The genome of the mesopolyploid crop species Brassica rapa.

Abstract

We report the annotation and analysis of the draft genome sequence of Brassica rapa accession Chiifu-401-42, a Chinese cabbage. We modeled 41,174 protein coding genes in the B. rapa genome, which has undergone genome triplication. We used Arabidopsis thaliana as an outgroup for investigating the consequences of genome triplication, such as structural and functional evolution. The extent of gene loss (fractionation) among triplicated genome segments varies, with one of the three copies consistently retaining a disproportionately large fraction of the genes expected to have been present in its ancestor. Variation in the number of members of gene families present in the genome may contribute to the remarkable morphological plasticity of Brassica species. The B. rapa genome sequence provides an important resource for studying the evolution of polyploid genomes and underpins the genetic improvement of Brassica oil and vegetable crops.

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Brassica nigra (B genome) and B. oleracea (C genome) having formed
the amphidiploid species B. juncea (A and B genomes), B. napus
(A and C genomes) and B. carinata (B and C genomes) by hybridiza-
tion. Comparative physical mapping studies have confirmed genome
triplication in a common ancestor of B. oleracea11 and B. rapa12 since
its divergence from the A. thaliana lineage at least 13–17 MYA6,7,13.
Using 72× coverage of paired short read sequences generated by
Illumina GA II technology and stringent assembly parameters, we
assembled the genome of the B. rapa ssp. pekinensis line Chiifu-401-42
and analyzed the assembly (Online Methods and Supplementary Note).
The final assembly statistics are summarized in Table 1. The assembled
sequence of 283.8 Mb was estimated to cover >98% of the gene space
(Supplementary Table 1) and is greater than the previous estimated
size of the euchromatic space, 220 Mb14. The assembly showed excellent
agreement with the previously reported chromosome A03 (ref. 15) and
with 647 bacterial artificial chromosomes (BACs)14 (Online Methods)
sequenced by Sanger technology. Integration with 199,452 BAC-end
sequences produced 159 super scaffolds representing 90% of the assem-
bled sequences, with an N50 scaffold (N50 scaffold is a weighted median
statistic indicating that 50% of the entire assembly is contained in scaf-
folds equal to or larger than this value) size of 1.97 Mb. Genetic mapping
of 1,427 markers in B. rapa allowed us to produce ten pseudo chromo-
somes that included 90% of the assembly (Supplementary Table 2).
We found the difference in the physical sizes of the A. thaliana
and B. rapa genomes to be largely because of transposable elements
(Supplementary Table 3). Although widely dispersed throughout the
genome, as shown in Figure 1, the transposon-related sequences were
most abundant in the vicinity of the centromeres. We estimated that
transposon-related sequences occupy 39.5% of the genome, with the
proportions of retrotransposons (with long terminal repeats), DNA
transposons and long interspersed elements being 27.1%, 3.2% and
2.8%, respectively (Supplementary Tables 4 and 5).
We modeled and analyzed protein coding genes (described in the
Online Methods and the Supplementary Note). We identified 41,174
protein coding genes, distributed as shown in Figure 1. The gene models
have an average transcript length of 2,015 bp, a coding length of 1,172
bp and a mean of 5.03 exons per gene, both similar to that observed in
A. thaliana16. A total of 95.8% of gene models have a match in at least
one of the public protein databases and 99.3% are represented among
the public EST collections or de novo Illumina mRNA-Seq data. Among
the total 16,917 B. rapa gene families, only 1,003 (5.9%) appear to be
lineage specific, with 15,725 (93.0%) shared with A. thaliana16 and 9,909
(58.6%) also shared by Carica papaya17 and Vitis vinifera18 (Fig. 2).
The genome of the mesopolyploid crop species Brassica rapa
The Brassica rapa Genome Sequencing Project Consortium
We report the annotation and analysis of the draft genome 
sequence of Brassica rapa accession Chiifu-401-42, a Chinese 
cabbage. We modeled 41,174 protein coding genes in the  
B. rapa genome, which has undergone genome triplication.  
We used Arabidopsis thaliana as an outgroup for investigating 
the consequences of genome triplication, such as structural 
and functional evolution. The extent of gene loss (fractionation) 
among triplicated genome segments varies, with one of the 
three copies consistently retaining a disproportionately large 
fraction of the genes expected to have been present in its 
ancestor. Variation in the number of members of gene families 
present in the genome may contribute to the remarkable 
morphological plasticity of Brassica species. The B. rapa 
genome sequence provides an important resource for studying 
the evolution of polyploid genomes and underpins the genetic 
improvement of Brassica oil and vegetable crops.
