structure is highly
conserved compared to its progenitor species, B. rapa and
B. oleracea, which diverged approximately 8 million years
ago, but significantly diverged relative to A. thaliana at the
sequence level [7,8]. Thus, investigation of the B. rapa
genome provides substantial opportunities to study the
divergence of gene function and genome evolution
associated with polyploidy, extensive duplication, and
hybridization. In addition, access to a complete and
highresolution B. rapa genome will facilitate research on other
Brassica crops with partially sequenced or larger genomes.
Despite the importance of Brassica crops in plant
biology and world agriculture, none of the Brassica species
have had their genomes fully sequenced. Cytogenetic
analyses have showed that the B. rapa genome is
organized into ten chromosomes, with genes concentrated in
the euchromatic space and centromeric repeat sequences
and rDNAs arranged as tandem arrays primarily in the
heterochromatin [9,10]. The individual mitotic
metaphase chromosome size ranges from 2.1 to 5.6 m, with
a total chromosome length of 32.5 m . An alternative
cytogenetic map based on a pachytene DAPI
(4,6-diamidino-2-phenylindole dihydrochloride) and fluorescent
in situ hybridization (FISH) karyogram showed that the
mean lengths of ten pachytene chromosomes ranged
from 23.7 to 51.3 m, with a total chromosome length of
385.3 m . Thus, chromosomes in the meiotic
prophase stage are 12 times longer than those in the mitotic
metaphase, and display a well-differentiated pattern of
bright fluorescent heterochromatin segments.
Sequencing of selected BAC clones has confirmed that the gene
density in B. rapa is similar to that of A. thaliana in the
order of 1 gene per 3 to 4 kb . Each of the gene-rich
BAC clones examined so far by FISH (> 100 BACs) was
found to be localized to the visible euchromatic region of
the genome. Concurrently, a whole-genome shotgun
pilot sequencing of B. oleracea with 0.44-fold genome
coverage generated sequences enriched in transposable
elements [12,13]. Taken together, these data strongly
point to a tractable genome organization where the
majority of the B. rapa euchromatic space (gene space)
can be sequenced in a highly efficient manner by a
cloneby-clone strategy. Based on these results, the
multinational Brassica rapa Genome Sequencing Project
(BrGSP) was launched, with the aim of sequencing the
euchromatic arms of all ten chromosomes . The
project aimed to initially produce a phase 2 (fully oriented
and ordered sequence with some small gaps and low
quality sequences) sequence with accessible trace files by
shotgun sequencing of clones so that researchers who
require complete sequences from a specific region can
To support genome sequencing, five large-insert BAC
libraries of B. rapa ssp. pekinensis cv. Chiifu were
constructed, providing approximately 53-fold genome
coverage overall . These libraries were constructed
using several different restriction endonucleases to
cleave genomic DNA (EcoRI, BamHI, HindIII, and
Sau3AI). Using these BAC libraries, a total of 260,637
BAC-end sequences (BESs) have been generated from
146,688 BAC clones (approximately 203 Mb) as a
collaborative outcome of the multinational BrGSP
community. The strategy for clone-by-clone sequencing was to
start from defined and genetically/cytogenetically
mapped seed BACs and build outward. Initially, a
comparative tiling method of mapping BES onto the A.
thaliana genome, combined with fingerprint-based
physical mapping, along with existing genetic anchoring
data provided the basis for selecting seed BAC clones
and for creating a draft tiling path [6,16,17]. As a result,
589 BAC clones were sequenced and provided to the
BrGSP as seed BACs for chromosome sequencing.
Integration of seed BACs with the physical map provided
gene-rich contigs spanning approximately 160 Mb.
These gene-rich contigs enabled the selection of clones
to extend the initial sequence contigs. Here, as the first
report of the BrGSP, we describe a detailed analysis of
B. rapa chromosome A3, the largest of the ten B. rapa
chromosomes, as assessed by both cytogenetic analysis
and linkage mapping (length estimated as 140.7 cM).
