Access to this full-text is provided by Springer Nature.
Content available from Nature Genetics
This content is subject to copyright. Terms and conditions apply.
Articles
https://doi.org/10.1038/s41588-021-00922-y
1College of Agronomy, Hunan Agricultural University, Changsha, China. 2Collaborative Innovation Center of Grain and Oil Crops in South China, Hunan
Agricultural University, Changsha, China. 3Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and
Genetic Improvement of Oil Crops, Ministry of Agriculture and Rural Affairs, Wuhan, China. 4Novogene Bioinformatics Institute, Beijing, China. 5Guizhou
Institute of Oil Crops, Guizhou Academy of Agricultural Sciences, Guiyang, China. 6Hunan Key Laboratory of Economic Crops Genetic Improvement and
Integrated Utilization, School of Life Science, Hunan University of Science and Technology, Xiangtan, China. 7Xinjiang Academy of Agricultural Sciences,
Urumqi, China. 8Department of Plant Breeding, Justus Liebig University Giessen, Giessen, Germany. 9Division of Biological Sciences, University of Missouri,
Columbia, MO, USA. 10Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ, USA. 11College of Life Sciences, Resources and
Environment Sciences, Yichun University, Yichun, China. 12Plant Breeding Department, University of Bonn, Bonn, Germany. 13These authors contributed
equally: Lei Kang, Lunwen Qian, Ming Zheng, Liyang Chen. ✉e-mail: huawei@caas.cn; zsliu48@hunau.net
Brassica juncea (L.) Czern & Coss is a diverse and important
agricultural species1. An allotetraploid (AABB, 2n = 36),
B. juncea derived from interspecific hybridization between
the diploid progenitors Brassica rapa (AA, 2n = 20) and Brassica
nigra (BB, 2n = 16)2. Four subspecies have been proposed based on
crop use and morphology: juncea (seed mustard), integrifolia (leaf
mustard), napiformis (root mustard) and tumida (stem mustard)3.
B. juncea has a wide geographic range as native plants, adapted crops
and introduced weeds, spanning the continents of Asia, Europe,
Africa, America and Australia4. B. juncea is an important oilseed
crop in India, Bangladesh, China and Ukraine, and is recently also
gaining importance in Canada and Australia5. Meanwhile, it is
grown as a condiment in Europe, North America, Argentina and
China. Root mustard is distributed in Mongolia and northeast-
ern China, whereas leaf mustards are most common in China and
Southeast Asia5,6.
Brassica juncea is regarded as one of the earliest domesticated
plants, with mustard mentioned as a condiment in Sanskrit and
Sumerian texts from as early as 3,000 BC7. However, its center of
origin is uncertain. Based on biogeographic explorations, Vavilov8
proposed Central Asia (Afghanistan and its contiguous regions)
as the primary center of the origin of B. juncea, and Asia Minor,
central/western China and eastern India as secondary centers of
diversity. By contrast, many investigators9–12 proposed that B. juncea
first evolved in the Middle East where its progenitor species, B. rapa
and B. nigra, are sympatric. Whether B. juncea has a monophyletic
or polyphyletic origin is controversial. Early morphological studies
proposed a single origin13,14, whereas more detailed investigations
implementing chemotaxonomy15, nuclear DNA markers16,17 and
chloroplast (CP) genomic markers18 suggested a polyphyletic origin.
Recently, a single origin was proposed once again based on genome
re-sequencing, using 109 B. juncea accessions19,20. More comprehen-
sive studies would accelerate our understanding of either the center
of origin of B. juncea, or the number of origin and/or domestication
events that gave rise to this important crop species.
Population genomics offers an opportunity to improve our under-
standing of the origin and domestication of crop plants21. To obtain
a comprehensive overview of the origin, domestication and diversi-
fication of B. juncea, we first generated a chromosome-scale de novo
assembly of a genome of the yellow-seeded B. juncea var. Sichuan
Yellow (SY), using PacBio long reads combined with BioNano opti-
cal mapping and Hi-C chromatin interaction maps. Subsequently,
we re-sequenced 480 B. juncea accessions from 38 countries,
leading to the identification of around 4.53 million SNPs and
0.97 million insertion–deletion polymorphisms (InDel; <50 bp).
Our combined analysis of CP, mitochondrial (MT) and nuclear
genome data supports a single origin of B. juncea in West Asia, fol-
lowed by at least three independent domestication events, and the
evolution of new forms through spontaneous gene mutations and
introgressions during its eastward spread. We furthermore scanned
Genomic insights into the origin, domestication
and diversification of Brassica juncea
Lei Kang 1,13, Lunwen Qian1,2,13, Ming Zheng 3,13, Liyang Chen 4,13, Hao Chen1, Liu Yang1,
Liang You1, Bin Yang1,5, Mingli Yan6, Yuanguo Gu7, Tianyi Wang4, Sarah-Veronica Schiessl8,
Hong An 9, Paul Blischak10, Xianjun Liu11, Hongfeng Lu4, Dawei Zhang6, Yong Rao5, Donghai Jia7,
Dinggang Zhou 6, Huagui Xiao5, Yonggang Wang7, Xinghua Xiong1, Annaliese S. Mason 8,12,
J. Chris Pires 9, Rod J. Snowdon 8, Wei Hua 3 ✉ and Zhongsong Liu 1 ✉
Despite early domestication around 3000 BC, the evolutionary history of the ancient allotetraploid species Brassica juncea (L.)
Czern & Coss remains uncertain. Here, we report a chromosome-scale de novo assembly of a yellow-seeded B. juncea genome by
integrating long-read and short-read sequencing, optical mapping and Hi-C technologies. Nuclear and organelle phylogenies of
480 accessions worldwide supported that B. juncea is most likely a single origin in West Asia, 8,000–14,000 years ago, via nat-
ural interspecific hybridization. Subsequently, new crop types evolved through spontaneous gene mutations and introgressions
along three independent routes of eastward expansion. Selective sweeps, genome-wide trait associations and tissue-specific
RNA-sequencing analysis shed light on the domestication history of flowering time and seed weight, and on human selection for
morphological diversification in this versatile species. Our data provide a comprehensive insight into the origin and domestica-
tion and a foundation for genomics-based breeding of B. juncea.
NATURE GENETICS | VOL 53 | SEPTEMBER 2021 | 1392–1402 | www.nature.com/naturegenetics
1392
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles
NATuRE GENETICS
for selective sweeps, performed genome-wide association studies
(GWAS) for flowering time and seed weight, and illuminated the
domestication history and artificial selection of genes implicated
in morphological diversification among diverse B. juncea subspe-
cies. Our results provide a comprehensive picture of the origin and
domestication history of this versatile and economically important
crop species.
Results
Chromosome-scale genome of a yellow-seeded Brassica juncea.
Yellow-seeded B. juncea is grown widely as a condiment and oil-
seed. For de novo assembly of the SY genome, we integrated four
sequencing and assembly technologies: PacBio long-read sequenc-
ing, Illumina short-read sequencing, BioNano optical mapping and
Hi-C data (Supplementary Fig.1 and Supplementary Table1). The
SY genome size was estimated to be 1056.53 Mb by k-mer analysis
(Table1 and Supplementary Fig.2), close to the 1,068 Mb estimated
by flow cytometry22. PacBio reads (~93×) were first assembled
using FALCON23, followed by contig correction using Illumina
reads (~130×) to generate a V.1 assembly (Supplementary Table2).