Model species have provided valuable insights into angiosperm
(flowering plant) genome structure, function and evolution. For example,
A. thaliana has experienced two genome duplications since its divergence
from Carica, with rapid DNA sequence divergence, extensive gene loss
and fractionation of ancestral gene order eroding the resemblance of
A. thaliana to ancestral Brassicales1. Compared with an ancestor at just
a few million years ago, A. thaliana has undergone a ~30% reduction in
genome size2 and 9–10 chromosomal rearrangements3,4 that differentiate
it from its sister species Arabidopsis lyrata. Whole-genome duplication
has been observed in all plant genomes sequenced to date. A. thaliana has
undergone three paleo-polyploidy events5: a paleohexaploidy (γ) event
shared with most dicots (asterids and rosids) and two paleotetraploidy
events (β then α) shared with other members of the order Brassicales.
B. rapa shares this complex history but with the addition of a whole-
genome triplication (WGT) thought to have occurred between 13 and
17 million years ago (MYA)6,7, making ‘mesohexaploidy’ a characteristic
of the Brassiceae tribe of the Brassicaceae8.
Brassica crops are used for human nutrition and provide opportuni-
ties for the study of genome evolution. These crops include important
vegetables (B. rapa (Chinese cabbage, pak choi and turnip) and Brassica
oleracea (broccoli, cabbage and cauliflower)) as well as oilseed crops
(Brassica napus, B. rapa, Brassica juncea and Brassica carinata), which
provide collectively 12% of the world’s edible vegetable oil production9.
The six widely cultivated Brassica species are also a classical example
of the importance of polyploidy in botanical evolution, described by
‘U’s triangle’10, with the three diploid species B. rapa (A genome),
A full list of members appears at the end of the paper.
Received 7 March 2011; accepted 3 August 2011; published online 28 August 2011; doi:10.1038/ng.919

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We analyzed the organization and evolution of the genome (as
described in the Online Methods and the Supplementary Note).
B. rapa’s close relationship to A. thaliana allows Arabidopsis to be used
as an outgroup for investigating the adaptation of the Brassica lineage
to the triplicated state. In total, 108.6 Mb (90.01%) of the A. thaliana
genome and 259.6 Mb (91.13%) of the B. rapa genome assembly were
contained within collinear blocks. We confirmed the almost complete
triplication of the B. rapa genome relative to A. thaliana (Fig. 3) and (by
inference) to the postulated Brassicaceae ancestral genome (n = 8). The
gene paralogues anchored in the triplicated segments (Supplementary
Fig. 1) and their orthologs (Supplementary Table 6) dated the meso-
hexaploidy event to between 5 and 9 MYA (Supplementary Fig. 2),
which is more recent than has been reported previously13.
The Brassica mesohexaploidy offers an opportunity to study gene
retention in triplicated genomes. Assuming an initial count of protein
coding genes similar to that of A. thaliana (around 30,000), the newly
formed hexaploid would have about 90,000 genes, of which we can now
identify only 41,174. This is typical of the substantial gene loss that occurs
following polyploid formation in eukaryotes19–21. We identified each of
the orthologous blocks in the B. rapa genome corresponding to ancestral
blocks using collinearity between orthologs on the genomes of B. rapa
and A. thaliana and found significant disparity in gene loss across the
triplicated blocks (Supplementary Fig. 3). Of the 21 regions of conserved
synteny, 20 showed significant deviations from equivalent gene frequen-
cies (P < 0.05) (Supplementary Fig. 4). To illustrate this variation, we
concatenated the least fractionated blocks (LF), the medium fractionated
blocks (MF1) and the most fractionated blocks (MF2) and calculated
the proportions of genes retained in each of these sub-genomes relative
to A. thaliana. The LF sub-genome retains 70% of the genes found in
A. thaliana, whereas the MF1 and MF2 sub-genomes retain substantially
lower proportions of retained genes (46% and 36%, respectively; Fig. 4).