The A3 linkage group also contains numerous
collinearity discontinuities (CDs) compared with A. thaliana, a
recent study into which  revealed greater complexity
than originally described for the segmental collinearity
of Brassica and Arabidopsis genomes [19,20]. In
accordance with the agreed standards of the BrGSP, we
aimed to generate phase 2 contiguous sequences for
B. rapa chromosome A3. We annotated these sequences
for genes and other characteristics, and used the data to
analyze genome composition and examine consequential
features of polyploidy, such as genome rearrangement.
Results and discussion
General features of chromosome A3
Chromosome A3 is acrocentric, with a heterochromatic
upper (short) arm bearing the nucleolar organizer region
(NOR) and a euchromatic lower (long) arm (Figure 1a).
The NOR comprises a large domain of 45S rDNA
repeats and a small fraction of 5S rDNA repeats
extending to the centromere. The centromere of chromosome
A3 is typically characterized by hybridization of the
176bp centromeric tandem repeat CentBr2, which resides
on only chromosomes A3 and A5 . The euchromatic
region of chromosome A3, the lower arm, has been
measured as 45.5 m in pachytene FISH (Figure 1b).
The sequence length of the lower arm from centromere
to telomere was estimated to be approximately 34 to 35
Mb based on measurement of the average physical
Figure 1 Features of B. rapa chromosome A3. (a) Mitotic metaphase structure of chromosome A3 with FISH signals of 45S (red), 5S (green)
rDNAs, and CentBr2 (magenta). (b) Image of DAPI-stained pachytene spread of chromosome A3 showing the heterochromatic NORs of the
short arm (bright blue) and euchromatic long arm (blue). (c) VCS (cv. VC1 cv. SR5) genetic map showing the positions of the BAC clones found
nearest the end of each contig. (d) Physical map showing the location of nine sequence contigs (blue). The chromosome is roughly 34.2 Mb
long, spans a genetic map distance of 140.7 cM with 243 kb/cM, and contains 6.4% of the unique sequence of the B. rapa genome. The
centromere is shown as a pink circle, the NOR of the rDNA repeat region in the short arm is represented as a brown bar, and telomeres are
light blue. The telomere, centromere, and NOR are not drown to scale. The sizes of eight unsequenced gaps measured by pachytene FISH are
given in kilobases. Red areas in (b, d) point to the position of the hybridization signal of KBrH34P23 in sequence contig 8.
length of sequenced contigs (1 m/755 kb).
Chromosome sequencing was initiated using BAC clones that
had been anchored onto the lower arm of chromosome
A3 by genetic markers. Subsequently, BES and physical
mapping of chromosome A3 allowed extension from
these initial seed points and completion of the entire
lower arm. However, no BAC clones were identified
from the upper arm, possibly owing to the lack of
appropriate restriction enzyme sites in these regions, the
instability of the sequences in Escherichia coli or a
complete lack of euchromatic sequences on that arm.
A total of 348 BAC clones were sequenced from the
lower arm of chromosome A3 to produce 31.9 Mb of
sequences of phase 2 or phase 3 (finished sequences)
standard. These were assembled into nine contigs that
span 140.7 cM of the genetic map (Figures 1c, d; Figure
S1 in Additional file 1). The lower arm sequence starts
at the proximal clone KBrH044B01 and terminates at
the distal clone KBrF203I22 (Table S1 in Additional file
2). Excluding the gaps at the centromere and telomere,
the pachytene spread FISH indicated that eight physical
gaps, totaling approximately 2.3 Mb, remain on the
pseudochromosome sequence. Despite extensive efforts,
no BACs could be identified in those regions. The total
length of the lower arm, from centromere to telomere,
was therefore calculated to be 34.2 Mb. Thus, the 31.9
Mb of sequences we obtained represents 93% of the
lower arm of the chromosome. The sequence and
annotation of B. rapa chromosome A3 can be found in
GenBank (see Materials and methods).