Using 202-fold coverage of BioNano data, we then generated an
optical consensus map, which was implemented to assemble 1,897
super-scaffolds with an N50 of 5.87 Mb (assembly V.2). These contigs
were categorized and ordered into 18 chromosome-scale scaffolds
using a 15,543-marker high-density linkage map (Supplementary
Fig. 3a and Supplementary Table 3). Finally, we used Hi-C data
to confirm the pseudo-chromosomes and manually adjusted 165
mis-joined contigs by Juicebox24 (Supplementary Fig. 3b,c and
Supplementary Table2). The final SY assembly captured 933.5 Mb
of genome sequence, with 867.3 Mb (~92.9%) anchored into chro-
mosomes (Fig.1 and Supplementary Table 4), which is superior
to previous assemblies of stem19 and Indian25 mustard in terms of
genome size, contiguity and anchorage. We simultaneously assem-
bled the CP (153,465 bp) and MT (219,803 bp) genomes of SY
(Supplementary Figs.4 and 5).
The high quality of the SY assembly was validated
(Methods) by BUSCO and CEGMA scores of more than 98.5%
(Supplementary Table 6), by alignment of over 95% identity
with 81 randomly selected BACs and 2,567 paired BAC-end
sequences26 (Supplementary Fig.6 and Supplementary Tables 7
and 8), by high long terminal repeat (LTR) Assembly Index (LAI)27
of 10.73 among the assembled Brassica genomes (Supplementary
Table9), by high consistency with our genetic and optical maps
(Supplementary Figs.3a and 7), by consistent syntenic gene order-
ing (Supplementary Fig.8) using genome-ordered graphical geno-
types28, and by the good collinearity of SY to those of B. rapa29 and
B. nigra30 and other previously reported Brassica genomes19,25,31
(Supplementary Fig.9).
The SY assembly contained 50.36% TEs (Table 1 and
Supplementary Table10), slightly more than the published genomes
of B. juncea T84-66 (43.5%)19 and Varuna (45.8%)25 and B. rapa
(37.51%)32, but less than B. nigra (53.73%)30. In accordance with
previous Brassica genomes19,25,29–33, LTR/gypsy retroelements were
the predominant TE family (Supplementary Table 10). We dis-
tinguished the chromosomal centromeric from pericentromeric
regions by specific repeats30,34–37 (Fig.1, Extended Data Fig.1 and
Supplementary Table 11), and remarkably lower recombination
frequencies (Supplementary Fig.3a). The centromere and pericen-
tromeric regions were enriched for LTR/copia and LTR/gypsy ele-
ments, respectively (Fig.1 and Supplementary Table12).
Among 92,878 predicted gene models (Supplementary Note and
Supplementary Table 13), 95.5% were functionally annotated in
public databases (Supplementary Table 14). Alignment to known
proteins and expression in at least one tissue type showed 82,723
gene models were high-confidence (HC) genes (Supplementary
Table15), with an average coding sequence length of ~1.13 kb and
an average of five exons per gene, similarly to predictions in other
Brassica genomes (Supplementary Table13). A total of 5,756 genes
(6.96% of the HC genes) encoded putative transcription factors
belonging to 58 different families (Supplementary Table 16). We
also identified 2,525 tRNAs, 8,363 rRNAs, 1,951 microRNAs and
4,691 small nuclear RNAs (Supplementary Table17).
Population structure and genomic variation. To explore genetic
variation in B. juncea, we re-sequenced 480 accessions representing
the four subspecies from 38 countries (Fig.2a and Supplementary
Table 18) with an average depth of 15× and 97.7% of the SY
genome. Using this dataset, we identified 4,529,618 high-quality
SNPs and 967,266 InDels (Supplementary Table 19) based on
four parameters (Methods), corresponding to 4.85 SNPs and 1.04
InDels per kb (Supplementary Table20). A total of 946,661 SNPs
(20.9%) and 50,955 InDels (5.27%) were located in coding regions.
Among them, 345,138 SNPs (7.62%) caused codon changes, elon-
gated transcripts or premature stop codons, while 27,420 InDels
(2.83%) led to frameshift mutations. The SNP distribution varied
across the genome depending on genome context and gene den-
sity, but was generally higher toward the telomeric chromosome
regions (Supplementary Fig.10). The A subgenome of B. juncea
had higher nucleotide diversity (π = 2.05 × 10−3) than the B subge-
nome (π = 1.45 × 10−3; Supplementary Fig. 11). Moreover, linkage
disequilibrium (LD) decayed faster in the A subgenome than in the
B subgenome (Supplementary Fig.12), indicating a higher degree of
genetic recombination in the A subgenome of B. juncea.
Next, we investigated the genetic structure of the B. juncea pop-
ulation for clusters (K) from 2 to 10 based on 4.53 million SNPs
among the 480 B. juncea accessions. When K = 6, clusters maxi-
mized the marginal likelihood (Supplementary Fig.13). To bet-
ter clarify the relationships within the population, 90 genetically
admixed accessions with main genetic components of less than 60%
were excluded from further analysis. Both phylogenetic and princi-
pal component analyses (PCAs) of the remaining 390 samples indi-
cated three distinct clades (Fig.2b,c). Clade І consisted only of root
mustard from Northeast Asia. Clade II consisted of seed mustard
from West Asia, Central Asia and Northwest China along the Steppe
Route, a trans-Eurasian trading route predating the Silk Road38.
Clade III included oilseed and vegetable mustards from the Indian
subcontinent and southern China, corresponding to the South Silk
Road connecting East and Central Asia39.
Our phylogenetic and genetic clustering analyses resolved six
B. juncea genetic groups (G1–G6), which largely corresponded
to morphologically distinct crops (Supplementary Fig. 14 and
Supplementary Table 21). G1, the root mustard group, showed
the slowest LD decay, especially in the B subgenome, and strong
genetic divergence from the other five groups (pairwise FST ≥ 0.33;
Table 1 | Summary statistics for the Brassica juncea var. Sichuan
Yellow genome assembly
Genomic feature SY
Estimated genome size (Mb) 1056.53
Total assembly size (bp) 933,496,244
Longest scaffold (bp) 76,001,744
Scaffold N50 (bp) 59,341,207
Contig N50 (bp) 1,926,153
Missing bases (%) 4.76
Sequences anchored to chromosome (%) 92.91
Annotated protein-coding genes (n) 82,723
TE proportion (%) 50.36
NATURE GENETICS | VOL 53 | SEPTEMBER 2021 | 1392–1402 | www.nature.com/naturegenetics 1393
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles NATuRE GENETICS
Fig. 2e and Supplementary Tables 22 and 23). G2 comprised
yellow-seeded mustard, and almost 60% of the G2 accessions with
known geographic origins were from northwestern China; other
G2 accessions sourced from the former Soviet Union, Canada
and Europe were documented introductions from China40–42.
G3 spanned wide geographic origins from Tibet, central and west-
ern Asia to Europe. G3 clustered close to but distinctly from G2
(FST = 0.07; Fig.2d). G4 comprised mainly accessions from south-
western China and clustered closest to the G5 group. The G5
group, including 96 leaf, 14 stem and 10 seed mustards originating
from southern China to Japan43 and the USA9,41, showed the high-
est nucleotide diversity (π = 1.54 × 10−3) and the greatest LD decay
(Fig.2d,e). The 59 accessions forming the group G6 were almost all
from South Asia. G6 showed a similarly slow LD decay to G1, and
it also exhibited the lowest nucleotide diversity (π = 0.93 × 10−3),
consistent with a narrow genetic base of Indian mustard44. All gen-
otypes belonging to G2 and G3 in Clade II and to G4 and G6 in
Clade III are grown for seed use, whereby G2 and G3 differentiate
less strongly from G4 (pairwise FST = 0.25 and 0.24, respectively)
than from G6 (pairwise FST = 0.42 and 0.39, respectively; Fig.2e
and Supplementary Table22).
Domestication and spread of Brassica juncea. To delineate
domestication and spread, we further constructed A and B subge-
nome phylogenies of B. juncea and its progenitors (Supplementary
Table24). Both subgenome phylogenetic trees confirmed six groups
of B. juncea and that the G1 group was the closest to the progenitor
species, although G4 and G6 had the opposite positions (Fig.3a and
Supplementary Figs.15 and 16). These nuclear phylogenies support
the hypothesis that B. juncea originated monophyletically19.