Based on the analysis of synonymous base substitution rates (Ks values),
the pairwise divergences between the three sub-genomes are indistin-
guishable from each other (Supplementary Table 7). Our observation of
differentially fractionated sub-genomes is consistent with the hypothesis
that the sub-genomes MF1 and MF2 underwent substantial fractiona-
tion in a tetraploid nucleus before fractionation commenced in the LF
genome in a more recently formed hexaploid. However, biased fractiona-
tion following tetraploidy (albeit less extreme than we observed) has
been reported in A. thaliana22 and maize23, where it was hypothesized
to be the result of differential epigenetic marking of the parent genomes
(resulting in differential gene silencing and consequential fraction), rep-
resenting an alternative hypothesis.
The retention of extensive collinear genome blocks provides a
potential opportunity for ectopic DNA recombination. By finding and
comparing homologous gene quartets, including two α or β duplicates
in Brassica and their respective orthologs in Arabidopsis, we noted that,
respectively, 25% and 30% of Brassica and Arabidopsis duplicates are
more similar to their intragenomic paralog than to their intergenomic
ortholog, suggesting appreciable gene conversion since the divergence of
these lineages (Supplementary Note). The sizes of the affected regions
vary from 10 bp to >2 kb, with a majority of these apparent conversion
events occurring in parallel in both species. Genes proximal to telo-
meres tend to have lower nucleotide substitution rates than distal genes
(P = 0.0004), which is likely to be a result of higher conversion rates in
the former and is consistent with prior findings in grasses24,25.
The gene dosage hypothesis26 predicts that gene functional categor-
ies encoding products that interact with one another or in networks
table 1 summary of the final assembly statistics
Contig sizeContig number Scaffold sizeScaffold number
N90
N80
N70
N60
N50
Total size
Total number
(>100 bp)
Total number
(>2 kb)
5,593
10,984
15,947
21,229
27,294
10,564
7,292
5,308
3,874
2,778
357,979
773,703
1,257,653
1,452,355
1,971,137
283,823,632
159
104
77
56
39
264,110,991
60,52140,549
14,207 794
A01
A02
A03
A04
A05
A06
A07
A08
A09
A10
0M10M 20M 30M
Retrotransposons
DNA transposons
Genes (introns)
Genes (exons)
Figure 1 Chromosomal distribution of the main B. rapa genome features.
Area charts quantify retrotransposons, genes (exons and introns) and DNA
transposons. The x axis denotes the physical position along the B. rapa
chromosomes in units of million (M) bases.
C. papaya
19,093
13,533
A. thaliana
29,139
16,985
B. rapa
32,543
16,917
821
3,660
72
1,228
1,043
9,909
71
70
891
249
1,113
118
1,412
48
V. vinifera
22,608
13,810
1,003
Figure 2 Venn diagram showing unique and shared gene families between
and among four sequenced dicotyledonous species (B. rapa, A. thaliana,
C. papaya and V. vinifera).

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Nature GeNetics  VOLUME 43 | NUMBER 10 | OCTOBER 2011
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should be over retained and genes with products that do not interact
with other gene products should be under retained. In accordance
with this hypothesis, we found B. rapa transcription factors with a
detectable ortholog in A. thaliana to be significantly over retained
(Supplementary Table 8 and Supplementary Note). We obtained
similarly consistent results for genes encoding known protein subunits
of cytoplasmic ribosomes and for genes known to be involved with the
proteosome. We found under retention of genes encoding products
with few interactions, specifically those associated with DNA repair,
nuclease activity, binding and the chloroplast (Supplementary Table 9).
The Gene Ontology annotation classes of over retained genes sug-
gests that genome triplication may have expanded gene families that
underlie environmental adaptability, as observed in other polyploid
species27. Genes with Gene Ontology terms associated with response
to important environmental factors, including salt, cold, osmotic
stress, light, wounding, pathogen (broad spectrum) defense and both
cadmium and zinc ions, were over retained (Fig. 5). Genes respond-
ing to plant hormones (jasmonic acid, auxin, salicylic acid, ethylene,
brassinosteroid, cytokinin and abscisic acid) were also over retained.