Characterization of the sequences
The distribution of genes and various repetitive DNA
elements along chromosome A3 are depicted in Figure
2, with details of the content of repetitive sequences
provided in Table S2 in Additional file 2. Overall, 11%
of the sequenced region in chromosome A3 is
composed of repetitive sequences, which are dispersed over
the lower arm. The distribution of repetitive sequences
along the chromosome was not even, with fewer
retrotransposons (long terminal repeats) and DNA
transposons towards the distal end. In addition, low complexity
repetitive sequences are relatively abundant in the lower
arm, indicating B. rapa-specific expansion of repetitive
sequences. These are the most frequently occurring
class of repetitive elements, accounting for 41% of the
total amount of repetitive sequence elements. Other
types of repeat do not show obvious clustering except
satellite sequences around 22 Mb from the centromere.
These sequences have high sequence similarity to a
350bp AT-rich tandem repeat of B. nigra .
Gene structure and density statistics are shown
in Table 1. The overall G+C content of chromosome
A3 is 33.8%, which is less than was reported for the
euchromatic seed BAC sequences (35.2%)  and
the entire A. thaliana genome (35.9%) . Gene
annotation was carried out using our specialized B. rapa
annotation pipeline. This modeled a total of 7,058
protein-coding genes, of which 1,550 have just a single
exon. On average, each gene model contains 4.7 exons
and is 1,755 bp in length. Consistent with the results of
more restricted studies , the average length of gene
models annotated on chromosome A3 is shorter than
those of A. thaliana genes due to reduction in both
exon number per gene and exon length. The average
gene density is 4,633 bp per gene, which is also lower
than in A. thaliana (4,351 bp per gene), indicating a
slightly less compact genome organization. The longest
gene model, which is predicted to encode a potassium
ion transmembrane transporter, consists of 8 exons
across 31,311 bp.
Potential alternative splicing variants, based upon a
minimum requirement for three EST matches, was
identified for only 2.3% of the gene models. This finding
suggests that alternative splicing may be rarer in B. rapa
than it is in A. thaliana, where it occurs at a frequency
of 16.9% . Additional EST data will enable more
precise identification of alternative spliced variants on the
B. rapa genome.
We identified 5,825 genes as known based upon EST
matches, protein matches, or any detectable domain
signatures. The remaining 1,417 predicted genes were
assigned as unknown or hypothetical. The functions of
known genes were classified according to Gene
Ontology (GO) analysis (Figure 3). We compared the results
of GO-based classification of gene models from
chromosome A3 with a similar analysis of gene models from
the 65.8 Mb of genome-wide seed BAC sequences .
This revealed several categories for which the functional
complement of genes on chromosome A3 is atypical of
the genome as a whole. For example, it has higher
proportions of genes classified as related to stress or
developmental process under the GO biological process
category compared to the collection of seed BAC
sequences (P < 0.0001). In addition, there are differences
in terms pertaining to membrane related genes and
chloroplast of the GO cellular component category
between the two data sets (P < 0.2).
The predicted proteins found on chromosome A3
were categorized into gene families by BLASTP (using a
minimum threshold of 50% alignment coverage at a
cutoff of E-10). The chromosome contains 384 families of
tandemly duplicated genes with 1,262 members,
comprising 17.9% of all genes (Figure S2 in Additional file
1). This is lower than found in A. thaliana, which has
27% of genes existing as tandem duplicates in the
genome. The most abundant gene family was the protein
kinase family, with 249 members, followed by F-box
Figure 2 Distribution of various repeats and features on chromosome A3. The long arm of chromosome A3 is shown on the x-axis and is
numbered from the beginning of contig 1 to the end of contig 9 by joining up the physical gaps. The y-axis represents genes, ESTs, and the
various repeats plotted relative to the nucleotide position on the chromosome. The densities of genes, ESTs, and the repeats were obtained by
analyzing the sequence every 100 kb using a 10-kb sliding window. LINE, long interspersed nuclear element.
Table 1 Statistics of B. rapa chromosome A3
B. rapa chromosome A3
A. thaliana whole genome
The B. rapa chromosome A3 statistics were generated in this study. The Arabidopsis genome features are from The Arabidopsis Information Resource database
(release TAIR9) .
proteins (170 members) and transcription factors (143
members). These families are distributed throughout the
chromosome (Figure 4). The highest number of tandem
duplicates detected at a single site was a cluster of 18
copies of the cysteine-rich receptor-like protein kinase
gene family, located around coordinate 7 Mb.