We assembled 478 CP and 10 MT genomes to study cytoplasmic
relationships between B. juncea and its progenitors (Supplementary
Tables18 and 25). Based on the assembled CP genomes, we found
two InDel variants and divided the B. juncea CP genomes into three
types (CPs 1–3; Extended Data Fig.2a and Supplementary Table18).
Meanwhile, we classified the MT genomes into three types (MTs
1–3) using an InDel and a SNP locus45 (Extended Data Fig.2b and
Supplementary Table 18). These three MT types corresponded
Flowering time
Pod shattering
Seed weight
Fatty acid synthesis
Glucosinolates synthesis
Disease resistance
Other
A01
0
35
A02
0
34
A03
0
39
A04
0
23
A05
0
33
A06
0
36
A07
0
30
A08
0
25
A09
0
70
A10
0
22
B01
0
58
B02
0
76
B03
0
61
B04
0
57
B05
0
66
B06
0
72
B07
0
59
B08
0
71
a. Chromosome
b. Centromere
c. HC gene
d. HC gene expression
e. LTR/gypsy
f. LTR/copia
g. DNA retrotransposon
h. Known genes for agronomic traits
b
c
a
d
e
f
g
h
Fig. 1 | Chromosomal features and functional and synteny landscape of the yellow-seeded B. juncea var. SY genome. Tracks from outer (a) to inner (h)
rings indicate the following: a, Chromosome size with units in Mb; b, Density of centromere-specific repeats in 5-Mb bins; c, Density of HC genes in 5-Mb
bins; d, Expression of HC genes from nine tissues, calculated as the fragments per kilobase of transcript per million mapped reads (FPKM) in 5-Mb bins and
normalization of FPKM by log10(FPKM + 1). e, LTR/Gypsy density (Gypsy length/5 Mb). f, LTR/Copia density (Copia length/5 Mb). g, DNA retrotransposon
density (DNA retrotransposon length/5 Mb). h, Location of known genes (Supplementary Table5) for major phenotypic traits. Lines in the center linking
different chromosomal regions show the syntenic relationships between the A and B subgenomes.
NATURE GENETICS | VOL 53 | SEPTEMBER 2021 | 1392–1402 | www.nature.com/naturegenetics
1394
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles
NATuRE GENETICS
b
e
c
K = 2
K = 3
K = 4
K = 5
K = 6
a
0 100 200 300 400 500
0
0.2
0.4
0.6
0.8
r2
Physical distance (kb)
G2
G1
G3
G5
G6
G4
G4G2
G1
G5 G6G3
−0.06 −0.04 −0.02 0 0.02 0.04 0.06
−0.20
−0.15
−0.10
−0.05
0
PC1 (17.53%)
PC2 (9.38%)
Clade I Clade II Clade III
Clade I
Clade II Clade III
d
1.04
0.11
0.28 0.24
0.39
0.37
0.35
0.39
0 .49
0.30
0.25 0.42
0.180.18
0.26
0.33
0.07
0.38
0.41
0.90
1.51
1.01
1.17
2.29
0.43
0.42
1.12
0.110.28
0.65
G2
1.21 × 10
–3
G1
1.19 × 10
–3
G3
1.45 × 10
–3
G5
1.54 × 10
–3
G4
1.40 × 10
–3
G6
0.93 × 10
–3
N412
N430
N429
N277
N409
N414
N415
N413
N427
N442
N393
N411
N392
N329
N331
N448
N449
N391
N410
N439
N400
N431
N432
N390
N416
N344
J378
J206
J282
J299
J315
J472
J236
J203
J152
J202
J235
J153
J019
J447
J020
J333
J117
J058
J309
J023
J312
J207
J304
J316
J314
J462
J279
J057
J305
J283
J281
J280
J317
J303
J150
J220
J130
J357
J131
J322
J456
J133
J132
J480
J208
J209
J210
X002
J129
J125
J471
J241
J123
J380
J211
J124
J134
J445
J356
J325
J326
J319
J477
J126
J127
J470
J476
J460
J475
J458
J457
J327
J128
J452
J022
J461
J479
J478
J459
J074
J240
J222
J358
J361
J381
J330
J071
J363
J242
J272
J446
J375
J359
J262
J453
J212
J122
J451
J306
J307
J310
J313
J311
J157
J218
J084
J085
J081
J078
J082
J076
J075
J159
J297
J007
J083
J264
J215
J376
J377
J151
J219
J366
J237
J147
J148
J365
J008
J239
J371
J369
J370
J373
J352
J216
J364
J244
J379
J172
J175
J174
J169
J168
J328
J266
J362
J173
J038
J256
J072
J184
J382
J156
J105
J104
J088
J103
J069
J086
J335
J089
J093
J106
J021
J338
J340
J337
J044
J102
J095
J384
J278
J113
J101
J136
J092
J090
J094
J079
J116
J118
J099
J135
J046
J096
J383
J100
J001
J355
J091
J334
I489
I141
I030
I389
I145
J404
I408
I407
I143
I056
I406
I063
I054
I065
I233
I144
I405
I160
I182
I015
I011
I255
I014
I473
I349
J158
I033
I005
J111
J112
J059
J049
I342
J114
I032
I017
I418
I417
I426
I225
I421
I420
I450
I425
I139
I138
I098
I097
I482
I155
I154
I481
I137
I444
T395
I488
I010
I034
J291
T000
T486
T273
J290
T028
I041
T052
T275
T274
T332
T394
T109
T387
T324
I140
I419
I487
I396
I142
I110
I422
I251
J080
J002
J025
I043
I428
I257
I259
I397
I029
I064
I188
I228
T070
I258
I401
I227
I234
I436
I232
I403
I424
I441
I402
I260
I440
I217
I040
I051
I060
I039
I226
I224
I433
I398
I399
I437
I443
I435
I434
J037
J193
J199
J165
J197
J171
J189
J214
J485
J162
J053
J012
J205
J198
J200
J463
J467
J468
J048
J464
J465
J466
J469
J246
J166
J213
J269
J247
J167
J179
J323
J187
J194
J190
J177
J195
J454
J455
J196
J180
J178
J170
J186
J204
J249
J191
J252
J164
J250
J192
J271
J270
J245
N412
N430
N429
N277
N409
N414
N415
N413
N427
N442
N393
N411
N392
N329
N331
N448
N449
N391
N410
N439
N400
N431
N432
N390
N416
N344
J378
J206
J282
J299
J315
J472
J236
J203
J152
J202
J235
J153
J019
J447
J020
J333
J117
J058
J309
J023
J312
J207
J304
J316
J314
J462
J279
J057
J305
J283
J281
J280
J317
J303
J150
J220
J130
J357
J131
J322
J456
J133
J132
J480
J208
J209
J210
X002
J129
J125
J471
J241
J123
J380
J211
J124
J134
J445
J356
J325
J326
J319
J477
J126
J127
J470
J476
J460
J475
J458
J457
J327
J128
J452
J022
J461
J479
J478
J459
J074
J240
J222
J358
J361
J381
J330
J071
J363
J242
J272
J446
J375
J359
J262
J453
J212
J122
J451
J306
J307
J310
J313
J311
J157
J218
J084
J085
J081
J078
J082
J076
J075
J159
J297
J007
J083
J264
J215
J376
J377
J151
J219
J366
J237
J147
J148
J365
J008
J239
J371
J369
J370
J373
J352
J216
J364
J244
J379
J172
J175
J174
J169
J168
J328
J266
J362
J173
J038
J256
J072
J184
J382
J156
J105
J104
J088
J103
J069
J086
J335
J089
J093
J106
J021
J338
J340
J337
J044
J102
J095
J384
J278
J113
J101
J136
J092
J090
J094
J079
J116
J118
J099
J135
J046
J096
J383
J100
J001
J355
J091
J334
I489
I141
I030
I389
I145
J404
I408
I407
I143
I056
I406
I063
I054
I065
I233
I144
I405
I160
I182
I015
I011
I255
I014
I473
I349
J158
I033
I005
J111
J112
J059
J049
I342
J114
I032
I017