Under selection, Brassica species have a remarkable propensity for the
development of morphological variants28; we analyzed factors poten-
tially involved in this development (Supplementary Note). One factor
may be a general acceleration of nucleotide substitution rates. For 2,275
orthologous groups of genes in B. rapa, A. thaliana, papaya and grape
(Supplementary Table 10), the nucleotide substitution rates in B. rapa
were greater than in the other plants, with average Ks (Ks is the ratio
of the number of synonymous substitutions per synonymous site) and
Ka (Ka is the ratio of the number of non-synonymous substitutions per non-
synonymous site) values 69% and 24%, respectively, greater than papaya
and 1% and 7%, respectively, greater than A. thaliana (Supplementary
Table 11). The much slower evolutionary rate in papaya may be explained
by its longer generation time as a perennial. Another factor may be expan-
sion of auxin-related gene families, as auxin controls many plant growth
and morphological developmental processes29–31. We identified 347
B. rapa genes related to auxin synthesis, transportation, signal transduc-
tion and inactivation, in contrast to 187 such genes present in A. thaliana
(Supplementary Tables 12 and 13 and Supplementary Figs. 5–14). The
TCP gene family is important in the evolution and specification of plant
morphology32. This family has been amplified in B. rapa, which contains
39 TCP genes, which is more than A. thaliana (24), grape (19) or papaya (21)
(Supplementary Fig. 15). The regulation of flowering is key to many
Brassica morphotypes. Mesohexaploidy has had contrasting effects on
the genes involved. FLC (FLOWERING LOCUS C)33 has three orthologs
in B. rapa as a consequence of the WGT (Supplementary Fig. 16).
Likewise, five of six B. rapa VRN1 (VERNALIZATION1) genes34
U
CHR5
CHR4
CHR3
CHR2
CHR1
90 Mb
70 Mb
50 Mb
30 Mb
10 Mb
10 Mb
A01
A02
A03 A04 A05A06A07A08A09
A10
30 Mb 50 Mb 70 Mb90 Mb110 Mb 130 Mb150 Mb 170 Mb 190 Mb 210 Mb230 Mb250 Mb
110 Mb
T NMDW
F
S RWE VKL QX R W JP
O
FUNIJJABF
C A BM XH QXBLKVH F B X N
E C TBUB AO TS OC
I
QX H H D
B VK VB N I AA W RH R
X
W
V
S
Q
R
U
T
P
O
N
M
L
F
J
I
H
G
K
E
D
C
B
A
Figure 3 Segmental collinearity of the genomes of B. rapa and A. thaliana. Conserved collinear blocks of gene models are shown between the ten
chromosomes of the B. rapa genome (horizontal axis) and the five chromosomes of the A. thaliana genome (vertical axis). These blocks are labeled A to
X and are color coded by inferred ancestral chromosome following established convention.
100
LF
MF1 and MF2
80
60
Percent of orthologs retained
40
20
0
0 20
Chr1Chr2 Chr3Chr4Chr5
40
A. thaliana chromosome (Mb)
60 80100 120
Figure 4 The density of orthologous genes in three subgenomes (LF, MF1
and MF2) of B. rapa compared to A. thaliana. The x axis denotes the
physical position of each A. thaliana gene locus. The y axis denotes the
percentage of retained orthologous genes in B. rapa subgenomes around
each A. thaliana gene, where 500 genes flanking each side of a certain
gene locus were analyzed, giving a total window size of 1,001 genes.

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produced by the WGT have been preserved (Supplementary Fig. 17).
However, GI (GIGANTEA) genes35 have been limited to only one copy
(Supplementary Fig. 18), as have the SVP (SHORT VEGETATIVE
PHASE) genes36 (Supplementary Fig. 19) and each of the three COL
(CONSTANS-LIKE) genes37 (Supplementary Fig. 20).
The comparison of the genomes of B. rapa and A. thaliana, as for pre-
vious comparisons of the cereals sorghum and rice38, sheds new light on
the evolution of genome evolution in plants important for human nutri-
tion. Our growing understanding of the processes shaping the triplicated
genome of the mesopolyploid B. rapa is of relevance not only for closely
related crops species, such as B. oleracea and B. napus, but also for other
important crops with triplicated genomes, such as bread wheat.
URLs. Brassica info, http://www.brassica.info/; GenoScope database,
http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/; Hawaii
Papaya Genome Project, http://asgpb.mhpcc.hawaii.edu/papaya/;
Arabidopsis Information Resource, http://www.arabidopsis.org/.
MeThods
Methods and any associated references are available in the online
version of the paper at http://www.nature.com/naturegenetics/.