The chromosome contains 164 tRNAs and 3 small
nuclear RNAs. The tRNAs are evenly distributed along
chromosome A3 except for one region where they
cluster. This cluster, at 23.9 Mb, contains 12 tandem
tRNAPro genes, which are the most abundant tRNA genes on
the chromosome (Figure S3 in Additional file 1). A
tRNAPro cluster was previously detected also on A.
thaliana chromosome 1 . A computational search
coupled with prediction of secondary structure using
reported mature microRNA (miRNA) sequences
identified 26 miRNA genes, which outnumber the total
number of B. rapa (17) recorded in miRBase (release 15.0;
April 2010; Table S3 in Additional file 2). Abundant
miRNAs on chromosome A3 included miR2111 and
miR399. These have been implicated in regulating
nutritional balance in B. rapa based upon observation of
their induction during phosphate limitation in A.
thaliana and rapeseed [25,26].
A sequence similarity search showed that 2.5% of the
genes identified on chromosome A3 are of mitochondrial
(98 genes) or chloroplast (78 genes) origin. The
widespread distribution observed for organellar insertions
across the chromosome indicates that mitochondrial and
chloroplast gene transfer occurred independently.
Synteny between chromosome A3 and the A. thaliana
To investigate detailed syntenic relationships between
chromosome A3 and the five chromosomes of A.
thaliana, we compared the proteomes predicted from the two
genomes using BLASTP analysis (Table S4 in Additional
file 2). Approximately 75.4% of the genes of chromosome
A3 have similarity to genes in the A. thaliana genome.
Figure 5 represents a dot matrix plot showing the
largescale blocks of collinearity between the two genomes.
The collinearity blocks, identified by the red dots, extend
the whole length of chromosome A3 and correspond to
parts of four A. thaliana chromosomes (2, 3, 4, and 5) in
a mosaic pattern. The collinearity blocks contain 6,551
gene models in B. rapa and 12,783 gene models in
A. thaliana. Comparative analysis showed that 79.7% of
gene models on chromosome A3 show similarity with
Figure 3 Functional classification of the proteins encoded on chromosome A3 or seed BAC sequences through annotation using Gene
Ontology. Assignments are based on the annotations to terms in the GO biological process, cellular component, and molecular function categories.
counterparts in the collinear A. thaliana genome
segments, whereas only 32.4% of A. thaliana genes show
similarity with counterparts on chromosome A3. This is
indicative of extensive and interspersed gene loss from
B. rapa since divergence of the Brassica and Arabidopsis
lineages, as described previously [5,27,28]. We found
little evidence to support the presence of paralogous
segments on chromosome A3 using self-syntenic
comparison (Figure S4 in Additional file 1).
Recombination and evolution of chromosome A3
Comparison of chromosome sequences between B. rapa
chromosome A3 and A. thaliana allows complete
mapping of the inferred ancient karyotype (AK) genome
building blocks. According to genome mapping of AK
blocks on the A. thaliana genome [20,29] and pairwise
information for chromosome A3 and A. thaliana genome
collinearity blocks, we defined conserved AK genome
building blocks with pairwise boundary delineations of
each block on the two genomes (Figure 6; Table S4 in
Additional file 2). The order and boundaries of AK
blocks on chromosome A3 were fundamentally similar to
those of our previous report using seed BAC sequences
. Chromosome A3 is highly rearranged relative to A.
thaliana chromosomes and compared with the AK.
Overall, 14 blocks derived from 6 AK chromosomes
(AK3, AK4, AK5, AK6, AK7, and AK8) were aligned with
chromosome A3. All the AK blocks on chromosome A3
were shorter than those on the A. thaliana genome and
seven CD regions were found between the blocks,
suggesting that a complicated recombination of six AK
chromosomes resulted in the emergence of chromosome A3.