I418
I417
I426
I225
I421
I420
I450
I425
I139
I138
I098
I097
I482
I155
I154
I481
I137
I444
T395
I488
I010
I034
J291
T000
T486
T273
J290
T028
I041
T052
T275
T274
T332
T394
T109
T387
T324
I140
I419
I487
I396
I142
I110
I422
I251
J080
J002
J025
I043
I428
I257
I259
I397
I029
I064
I188
I228
T070
I258
I401
I227
I234
I436
I232
I403
I424
I441
I402
I260
I440
I217
I040
I051
I060
I039
I226
I224
I433
I398
I399
I437
I443
I435
I434
J037
J193
J199
J165
J197
J171
J189
J214
J485
J162
J053
J012
J205
J198
J200
J463
J467
J468
J048
J464
J465
J466
J469
J246
J166
J213
J269
J247
J167
J179
J323
J187
J194
J190
J177
J195
J454
J455
J196
J180
J178
J170
J186
J204
J249
J191
J252
J164
J250
J192
J271
J270
J245
N412
N430
N429
N277
N409
N414
N415
N413
N427
N442
N393
N411
N392
N329
N331
N448
N449
N391
N410
N439
N400
N431
N432
N390
N416
N344
J378
J206
J282
J299
J315
J472
J236
J203
J152
J202
J235
J153
J019
J447
J020
J333
J117
J058
J309
J023
J312
J207
J304
J316
J314
J462
J279
J057
J305
J283
J281
J280
J317
J303
J150
J220
J130
J357
J131
J322
J456
J133
J132
J480
J208
J209
J210
X002
J129
J125
J471
J241
J123
J380
J211
J124
J134
J445
J356
J325
J326
J319
J477
J126
J127
J470
J476
J460
J475
J458
J457
J327
J128
J452
J022
J461
J479
J478
J459
J074
J240
J222
J358
J361
J381
J330
J071
J363
J242
J272
J446
J375
J359
J262
J453
J212
J122
J451
J306
J307
J310
J313
J311
J157
J218
J084
J085
J081
J078
J082
J076
J075
J159
J297
J007
J083
J264
J215
J376
J377
J151
J219
J366
J237
J147
J148
J365
J008
J239
J371
J369
J370
J373
J352
J216
J364
J244
J379
J172
J175
J174
J169
J168
J328
J266
J362
J173
J038
J256
J072
J184
J382
J156
J105
J104
J088
J103
J069
J086
J335
J089
J093
J106
J021
J338
J340
J337
J044
J102
J095
J384
J278
J113
J101
J136
J092
J090
J094
J079
J116
J118
J099
J135
J046
J096
J383
J100
J001
J355
J091
J334
I489
I141
I030
I389
I145
J404
I408
I407
I143
I056
I406
I063
I054
I065
I233
I144
I405
I160
I182
I015
I011
I255
I014
I473
I349
J158
I033
I005
J111
J112
J059
J049
I342
J114
I032
I017
I418
I417
I426
I225
I421
I420
I450
I425
I139
I138
I098
I097
I482
I155
I154
I481
I137
I444
T395
I488
I010
I034
J291
T000
T486
T273
J290
T028
I041
T052
T275
T274
T332
T394
T109
T387
T324
I140
I419
I487
I396
I142
I110
I422
I251
J080
J002
J025
I043
I428
I257
I259
I397
I029
I064
I188
I228
T070
I258
I401
I227
I234
I436
I232
I403
I424
I441
I402
I260
I440
I217
I040
I051
I060
I039
I226
I224
I433
I398
I399
I437
I443
I435
I434
J037
J193
J199
J165
J197
J171
J189
J214
J485
J162
J053
J012
J205
J198
J200
J463
J467
J468
J048
J464
J465
J466
J469
J246
J166
J213
J269
J247
J167
J179
J323
J187
J194
J190
J177
J195
J454
J455
J196
J180
J178
J170
J186
J204
J249
J191
J252
J164
J250
J192
J271
J270
J245
N412
N430
N429
N277
N409
N414
N415
N413
N427
N442
N393
N411
N392
N329
N331
N448
N449
N391
N410
N439
N400
N431
N432
N390
N416
N344
J378
J206
J282
J299
J315
J472
J236
J203
J152
J202
J235
J153
J019
J447
J020
J333
J117
J058
J309
J023
J312
J207
J304
J316
J314
J462
J279
J057
J305
J283
J281
J280
J317
J303
J150
J220
J130
J357
J131
J322
J456
J133
J132
J480
J208
J209
J210
X002
J129
J125
J471
J241
J123
J380
J211
J124
J134
J445
J356
J325
J326
J319
J477
J126
J127
J470
J476
J460
J475
J458
J457
J327
J128
J452
J022
J461
J479
J478
J459
J074
J240
J222
J358
J361
J381
J330
J071
J363
J242
J272
J446
J375
J359
J262
J453
J212
J122
J451
J306
J307
J310
J313
J311
J157
J218
J084
J085
J081
J078
J082
J076
J075
J159
J297
J007
J083
J264
J215
J376
J377
J151
J219
J366
J237
J147
J148
J365
J008
J239
J371
J369
J370
J373
J352
J216
J364
J244
J379
J172
J175
J174
J169
J168
J328
J266
J362
J173
J038
J256
J072
J184
J382
J156
J105
J104
J088
J103
J069
J086
J335
J089
J093
J106
J021
J338
J340
J337
J044
J102
J095
J384
J278
J113
J101
J136
J092
J090
J094
J079
J116
J118
J099
J135
J046
J096
J383
J100
J001
J355
J091
J334
I489
I141
I030
I389
I145
J404
I408
I407
I143
I056
I406
I063
I054
I065
I233
I144
I405
I160
I182
I015
I011
I255
I014
I473
I349
J158
I033
I005
J111
J112
J059
J049
I342
J114
I032
I017
I418
I417
I426
I225
I421
I420
I450
I425
I139
I138
I098
I097
I482
I155
I154
I481
I137
I444
T395
I488
I010
I034
J291
T000
T486
T273
J290
T028
I041
T052
T275
T274
T332
T394
T109
T387
T324
I140
I419
I487
I396
I142
I110
I422
I251
J080
J002
J025
I043
I428
I257
I259
I397
I029
I064
I188
I228
T070
I258
I401
I227
I234
I436
I232
I403
I424
I441
I402
I260
I440
I217
I040
I051
I060
I039
I226
I224
I433
I398
I399
I437
I443
I435
I434
J037
J193
J199
J165
J197
J171
J189
J214
J485
J162
J053
J012
J205
J198
J200
J463
J467
J468
J048
J464
J465
J466
J469
J246
J166
J213
J269
J247
J167
J179
J323
J187
J194
J190
J177
J195
J454
J455
J196
J180
J178
J170
J186
J204
J249
J191
J252
J164
J250
J192
J271
J270
J245
MT2
MT3
Group
G2
G1
G3
G5
G6
G4
MT1
Plasmon
Fig. 2 | Geographic distribution, population structure and genomic diversity of Brassica juncea accessions. a, Geographic distributions of 480 B. juncea
accessions. The geographic map was drawn using R ggplot2. b, The maximum-likelihood phylogeny of 390 B. juncea accessions with over 60% genetic
components to the group and model-based clustering with K from 2 to 6. The five other Brassicaceae species used to root the phylogenetic tree are shown
as a single branch. Branch colors indicate different groups based on the population structure. Scale bars, 5 cm for G1 and G5; 5 mm for G2, G3, G4 and G6.
c, PCA plots showing three divergent clades of 390 B. juncea accessions. d, Nucleotide diversity (π), population divergence (FST) and genetic distance (D)
across the six groups. The value in each circle represents a measure of nucleotide diversity for each group; values in red on each line indicate pairwise
population divergence between groups, while values in black on each line indicate pairwise genetic distances among groups. e, Group-specific LD decay plots.