Accession codes. This whole-genome shotgun project has been depos-
ited at DDBJ/EMBL/GenBank under the accession AENI00000000. The
version described in this paper is the first version, AENI01000000. Full
annotation is available at http://brassicadb.org/.
Note: Supplementary information is available on the Nature Genetics website.
ACknowledGmenTS
This work was primarily funded by the Chinese Ministry of Science and
Technology, Ministry of Agriculture, Ministry of Finance, the National Natural
Science Foundation of China. Other funding sources included: Core Research
Budget of the Non-profit Governmental Research Institution; the European Union
7th Framework Project; funds from Shenzhen Municipal Government of China;
the Danish Natural Science Research Council; National Academy of Agricultural
Science and the Next-Generation Biogreen21 Program, Rural Development
Administration, Korea; the Technology Development Program for Agriculture and
Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Korea; United
Kingdom’s Biotechnology and Biological Sciences Research Council; Institute
National de la Recherche Agronomique, France; Japanese Kazusa DNA Research
Institute Foundation; National Science Foundation, USA; Bielefeld University,
Germany; the Australian Research Council; the Australian Grains Research
and Development Corporation; Agriculture and Agri-Food Canada; and the
National Research Council of Canada’s Plant Biotechnology Institute. See the
Supplementary Note for a full list of support and acknowledgments.
AUTHoR ConTRIBUTIonS
Principal investigators: Xiaowu Wang, J. Wu, S.L., Y.B., J.-H.M. and I.B.
DNA and transcriptome sequencing: Bo Wang (group leader), Xiaowu Wang
(group leader), B.C. (group leader), Jun Wang (BGI), K.W., J. Wu, S.L., W.H.,
B.-S.P., I.B., D.E., I.A.P.P., J.-H.M., H.A., Bernd Weisshaar, Shusei Sato, H.H., S.T.,
A.G.S., Y. Lim, G.B., J.B., C.L., C.G., J.P., S.-J.K., J.A.K., M.T., F.F., E.S., M.G.L., C.K.,
K.H., Y.N., P.J.B. and C.D. Sequence assembly: Junyi Wang (group leader), Jun Wang
(BGI), D.M., Y. Li, X.X., Bo Liu, Silong Sun, Z.Z., Z.L., Binghang Liu, Q.C., Shu
Zhang, Y.B., Zhiwen Wang, X.Z., C.S., J.Y. and J.J. Anchoring to linkage maps:
J. Wu (group leader), W.H. (group leader), G.J.K., Y. Lim, B.-S.P., I.B., J.B., D.E.,
Yan Wang, Bo Liu, Silong Sun, Jun Wang (Rothamsted), I.A.P.P., J. Meng, Hui
Wang, J.D., Y. Liao, Y.B., Haiping Wang, M.J., J.-S.K., S.-R.C., N.R. and A.H.
Annotation: Y.B. (group leader), S.L. (group leader), R.L., W.F., Q.H., F.C., Bo Liu,
D.E., J. Min, Jianwen Li, C.P., H.Z., Shunmou Huang, B.C., J.J., H.B., G.L., N.D. and
M.T. Stabilizing the genome of a polyploidy dicotyledonous species: F.C. (group
leader), Sanwen Huang (group leader), Y.B., Xiaowu Wang, B. Li, S.C., Y.Y., J.X. and
C.T. Comparative genomics: Xiaowu Wang (group leader), J.C.P. (group leader),
Xiyin Wang (group leader), I.B., F.C., H.T., G.C., H.G., T.-H.L., Jinpeng Wang and
Zhenyi Wang. Retention of genes duplicated by polyploidy: M.F. (group leader),
A.H.P. (group leader), F.C., H.T., Bo Liu, Silong Sun, L.F., Z.X., M.Z., Jingping Li,
H.J. and X.T. Characteristics of a crop genome: J. Wu (group leader), X.L. (group
leader), R.S., Hanzhong Wang, Y.D., Xiaowu Wang, Hui Wang, J.D., D.S., Y.Q.,
Shujiang Zhang, F.L., L.W. and Yupeng Wang.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturegenetics/.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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1.0
ab
1,021
535
298
181
304
79
63
85
1,071
187
15,976
2,346
0.8
0.6
0.4
Ratio
0.2
0
4.78 × 10–21
RE
1.01 × 10–18
RH
5.24 × 10–20
TF
3.52 × 10–20
GO category
CR
3.29 × 10–15
CW
/ Total
One- or two-copy genes
Three-copy genes
1.0
492
247
827
469
898
31
111
83
477
189
9,293
9,029
0.8
0.6
0.4
Ratio
0.2
0
1.96 × 10–21
RE
6.46 × 10–18
RH
4.82 × 10–31
TF
1.70 × 10–12
GO category
CR
1.61 × 10–11
CW
/ Total
One-copy genes
Two- or three-copy genes
Figure 5 The over retention genes in B. rapa showing strong bias. The x axis
denotes the gene category. The y axis denotes the ratio of different copies
in each category. The number of B. rapa orthologs of each class is indicated
above each bar. RE, response to environment; RH, response to hormone;
TF, transcription factor; CR, cytosolic ribosome; CW, cell wall. (a) The orange
bar is the ratio of one- or two-copy orthologs, and the light green bar is the
ratio of three copies. (b) The yellow bar is the ratio of one-copy orthologs,
and the dark green bar is the ratio of two- or three-copy orthologs. The last
category is the total sets of all orthologs listed as a control. The P value of
each category is indicated under the bars. GO, Gene Ontology.