The combined analysis of AK mapping and
identification of CDs on chromosome A3 enable us to hypothesize
Figure 4 Distribution patterns of the top six gene categories on chromosome A3. Width of the vertical bars is proportional to the number
of genes located at that position.
how parts of this chromosome have evolved from the AK.
One hypothetical model for the reconstruction of the
chromosome from the AK is presented in Figure 7.
Chromosome A3 appears to have been derived from at least
six AK chromosomes that were recombined in the
progenitor of B. rapa by genome rearrangements, including
inversion, translocation, fusion, and recombination. The
detection of sequences from the W block of AK8 at both
ends of the AK4 block indicates that there might have
been a circular intermediate derived from fusion
chromosome AK8/4 that was then integrated into AK6.
Rearrangement of the AK seems to have taken place in the
Figure 5 Synteny between B. rapa chromosome A3 and the A. thaliana genome. Chromosome correspondence between the genomes is
represented by a dot-plot. Each dot represents a reciprocal best BLASTP match between gene pairs at an E value cutoff of < E-20. Red dots
show regions of synteny with more than 50% gene conservation as identified by DiagHunter. Color bars on the upper and left margins of the
dot plot indicate individual chromosomes of A. thaliana and B. rapa, respectively, demonstrating corresponding similarity. Black dots on the
chromosomes are centromeres. Color bars on the bottom and right margins of the dot plot show ancestral karyotype genome building blocks
mapped on the reduced karyotypes of A. thaliana and B. rapa, respectively. Bars of the same color are putative homologous counterparts.
Figure 6 Genome building blocks and block boundaries of the
ancestral karyotype mapped onto B. rapa chromosome A3. The
position of AK genome building blocks in chromosome A3 was
defined by a comparison of B. rapa-A. thaliana syntenic relationships
and the A. thaliana-AK mapping results [20,29]. AK segments are
labeled and oriented by arrows. Putative orthologs delineating the
boundaries of recombination events are designated. CDs between
AK blocks are indicated by dotted arrows. CEN, centromere.
B. rapa genome after whole genome triplication, as none
of the other chromosomes in the B. rapa genome show a
Figure 7 Hypothetical derivation of chromosome A3. Chromosome
A3 has originated due to inversion (i), translocation (t), fusion (f), and
recombination (r) of six AK chromosomes (AK3, AK4, AK5, AK6, AK7, and
AK8). The ancestral chromosomes are presumed to bear NORs (black
rectangles) and centromeres are represented as empty spheres. The
minichromosomes consisting of a NOR and a centromere that resulted
from translocation events have presumably been lost.
similar arrangement of AK blocks. Furthermore, this
study suggests that rearrangement events were involved
in reduction of the basic chromosome number of B. rapa
to ten. It remains uncertain, however, which group of
linked events occurred earlier or later because multiple
rounds of polyploidy followed by complex genome
recombination yielded the current chromosome structure
of B. rapa.
Polyploid ancestry greatly complicates efforts to sequence
genomes because of the presence of related sequences.
Nevertheless, we have successfully sequenced, almost in
its entirety, the largest chromosome of B. rapa, A3, using
a clone-by-clone strategy. Annotation of the 31.9 Mb of
sequences representing the gene space of chromosome
A3 resulted in the development of models for 7,058
protein-coding genes and revealed the gene density to be
only slightly lower than that observed for the related
species A. thaliana, which is considered to have an
exceptionally compact genome . Comparative
analysis of collinear genome segments with A. thaliana
revealed extensive chromosome-wide interspersed gene
loss from B. rapa since divergence of the Brassica and
Arabidopsis lineages, as described previously only for
small genomic regions [5,27,28]. The alignment of
genome segments that the whole chromosome sequence
permitted, relative to both the A. thaliana genome and
the inferred AK of a common progenitor of Brassica and
Arabidopsis, enabled the development of a model for the
derivation of chromosome A3. The results confirm that
the complete genome sequence of B. rapa, provided that
it is of an appropriate standard, will have a major impact
on comparative genomics and gene discovery in Brassica
Materials and methods
The B. rapa chromosome A3 was sequenced using a
clone-by-clone sequencing strategy with a BAC-based
physical map framework that was genetically anchored to
the B. rapa genome . We sequenced chromosome A3
of B. rapa ssp. pekinensis cultivar Chiifu from 348
overlapping BAC clones. Initially, we isolated seed BAC
clones using a comparative BES tiling method and
sequenced them by shotgun sequencing . Seed BAC
clones were then extended in both directions by
searching for sequence identity in the BES database, which was
then cross-examined with a physical map constructed
using the KBrH, KBrB, and KBrS1 BAC libraries . We
also used KBrE and KBrS2 BAC libraries for additional
extension and gap filling in particular. We carried out
shotgun sequencing of the BAC clones to generate
sequence data with eight- to ten-fold coverage of each
clone using the ABI3730l sequencer (Applied
Biosystems, Foster City, CA, USA). According to the BrGSP
, the minimal sequence goal was five phase 2 contigs.