NATURE GENETICS | VOL 53 | SEPTEMBER 2021 | 1392–1402 | www.nature.com/naturegenetics 1395
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles NATuRE GENETICS
a
A subgenome B subgenome
b
Drift parameter
0 0.05 0.10 0.15
G1
G4
G6
G5
G2
G3
0
0.5
Migration
weight
G1 G2 G3 G4 G5 G6
AA × BB
0
500
–
1,200
600
–
1,300
1,200
–
2,500
1,800
–
4,600
2,500
–
5,120
8,000
–
14,000
c
e
Years ago
0
5
10
15
20
0 0.25 0.50 0.75 1.00
Ks
Gene pairs (%)
~
11.33–18.89 Ma
(Arabidopis–Brassica)
~
5.33–8.89 Ma
(Ar–Bn)
~
8,333
–
13,889 years ago
(AjBj–ArBn)
d
Bra
G1
G2
G3
G6
G5
G4
Clade I
Clade II
Clade III
Clade I
Bni
Clade II
G1
G3
G2
G6
G5
G4
Clade III
H
Wari-Bateshwar
(400–100 BC)
Raja-Nal-Ka-Tila
(1300–700 BC)
New Delhi
G6
Jerf el Ahmar
(9500–8700 BC)
Athens
G3
Urumqi
Ulan Bator
Beijing
Lhasa
Harappa
(2400–1700 BC)
G3
G2
Banpo
(4800 BC)
Mawangdui
(138 BC)
Datong
G4
G5
Dali
Chengdu
G1
Fig. 3 | Speciation and demographic history of Brassica juncea. a, Maximum-likelihood phylogenies of the subgenomes of 390 B. juncea accessions
compared to 68 B. rapa accessions (left), and 11 B. nigra accessions (right). b, Estimates of molecular divergence between B. juncea (AjBj) and its
pseudo-ancestor (ArBn, pooled by two progenitors, B. rapa and B. nigra). c, Divergence time for six groups was estimated using SMC++. d, Detection
of gene flows among B. juncea groups by TreeMix analysis. Arrows represent the direction of migrations. Horizontal branch length is proportional to the
amount of genetic drift that has occurred on the branch. Scale bar shows ten times the average standard error of the entries in the sample covariance
matrix. e, Putative spread routes of B. juncea. Archaeological evidence showing that seed cakes or carbonized mustard seeds were excavated from
Jerf el Ahmar (9500–8700 BC)54, Banpo site (about 4800 BC)55, Harappa (2400–1700 BC)59, Raja-Nal-ka-Tila site (1300–700 BC)60, Wari-Bateshwa
(400–100 BC)61 and Mawangdui site (about 138 BC)64. The geographic map was adapted from NASA (https://visibleearth.nasa.gov/images/147190/
explorer-base-map/147191w/). Ma, million years ago.
NATURE GENETICS | VOL 53 | SEPTEMBER 2021 | 1392–1402 | www.nature.com/naturegenetics
1396
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles
NATuRE GENETICS
to the three specific CP classifications, and were subsequently
named plasmotypes I–III. All G1 accessions carried plasmotype І,
whereas all G6 and most (94.2%, 113/120) G5 accessions harbored
plasmotype III. The remaining three groups contained all three
plasmotypes, with plasmotype II predominating (G2 91.3%, G3
71.2%, G4 70.0%; Supplementary Table 21). In the CP phylog-
eny, most (467/478) of the B. juncea accessions were rooted in the
B. rapa lineage (Supplementary Fig.17), consistent with the conclu-
sion that B. rapa is the maternal ancestor of B. juncea46,47. CP and
MT phylogenies (Supplementary Figs.17 and 18) and PCR analy-
sis (Extended Data Fig.2) indicated that plasmotype І of B. juncea
descended from B. rapa and evolved into plasmotype II and III via
insertion/deletions and a base substitution. From the perspective of
cytoplasmic inheritance, B. juncea shows a single origin.
The progenitor species of B. juncea are sympatric in the Middle
East48. Wild B. juncea forms have been observed to grow on the pla-
teaus in Asia Minor and southern Iran10,49–52. The group G3, includ-
ing Turkish accessions, possessed not only all three plasmotypes
(Fig. 2a and Supplementary table 21) but also higher nucleotide
diversity (Fig.2d), implying that the place where the G3 accessions
were collected is a plausible center of origin. Collectively, these
data support that B. juncea most likely originated in West Asia (the
Middle East).
Importantly, we estimated that B. juncea formed ~8,000–14,000
years ago by natural hybridization between both progenitors
(Fig.3b). A demographic history model of the B. juncea groups
favors at least three independent evolutionary routes (Fig. 3c).
Four gene flows were detected among the six groups by Treemix
and D-statistic analyses: from root mustard (G1) to leafy mustard
(G5), from Indian mustard (G6) to West and Central Asia mus-
tard (G3), from northwestern China (G2) to southwestern China
yellow-seeded mustard (G4) and, with a lower weight, in the recip-
rocal direction from G4 to G2 (Fig.3d and Supplementary Table26).
Root mustard first diverged from wild B. juncea, approximately
2,500–5,120 years ago (Fig.3c). We speculate that root mustard was
domesticated in Mongolia and northeastern China according to its
current geographic distribution and historical records53, although
how it spread into East Asia remains elusive (Fig.3e). Additionally,
wild B. juncea was domesticated into the seed mustard (G3), and
a diverse range of B. juncea accessions developed (Fig. 3c,e and
Supplementary Table 18). The G3 mustard spread eastward from
northern Afghanistan along the Steppe Route and entered Tibet via
the Hexi corridor. During the dissemination process of G3, a new
yellow-seed mustard (G2) evolved about 500 years ago from sponta-
neous gene mutations56,57, probably in Xinjiang58 (Fig.3e). In parallel,
the G3 mustard spread from southern Afghanistan into the Indian
subcontinent12 where it was domesticated into Indian mustard (G6),
which is supported by archaeological excavations59. Indian mustard
then spread eastward60,61 to form a new type of broad-leaf mustard
(var. rugosa)13, probably around 300 BC62. These broad-leaf mustards
spread further east into southwestern China, where they were grown
as vegetables and oilseed before the sixth century AD63. Historical
records documented the subsequent derivation of stem mustard from
broad-leaf mustard in the Sichuan Basin in the eighteenth century6.
Accordingly, we observed very low genetic diversity in stem mustard
and a closer relationship to leaf mustard (G5) than G4 accessions
from the same geographic region (Supplementary Table27).
The G4 group inherited yellow-seed color and plasmotype II
from G2, and early maturity from G5. Migration weight, f-branch
and fd values showed more genetic components were introgressed
into the B subgenome than into the A subgenome from G2 to G4
(Extended Data Fig.3), which can explain the opposite position of
G4 and G6 in the A and B subgenome phylogenies (Fig.3a). The
proportions of introgressed fragments from G2 detected in the
G4 accessions varied from 0.07 to 0.26, with an average of 0.159
(Supplementary Fig. 19 and Supplementary Table 28). The five
largest introgressed genomic blocks (relative IBD rate > 0.7;
Methods) included the regions from 49.8 to 50.8 Mb on chromo-
some A09 and from 39.8 to 41.8 Mb on chromosome B08, which
carry Arabidopsis thaliana TT8 (TRANSPARENT TESTA 8) orthol-
ogous genes (BjuA09g45700S and BjuB08g18790S) that are non-
functional in yellow-seed B. juncea56,57. Therefore, we concluded
that G4 is a genetic admixture from the natural hybridization of G2
with G5, implying that the combination of gene mutations by natu-
ral hybridization played a significant role in the domestication and
spread of yellow-seeded B. juncea.