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Xiaowu wang1, Hanzhong wang2, Jun wang3,4, Rifei Sun1, Jian wu1, Shengyi liu2, Yinqi Bai3, Jeong-Hwan mun5,
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1Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (IVF, CAAS), Beijing, China. 2Oil Crops Research Institute, Chinese Academy
of Agricultural Sciences, Wuhan, Hubei, China. 3BGI-Shenzhen, Shenzhen, China. 4Department of Biology, University of Copenhagen, Copenhagen, Denmark.
5Department of Agricultural Biotechnology, National Academy of Agricultural Science, Rural Development Administration, Suwon, Korea. 6John Innes Centre,
Norwich Research Park, Colney, Norwich, UK. 7Center for Genomics and Computational Biology, School of Life Sciences, Hebei United University, Tangshan, Hebei, China.
8School of Sciences, Hebei United University, Tangshan, Hebei, China. 9Plant Genome Mapping Laboratory, University of Georgia, Athens, Georgia, USA.
10Department of Plant and Microbial Biology, University of California, Berkeley, California, USA. 11Division of Biological Sciences, Bond Life Sciences Center,
University of Missouri, Columbia, Missouri, USA. 12Organization and Evolution of Plant Genomes, Unité de Recherche en Génomique Végétale, Unité Mixte de
Recherché 1165, (Inland Northwest Research Alliance-Centre National de la Recherche Scientifique, Université Evry Val d’Essonne), Evry, France. 13University
of Queensland, School of Agriculture and Food Sciences, Brisbane, Queensland, Australia. 14Australian Research Council Centre of Excellence for Integrative
Legume Research, Brisbane, Queensland, Australia. 15National Research Council-Plant Biotechnology Institute, Saskatoon, Saskatchewan, Canada. 16Center for
Biotechnology, Bielefeld University, Bielefeld, Germany. 17Australian Centre for Plant Functional Genomics, Brisbane, Queensland, Australia. 18Division of Animal
Sciences, University of Missouri, Columbia, Missouri, USA. 19Inland Northwest Research Alliance-Agrocampus Rennes–University of Rennes 1, Unité Mixte de
Recherché 118 Amélioration des Plantes et Biotechnologies Végétales, Le Rheu Cedex, France. 20Centre for Crop Genetic Improvement, Rothamsted Research,
West Common, Harpenden, UK. 21Droevendaalsesteeg 1, Wageningen University, Wageningen, The Netherlands. 22Kazusa DNA Research Institute, 2-6-7 Kazusa-
Kamatari, Kisarazu, Chiba, Japan. 23Experimental Plant Division, RIKEN BioResource Center, Tsukuba, Japan. 24Agriculture and Agri-Food Canada, Saskatoon,
Saskatchewan, Canada. 25National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China. 26Genoscope, Institut de
Génomique du Commissariat à l’Energie Atomique, 2 rue Gaston Crémieux, Evry, France. 27National Institute of Vegetable and Tea Science, Tsu, Japan. 28Molecular
Genetics and Genomics Lab, Department of Horticulture, Chungnam National University, Daejeon, Republic of Korea. 29Research Institute for Biological Sciences,
Okayama, Japan. Correspondence should be addressed to Xiaowu Wang (wangxw@mail.caas.net.cn), Jun Wang (wangj@genomics.org.cn), Hanzhong Wang
(wanghz@oilcrops.cn) or R.S. (rifei.sun@caas.net.cn).