Individual BACs were assembled from the shotgun
sequences using the PHRED/PHRAP [31,32] and the
Consed  programs. The sequence contig assembly
was created based on overlapping sequences using
Sequencher (Gene Codes, Ann Arbor, MI, USA)
program. To evaluate the accuracy of the assembly,
alignment of EST unigenes, PCR amplification of the
assembled sequences, and sequence comparison with
fosmid clone links were performed. Contigs were ordered
using sequence tagged site markers mapping to the long
arm of the chromosome using VCS and Jangwon linkage
maps , followed by estimation of non-overlapping
gaps between contigs based on the results of FISH
experiments. Pseudochromosome sequences were
created by connecting sequence contigs with addition of
filler sequences according to the estimated gap size; 10 k
addition for gap sizes < 100 kb or 100 k addition for gap
sizes > 100 kb. All the sequence information has been
deposited in the National Center for Biotechnology and
Information (NCBI) with accession numbers [NCBI:
AC189184] to [NCBI:AC241201] (Table S1 in Additional
We carried out gene prediction using our in-house
automated gene prediction system . The assembled
sequences were masked using RepeatMasker  based
on a dataset combining the plant repeat element database
of The Institute for Genomic Research , Munich
Information Center for Protein Sequences , and our
specialized database of B. rapa repetitive sequences.
Gene model prediction was performed using
EVidenceModeler . Putative exons and open reading frames
(ORFs) were predicted ab initio using FGENESH ,
AUGUSTUS , GlimmerHMM , and SNAP 
programs with the parameters trained using the B. rapa
matrix. Putative gene splits predicted on the unfinished
gaps were removed. To predict consensus gene
structures, 152,253 B. rapa ESTs plus full-length cDNAs we
have generated, A. thaliana coding sequences (release
TAIR9), plant transcripts, and plant protein sequences
were aligned to the predicted genes using PASA  and
AAT  packages. The predicted genes and evidence
sequences were then assembled according to the weight
of each evidence type using EVidenceModeler. The
highest scoring set of connected exons, introns, and
noncoding regions was selected as a consensus gene model.
Proteins encoded by gene models were searched against
the Pfam database  and automatically assigned a
putative name based on conserved domain hits or
similarity with previously identified proteins. Annotated gene
models were also searched against a database of plant
transposon-encoded proteins . Predicted proteins
with a top match to transposon-encoded proteins were
excluded from the annotation and gene counts. Transfer
RNAs were identified using tRNAscan-SE . To scan
miRNA genes, the nonredundant miRNA sequences in
miRBase v15 were mapped using BLASTN (up to two
mismatches) . A search of potential precursor
structures was performed by extracting the genomic context
(400 bp upstream and downstream) surrounding the
position of the miRNA sequence predicted and by
analyzing those regions with Vienna RNA package .