Ecogeographic adaptation of Brassica juncea flowering time. We
observed flowering time variation across 390 B. juncea accessions
grown under four contrasting environments: 94 to 194 d in Guiyang,
71 to 200 d in Xiangtan, 29 to 78 d in Kunming and 25 to 65 d in
Urumqi (Supplementary Fig. 20 and Supplementary Table 29).
The flowering time of 390 accessions was positively correlated
across different environments (r2 = 0.46 to 0.95; Supplementary
Fig.21). The broad-sense heritability of flowering time reached 0.74
(Supplementary Table29). Most of the root mustards and some leaf
mustards did not flower in Kunming, indicating vernalization fail-
ure due to insufficiently low temperatures.
We identified 43 and 38 putative selective sweeps in G6/G1 and
G6/G2, respectively, containing 63 flowering time candidate genes
(Fig.4a and Supplementary Table30). Of these genes, 30 and 7 have
known roles in the photoperiod and vernalization pathways, respec-
tively. We also scanned selective sweeps for flowering time by com-
paring G1 with group G2, G3, G4 or G5 and identified 42 candidate
genes for flowering time (Supplementary Fig.22). Simultaneously,
a total of 56 candidate genes showed significant association to
flowering time across the four environments by GWAS analysis
(Supplementary Fig. 23 and Supplementary Table 31). Of these
genes, 12 also detected by the selective-sweep scan were investi-
gated in more detail as potential contributors to domestication
(Supplementary Fig.24).
Notably, two SNPs in the region of BjuA10g14550S (SRR1,
SENSITIVITY TO RED LIGHT REDUCED 1) and five SNPs in
BjuB05g31990S (VIN3, VERNALIZATION INSENSITIVE 3) were
found to be significantly associated with flowering time (Fig.4b,e
and Supplementary Table31). SRR1 is a pioneer protein involved
in the regulation of the circadian clock and phytochrome B signal-
ing65, while VIN3 is a crucial gene involved in vernalization66. We
found strong LD between SRR1 on chromosome A10 and VIN3 on
B05 (Extended Data Fig.4a). The combinations of both SRR1 and
VIN3 haplotypes were consistent with the haplotypes of either gene
(Extended Data Fig.4b,c). SRR1-A10-Hap1 and VIN3-B05-Hap1
were present in late-flowering or non-flowering accessions of the
G1 group, which was domesticated in cold, long-day environ-
ments. SRR1-A10-Hap2 and VIN3-B05-Hap2 were present mostly
in accessions from G2 and G3 with moderate flowering time.
These seed mustard groups were domesticated under long-day
conditions with large diurnal temperature variations (20–30 °C).
Finally, SRR1-A10-Hap3 and VIN3-B05-Hap3 were present in
the earliest-flowering accessions, mainly from G4, G5 and G6
(Fig.4c,d,f,g and Supplementary Table32). These results demon-
strate the coevolution of SRR1 and VIN3 during the domestication
of B. juncea, and support the conclusion that B. juncea underwent
three independent domestication events.
Furthermore, a 4,597-bp insertion was found in the exon of
SRR1. All SRR1-A10-Hap3 accessions have this insertion, whereas
it is carried only by some (50/118) SRR1-A10-Hap2 accessions
(Supplementary Fig.25a,b). Comparing flowering time, we found
that SRR1-A10-Hap2 accessions with the insertion flower earlier
than those without the insertion, suggesting that this gene lost its
function because of the premature termination codon produced
by the insertion (Supplementary Fig.25b,c). A 13-bp insertion in
NATURE GENETICS | VOL 53 | SEPTEMBER 2021 | 1392–1402 | www.nature.com/naturegenetics 1397
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles NATuRE GENETICS
the third intron and 6-bp deletion in the fifth exon of VIN3 were
detected in VIN3-B05-Hap1 and VIN3-B05-Hap2 (Supplementary
Fig. 26a). VIN3-B05-Hap3 accessions have the highest relative
expression level and flower earliest, while VIN3-B05-Hap1 and
VIN3-B05-Hap2 accessions flower latest and show a moderate, but
not significantly different, gene expression level (Supplementary
Fig.26b) because these two haplotypes differ at only a single SNP
(Supplementary Fig.27).
In addition, we identified 15 genes significantly associated
with flowering time by both GWAS and selective-sweep scan
f
e
b
XP-CLR
(G6/G1)
100
150
200
50
0
XP-CLR
(G6/G2)
300
200
100
400
0
14.35
14.45 Mb
150
45.85 45.89 Mb
XP-CLR
(G6/G1)
200
100
50
100
0
0
300
XP-CLR
(G6/G2)
Hap3
(n = 193) (n = 200)
(n = 139)
(n = 23)
(n = 131)
(n = 25)
5
′
1/1
Allele code 0/1 0/0
3
′
Hap2
SRR1 (BjuA10g14550S)
4,597-bp insertion
36/40
52/59
106/120
G5
51/53
G6
G4
G1
25/26
G2
79/92
G3
Chr. A10
SRR1
–log
10
P
12
8
6
4
0
10
2
16.0014.5013.00 Mb
0
100
300
200
G18 X18 K18 U18
P = 4.9 × 10
−25
P = 4.2 × 10
−13
P = 5.2 × 10
−11
P = 2.6 × 10
−13
P = 8.9 × 10
−15
P = 5.8 × 10
−33
250
200
150
100
50
0
0
210
420
630
840
1,050
XP-CLR (G6/G2)
GA3OX2 SPY MSI1
VIN3
COL9
RVE8
PRR5
LHY
SRR1
PIE1
ELF3
AP2 MMP
JMJ14
ELF4
VIN3
SOC1
FIS3
COL4
FTIP1
AGL16
GA3OX2
SPY
VIN3
FLC
FT FPA
PRR5
SRR1
CO ELF4 VIN3 FTIP1
COL4
a
A01 A02 A03 A04 A05 A06 A07 A08 A09 A10 B01 B02 B03 B04 B05 B06 B07 B08
MSI1
COL9
RVE8
TCP11 FPA
LHY
PIE1
ELF3 MMP
SOC1
XP-CLR (G6/G1)
0
0.2
0.4
0.6
0.8
1.0
r
2
15
Hap3
0
Mb
Chr. B05
45.00 45.70 46.40
g
Hap2
200
300
G18 X18 K18 U18
NA
0
100
G2
1/1
Allele code 0/1 0/0
G1
Exon
Insertion
Flowering time
(d)
Flowering time
(d)
Intron
VIN3 (BjuB05g31990S)
23/26
81/92
G3
34/40
G4
58/59
114/120
G5
52/53
G6
VIN3-B05-
Hap1
10
5
VIN3
5
′
3
′
c
d
SRR1-A10-
Hap1
P = 2.2 × 10
–11
P = 4.8
× 10
–25
P = 1.7 × 10
–13
P = 2.2 × 10
–12
P = 5.8 × 10
–33
P = 4 × 10
–15
NA
P = 2.0 × 10
−6
P = 3.9 × 10
−5
P = 0.12
P = 0.32 P = 0.36
P = 1.4 × 10
–3
P = 5.3 × 10
–4
P = 0.26
Fig. 4 | Genome-wide screening of selective sweeps and GWAS for flowering time in Brassica juncea. a, Genome-wide distribution of selective sweeps
identified through comparisons between G1 or G2 with G6 using XP-CLR (cross-population composite likelihood-ratio test) values (sliding window = 10 kb,
step = 1 kb). The flowering time candidate genes in the selective regions are labeled. b,e, Local Manhattan plot showing the 14.35–14.45 Mb and 45.85–
45.89 Mb regions on chromosomes A10 and B05, respectively. The green plots represent the position of these SNPs in SRR1 (BjuA10g14550S) and VIN3
(BjuB05g31990S). Two and five SNPs in the gene regions of SRR1 and VIN3 were significantly associated with flowering time, respectively. Heat maps
spanning the SNP markers in LD with the most strongly associated SNPs in VIN3 and SRR1 gene regions. The red lines indicate the significance threshold
(−log10P = 6.0). c,f, Three haplotypes with a frequency greater than 0.01 were identified in the SRR1 and VIN3 gene regions, respectively. Box plot showed
three haplotypes corresponding to flowering time in SRR1 and VIN3 gene regions, respectively. d,g, Box plots for flowering time based on the haplotypes
(Hap.) for SRR1 (d) and VIN3 (g) under four different environments. Box edges represent the 0.25 and 0.75 quartiles, with the median values shown by
bold lines. Whiskers extend to data no more than 1.5 times the interquartile range, and remaining data are indicated by dots. P values were calculated
using two-sided t-tests. NA, data missing (G1 group did not flower in Kunming).