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© 2011 Nature America, Inc. All rights reserved.
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Genome sequencing and assembly. Approximately 72-fold shotgun coverage was
generated using Illumina GA II sequencing from short (~200 bp), medium (~500 bp)
and long (~2 kb, 5 kb and 10 kb) insert libraries (Supplementary Note and
Supplementary Table 14). The raw Illumina reads were filtered for duplicates,
adaptor contamination and low quality before assembly into preliminary scaffolds
using SOAPdenovo39 run with default parameters. We first assembled the reads
from the short insert size (≤500 bp) libraries into contigs using Kmer (de bruijn
graph kmer) overlap information and ensured the resulting contigs were unique by
determining an unambiguous path in the de bruijn graph. This resulted in contigs
with an N50 length of 1.1 kb, achieving a total length of 222 Mb; the long insert size
mate-paired libraries (≥2 kb) were not used initially because the chimaeric reads
common to such libraries can generate incorrect sequence overlaps. After obtaining
the unique contigs, we mapped all available paired-end reads to these contigs to
connect adjacent contigs. In order to avoid interleaving and to reduce the impact of
the insert-size deviation of any sequencing library, we used a hierarchical assembly
method, constructing the scaffolds step by step by adding data from each library
separately ranked according to insert size from smallest to largest. This obtained
scaffolds with an N50 length of 347 kb and a total genome length of 288 Mb. Most
of the remaining gaps between contigs probably occur in repetitive regions, so we
identified the paired-end reads with only one end mapped to a unique contig and
performed local assembly with the unmapped end to fill small gaps within the scaf-
folds. The resulting assembly had a final contig N50 length of 27 kb (Supplementary
Table 15). In total, 32-Mb gaps were closed. A total of 199,452 BAC-end Sanger
sequences retrieved from http://www.brassica-rapa.org/BRGP/bacEndList.jsp were
used to construct the super scaffolds. The gaps within the scaffolds were filled in as
previously described40. The expected genome size of B. rapa was estimated from the
distribution of 17-mer depth as assessed from the filtered sequence data using meth-
ods previously described40. The peak depth of 17-mers was at 15-folds and a total
7,287,899,150 17-mers were obtained. We obtained an estimated genome size of
485 Mb by dividing the total number of 17-mers by the peak depth.
Validation of assembly. NUCmer41 was used to compare the sequence of chromo-
some A03 assembled here by whole-genome shotgun sequencing (WGS A03) to the
same chromosome assembled by BAC Sanger sequencing (BAC A03) previously
reported15 (Supplementary Note and Supplementary Fig. 21). The total sizes of
WGS A03 and BAC A03 are approximately 31.72 Mb and 32.70 Mb, respectively,
with slightly more repeat sequences assembled using the BAC approach (9.82 Mb
in BAC A03 and 5.68 Mb in WGS A03) (Supplementary Table 16). There were
more gaps observed in BAC A03 (1,035/1,358,889 bp, number of gaps/total size of
gaps) than in WGS A03 (858/844,319 bp) (Supplementary Table 17). We identi-
fied 44 obvious inversions (>1 kb) between the two assemblies. Evidence provided
by studying the mapped paired ends, the depth of the mapped reads and gaps at
the boundaries for 38 inversions supported the WGS assembly (Supplementary
Fig. 22a,b), and 6 inversions remained ambiguous (Supplementary Fig. 23c).
To evaluate the accuracy of the assembly on a local scale, the sequence of
647 complete BAC clones (phase 2 and phase 3) that had been deposited in NCBI
and had been genetically mapped (see URLs) were compared with their equivalent
WGS sequence (Supplementary Table 18).
Integration of shotgun assembly with genetic maps. The scaffolds were
anchored to the B. rapa genetic linkage map using 1,427 uniquely aligned
markers from an integrated linkage map developed from four populations
(Supplementary Table 19). In addition, 1,054 markers mapped to the B. napus
A genome were used to verify and aid the alignment. Chromosomes were ori-
entated by alignment to the reference A genome linkage groups from Parkin
et al.42 (equivalent to N1-N10). Where genetic information was not available
from Brassica maps, scaffold order and/or orientation was inferred based on
evidence of conserved collinearity with the A. thaliana gene order.