Only the putative pre-miRNA precursors with a folding
energy lower than -20 kcal/mol were selected. Organellar
insertions were determined using BLASTN with the A.
thaliana mitochondrion and the B. rapa chloroplast
genome sequence using a cutoff of 95% identity plus 90%
Comparative genome analysis
Syntenic regions between chromosome A3 of B. rapa and
the A. thaliana genome were identified by a proteome
comparison based on BLASTP analysis . The entire
proteomes of the two genomes were compared, and only
the top reciprocal BLASTP matches per chromosome
pair were selected (minimum of 50% alignment coverage
at a cutoff of < E-20). Chromosome scale synteny blocks
were inferred by visual inspection of dot-plots using
DiagHunter with parameters as described in the previous
reports [6,49]. At least four genes with the same
respective orientations in both genomes were required to
establish a primary candidate synteny block. To distinguish
highly homologous real synteny blocks from false
positives due to multiple rounds of polyploidy followed by
genome rearrangement, we manually evaluated the
degree of gene conservation in all the primary candidate
blocks and selected real syntenic regions showing a gene
conservation index of greater than 50% (the number of
conserved matches divided by the total number of genes
in the blocks). Self comparison of chromosome A3 with
other chromosomes of the B. rapa genome was also
conducted using seed BAC sequences .
Additional file 1: Figures S1, S2, S3, and S4. Figure S1: genetic versus
physical distance on chromosome A3. The genetic map was constructed
using the VCS population. Figure S2: frequency distribution of genes in
multigene families with tandem duplicated paralog arrangements.
Tandem duplicated paralogs on chromosome A3 were identified using
BLASTP analysis with a minimum threshold of 50% alignment coverage
at a cutoff of E-10 in a 100-kb window interval. Figure S3: clusters of
tRNAPro genes on chromosome A3. The tRNAPro repeat clusters at 23.68
Mb is located on BAC clone KBrH72P15. Figure S4: dot plot of
chromosome A3 compared with itself. Each dot in the dot plot
represents a reciprocal best BLASTP match between gene pairs at a
cutoff value of < E-20. Black dots show the regions of synteny identified
Additional file 2: Tables S1, S2, S3, and S4. Table S1: summary of
sequence contigs along with constituent BAC associations on minimum
tiling path for chromosome A3. Table S2: comparison of repetitive
sequences identified on chromosome A3 and seed BAC sequences of B.
rapa. Table S3: miRNAs identified on chromosome A3. Table S4: synteny
alignment between B. rapa chromosome A3 and the A. thaliana genome
along with mapping of AK genome building blocks.
AK: ancestral karyotype; BAC: bacterial artificial chromosome; BES: BAC-end
sequence; bp: base pair; BrGSP: Brassica rapa Genome Sequencing Project;
CD: collinearity discontinuity; DAPI: 4:6-diamidino-2-phenylindole
dihydrochloride; EST: expressed sequence tag; FISH: fluorescent in situ
hybridization; GO: Gene Ontology; kb: kilobase; miRNA: microRNA; NOR:
nucleolar organizer region.
We thank the many participants in the Korean Brassica rapa Genome Project
and Dr Xiaowu Wang of IVF, China for discussion. This work was supported
by the National Academy of Agricultural Science (05-1-12-2-1 and PJ006759)
and the BioGreen 21 Program (20050301034438), Rural Development
Administration, Korea, the UK Biotechnology and Biological Sciences
Research Council (BB/E017363), and the Australian Research Council (Projects
LP0882095 and LP0883462).
JHM conceived the project, designed research, analyzed data, and wrote the
manuscript. SJK designed research, performed the experiments, and
analyzed data. JHM, SJK, JAK, MHL, SIL, JKH, THP, SCL, MJL, JYP, JL, TJY, and
IYC contributed to shotgun sequencing, sequence assembly, and data
acquisition. MJ and JSK performed genetic mapping. YJH and KBL
contributed to FISH. YJS and JHH contributed to annotation and database
development. YJS, BJK, SB, JYS, MSS, HJY, and BSC analyzed data. SRC, NR,
YPL, FF, ND, ES, MT, IB, AGS, IAPP, JB, and DE participated in BAC-end
sequencing. HJY and IB participated in manuscript preparation. BSP
conceived the project.
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