NATURE GENETICS | VOL 53 | SEPTEMBER 2021 | 1392–1402 | www.nature.com/naturegenetics
1398
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles
NATuRE GENETICS
(Supplementary Table33). These genes included transcription fac-
tors, SUVR and WD-40 repeat proteins, and gibberellic acid signal-
ing, which warrant further investigation.
Genetics of morphological diversification in Brassica juncea.
Domestication and artificial selection of B. juncea imparted major
morphotype changes, including the increase in seed size, root
expansion and stem swelling. We aimed to identify selective sweeps
and genomic regions associated with each of these traits in the
B. juncea panel.
Seed size is a primary agronomic trait that contributes to seed
yield in condiment and oilseed mustards. We observed signifi-
cant variation in thousand seed weight (TSW), ranging from 0.29
to 2.48 g, 0.52 to 2.94 g, 0.66 to 3.16 g and 0.96 to 4.30 g across
the four environments, respectively (Supplementary Fig. 21 and
Supplementary Table29). A high broad-sense heritability of 0.92
was calculated for TSW (Supplementary Table29). Significant posi-
tive correlations were detected across the environments, with r2 val-
ues of 0.44–0.82 (Supplementary Fig.21).
We identified 33 and 51 putative selective sweeps in G5/G2
and G6/G2, respectively, which contained 65 candidate genes
for TSW. Among these genes, 19 overlapped between G5/G2 and
G6/G2 (Supplementary Table34). We detected 22 significantly asso-
ciated candidate genes using GWAS (Supplementary Fig. 28 and
Supplementary Table35), of which 7 were also detected by selective
sweeps (Supplementary Fig.28). The two genes detected by both
approaches, BjuA04g00760S (CYP78A9, CYTOCHROME P450
78A9) and BjuB05g28000S (CAM7, CALMODULIN 7; Extended
Data Fig.5b,e and Supplementary Table35), were previously shown
to regulate seed weight in Brassica napus67 and Gossypium hirsutum68.
Four haplotypes were detected in CYP78A9. CYP78A9-A04-Hap4
was present in 7 G3 accessions with the highest TSW, whereas
CYP78A9-A04-Hap1 was present in 11 G5 vegetable accessions with
the lowest TSW under four environments. CYP78A9-A04-Hap2
was mainly present in accessions from G1, G2 and G3, while
CYP78A9-A04-Hap3 was present mainly in accessions from G4, G5
and G6. We also detected four haplotypes for CAM7. CAM7-B05-Hap1
corresponded to the G1 root mustard types with the lowest TSW,
whereas CAM7-B05-Hap4 corresponded to 10 G2 oilseed accessions
which had the highest TSW across environments. The accessions
with CAM7-B05-Hap2 and CAM7-B05-Hap3 corresponded well to
those with CYP78A9-A04-Hap2 and CYP78A9-A04-Hap3, respec-
tively (Extended Data Fig.5f,g and Supplementary Table36).
Interestingly, Hap2 of CYP78A9 and CAM7 was sensitive
to environments. For example, the G2 and G3 accessions of
CYP78A9-A04-Hap2 produced heavier seeds under long-day
than under short-day conditions (Supplementary Fig. 29 and
Supplementary Table36). However, they showed delayed flowering
under short-day environments and produced lighter seeds than the
G4, G5 and G6 accessions of CYP78A9-A04-Hap3. The significant
increase in TSW of G2 and G3 accessions under long-day environ-
ments is a major factor causing opposing phenotypes in accessions
EXLB1 (BjuB02g61740S)
0
280
560
700
140
420
XP-CLR
A01 A02 A03 A04 A05 A06 A07 A08 A09 A10 B01 B02 B03 B04 B05 B06 B07 B08
CYCB1-2
ARF7
CDC48A, EXPA17, STP7, XTH9
PIF4
IAA9
EXPA16
BHLH80
SMR3
KRP6
CYCD6-1
G1
G2
G3
G4
G5
G6
G1
G2
G3
G4
G5
G6
CDC48A (BjuA03g27650S)
EXLB1
20
30
40
CDC48 A
a
bc
d
UTR
5′3′
5′3′
Expression level (FPKM)
Expression level (FPKM)
0
1
2
3
4
EXLB1
Allele code 0/0 0/1 1/1 Allele code 0/0 0/1 1/1
P = 0.045
P = 0.0021
P = 0.047
P = 1.6 × 10–5
(n = 4) (n = 4) (n = 6)
P = 0.0028
P = 0.92
CDS Intron UTR CDS Intron
Fig. 5 | Identification of candidate genes for root enlargement in root mustard (Brassica juncea ssp. napiformis). a, Genome-wide distribution of selective
sweeps related to tuber root formation in B. juncea. b, Haplotypes for the candidate gene CDC48A (BjuA03g27650S). c, Haplotypes for the candidate
gene EXLB1 (BjuB02g61740S). d, Expression levels of CDC48A and EXLB1 in non-root and root mustard (before and 2 weeks after root enlargement) were
estimated based on FPKM values. Box edges represent the 0.25 and 0.75 quartiles, with the median values shown by bold lines. Whiskers extend to data
no more than 1.5 times the interquartile range, and remaining data are indicated by dots. P values were calculated using two-sided t-tests. Scale bars, 2 cm.
NATURE GENETICS | VOL 53 | SEPTEMBER 2021 | 1392–1402 | www.nature.com/naturegenetics 1399
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles NATuRE GENETICS
with these two haplotypes under long-day and short-day conditions.
Quantitative PCR with reverse transcription (RT–qPCR) analysis
showed that both CYP78A9 and CAM7 were upregulated in the
large-seeded accession ‘7981’ (TSW, 2.65–4.30 g) compared to the
small-sized seeds accession ‘SY’ (TSW, 1.40–2.46 g; Supplementary
Fig.30). Collectively, these results implicate CYP78A9 and CAM7 as
causal genes for TSW in B. juncea. Haplotype analysis suggests that
selection of these genes for local photoperiod adaptation induced
diversification of seed size in B. juncea.
Meanwhile, we detected 30 genes significantly associated with
TSW by both GWAS and selective-sweep scan (Supplementary
Table37). These genes included transcription factors, hormone sig-
naling pathways, lipid transporters and ribosomal proteins, which
require further investigations.
To investigate selection signatures putatively related to the
domestication of root mustard, we compared the root mustard
genomes to those of seed and leaf mustards using selective-sweep
scan. In total, 2,803 sweep regions were identified in root mustard,
covering 21.85 Mb with 5,756 genes (Supplementary Table 38).