Protein coding gene annotation. In addition to available Brassica EST data
(downloaded from dbEST at NCBI 10 July 2010), we generated a total of 27.1
million Illumina RNA-Seq paired-end reads, 19.9 million of which were from
Chiifu-401-42 and 7.2 million of which were from a Caixin accession, L58, to
verify the predicted gene models (Supplementary Fig. 24). For Chiifu-401-42,
equally mixed total RNA isolated from eight different tissues and growth con-
ditions was used: leaves, roots and floral stems from plants grown in pots;
2-week-old etiolated seedlings; shoots from plants grown hydroponically
under normal conditions; and leaves from plants treated with 0.5% NaCl at
4 °C and 37 °C for 24 h. For L58, equally mixed total RNA was isolated from
similar tissues with the addition of germinating seeds, callus and pods.
The genome assembly was premasked for class I and class II transposable
elements, and Genscan and Augustus were used to carry out de novo predic-
tions with gene model parameters trained from A. thaliana. Genes with less
than 150 bp of coding sequence were filtered out. For homology-based gene
prediction, we aligned A. thaliana, C. papaya, Populus trichocarpa, V. vinifera
and Oryza sativa protein sequences to the B. rapa genome using TBLASTN
(at an E value of 1 × 10−5) for fast alignment and Genewise43 for precise align-
ment. The Unigene sequences of B. rapa and the Brassica ESTs downloaded
from NCBI were aligned to the B. rapa genome using BLAT and assembled
by PASA44 based on genomic location. As the fragmental exons in ESTs data
might lead to pseudo alignments, we filtered out the results with intron(s)
more than 10,000 bp. GLEAN45 was used to combine de novo gene sets and
homology-based gene sets and incorporated the expressed sequence data
described above as supporting evidence (Supplementary Tables 20 and 21 and
Supplementary Figs. 24 and 25). In addition, those predicted B. rapa proteins
that aligned to the Repbase transposable element protein database (E value
1 × 10−5 at ≥50%) were filtered out.
The B. rapa predicted proteins were annotated based on alignment to the Swiss-
Prot and TrEMBL databases with BLASTP at E value 1 × 10−5. InterPro was used
to annotate motifs and domains by comparison with publicly available databases
including Pfam, PRINTS, PROSITE, ProDom and SMART. The Gene Ontology
information for each gene code was extracted from the InterPro results.
To identify and estimate the number of potential orthologous gene fami-
lies between B. rapa, V. vinifera, A. thaliana and C. papaya, we applied the
OrthoMCL pipeline46 using standard settings (BLASTP E value < 1 × 10−5) to
compute the all-against-all similarities.
Inter- and intra-genomic alignments. The synteny within and between spe-
cies was constructed by McScan (MATCH_SCORE: 50, MATCH_SIZE: 5,
GAP_SCORE: –3, E_VALUE: 1E–05). An all-against-all BLASTP comparison
provided the pairwise gene information and P value for a primary clustering.
Then, paired segments were extended by identifying clustered genes using
dynamic programming. This method was used to build the genome synteny
blocks of B. rapa versus B. rapa, A. thaliana, C. papaya, P. trichocarpa,
V. vinifera and O. sativa, and A. thaliana versus A. thaliana.
Phylogenetic analyses of biologically important gene families. Gene sequences
from grape, papaya and Arabidopsis were downloaded from the GenoScope
database, the Hawaii Papaya Genome Project and the Arabidopsis Information
Resource, respectively (see URLs). Previously reported Arabidopsis and Brassica
gene sequences were downloaded from GenBank. The protein sequences of the
genes were used to determine homologs in grape, papaya, A. thaliana and B. rapa
by performing BLASTP searches at E value 1 × 10−10. Alignment of each family
was performed by running MEGA with default parameters47 and subjected to
careful manual checking to remove highly divergent sequences from further
analysis. The retrieved protein sequences were used to reconstruct phylogenies
using the neighbor-joining approach implemented in MEGA47. A bootstrapping
test was performed using 100 repetitive samplings for each gene family.