Fourteen candidate genes implicated in the formation of storage
roots were identified (Fig.5a and Supplementary Table 39), with
putative functions in auxin signaling, sugar transport, cell divi-
sion, cell expansion and cell wall modification. Of these, CDC48A4
(BjuA03g27650S), participating in cell division and growth69, was
found to have three haplotypes corresponding to the three inde-
pendent domestication events (Fig.5b). Its expression was upregu-
lated during root enlargement in root mustard (Fig.5d). The root
and non-root mustards carried distinctly different haplotypes of
the expansin gene EXPB1 (BjuB02g61740S; Fig. 5c). Its expres-
sion was downregulated after root enlargement in root mustard
(Fig.5d), which is consistent with the expression patterns of EXPB1
in Raphanus sativus70 and Ipomoea batatas71 during storage root
development. We observed similar expression patterns in another
expansin gene, EXPA16 (BjuA09g18260S), and the cell elongation
gene XTH9 (BjuA03g32220S) after root enlargement in root mus-
tard (Supplementary Table39).
Stem mustard is characterized by its enlarged edible stem with
a diameter of > 20 cm, much bigger in diameter than leaf mustard
(usually <5 cm72). We compared genomes of stem and leaf mus-
tards and identified a total of 5,018 selective sweeps, spanning
46.51 Mb (Extended Data Fig. 6 and Supplementary Table 40).
Twelve candidate genes selected during stem mustard breed-
ing (Supplementary Table 41) are implicated in cell division,
cell expansion, regulation of auxin signaling and glucose trans-
port, functions with reported roles in storage organ formation in
Brassica73. Expression of BjuA05g02460S, orthologous to GRF7
(GROWTH-REGULATING FACTOR 7) regulating leaf and stem
development74, was upregulated during stem swelling (Extended
Data Fig.6b,d), while the genes encoding auxin-responsive protein,
IAA33 (BjuA10g12920S), and the auxin-response factor, MP (also
known as ARF5, (BjuB03g51870S), were downregulated after stem
swelling (Extended Data Fig.6c,d and Supplementary Table 41).
This result contrasts with reports in turnip (B. rapa ssp. rapa)75,
where expression of auxin-response genes did not change signifi-
cantly during hypocotyl expansion. Overall, a greater subgenomic
prevalence of selective sweeps related to root and stem swelling sug-
gests that the A subgenome has undergone stronger selection than
the B subgenome (Supplementary Tables38 and 40). This finding
is consistent with the high morphotype diversity of B. rapa73, which
putatively provides a better selective substrate than the narrower
range of variation present in B. nigra.
Discussion
SY is a yellow-seeded landrace of B. juncea and represents a new
form evolved from hybridization between two big gene pools.
Therefore, SY is different from previously sequenced stem19 and
Indian25 mustard. The chromosome-scale reference genome of
SY, in conjunction with re-sequencing of 480 accessions, captured
major genetic variation and allowed detailed reconstruction of the
evolutionary and domestication history of this diverse ancient crop
species. Plant genomics, together with archaeological evidence and
historical written records, likely indicated a monophyletic origin
of B. juncea in West Asia 8,000–14,000 years ago and at least three
subsequent independent domestication events in the last 500–5,000
years: seed mustard near Central Asia, oilseed mustard in the Indian
subcontinent and root mustard in East Asia. As B. juncea spread
eastward, yellow-seeded (Oriental) mustard arose in Northwest
China, stem mustard in the Sichuan Basin and probably broad-leaf
mustard in eastern India, by selection acting on via spontaneous
mutations. Hybridization of leaf mustard with yellow-seeded and
root mustard gave rise to early-maturing yellow-seeded mustard in
the Yunnan–Kweichow Plateau and lobed-leaf mustard (var. mul-
tisection Bailey) in eastern China, respectively. We also identified
underlying genes and causal alleles for morphological variants such
as root and stem swelling, flowering time and seed size variation
associated with domestication and diversification. Our results not
only elucidate the complex evolutionary and domestication history
of B. juncea, but also pave the way for future research and breeding
of this morphologically diverse condiment, oilseed, leaf, stem and
root vegetable species.
Online content
Any methods, additional references, Nature Research report-
ing summaries, source data, extended data, supplementary infor-
mation, acknowledgements, peer review information; details of
author contributions and competing interests; and statements of
data and code availability are available at https://doi.org/10.1038/
s41588-021-00922-y.
Received: 13 July 2020; Accepted: 23 July 2021;
Published online: 6 September 2021
References
1. Vaughan, J. G. & Hemingway, J. S. e utilization of mustards. Econ. Bot.
13, 196–204 (1959).
2. Nagaharu, U. Genomic analysis in Brassica with special reference to the
experimental formation of B. napus and peculiar mode of fertilization. Jpn.
J. Bot. 7, 389–452 (1935).
3. Gladis, T. & Hammer, K. e Brassica collection in Gatersleben: Brassica
juncea, Brassica napus, Brassica nigra and Brassica rapa. Feddes Rep. 103,
469–507 (1992).
4. Spect, C. E. & Diederichsen, A. Brassica in Mansfeld’s Encyclopedia of
Agricultural and Horticultural Crops (ed. Hanelt, P.) 3, 1453–1456 (Springer
Press, 2001).
5. Dixon, G. R. Origins and Diversity of Brassica and its relatives in Vegetable
Brassicas and Related Crucifers (ed. Dixon, G.R.) 1–34 (CABI Press, 2007).
6. Chen, S. R. e origin and dierentiation of mustard varieties in China.
Cruciferae Newsl. 7, 7–10 (1982).
7. Hemingway, J. e mustard species: condiment and food ingredients
use and potential as oilseed crops in Brassica Oilseeds: Production
and Utilization (eds. Kimber, D. S. & McGregor, D. I.) 373–383
(CAB Press, 1995).
8. Vavilov, N. I. Phytogeographic basis of plant breeding. Chronica Bot. 13,
14–56 (1951).
9. Bailey, L. H. e cultivated Brassicas. Second paper. Gentes Herb. 2,
211–267 (1930).
10. Mizushima, U. & Tsunoda, S. A plant exploration in Brassica and allied
genera. Tohoku J. Agric. Res. 17, 249–277 (1967).
11. Sun, V. G. Breeding plants of Brassica. J. Agron. Assoc. China 71,
141–152 (1970).
12. Hinata, K. & Prakash, S. Ethnobotany and evolutionary origin of Indian
oleiferous Brassicae. Indian J. Genet. 44, 102–112 (1984).
13. Prain, D. e mustards cultivated in Bengal. Agric. Ledger 5, 1–80 (1898).
14. Sinskaia, E. N. e oleiferous plants and root crops of the family Cruciferae.
Bull. Appl. Bot. Genet. Plant Breed. 19, 555–648 (1928).
15. Vaughn, J. G., Hemmingway, J. S. & Schoeld, H. J. Contributions to a
study of variation in Brassica juncea Czern and Coss. J. Linn. Soc. 58,
435–447 (1963).
NATURE GENETICS | VOL 53 | SEPTEMBER 2021 | 1392–1402 | www.nature.com/naturegenetics
1400
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles
NATuRE GENETICS
16. Song, K. M., Osborn, T. C. & Williams, P. H. Brassica taxonomy based on
nuclear restriction fragment length polymorphisms (RFLPs). 1. Genome
evolution of diploid and amphidiploid species. eor. Appl. Genet. 75,
784–794 (1988).
17. Chen, S. et al. Evidence from genome-wide simple sequence repeat markers
for a polyphyletic origin and secondary centers of genetic diversity of
Brassica juncea in China and India. J. Hered. 104, 416–427 (2013).
18. Kaur, P. et al. Polyphyletic origin of Brassica juncea with B. rapa and B.
nigra (Brassicaceae) participating as cytoplasm donor parents in
independent hybridization events. Am. J. Bot. 101, 1157–1166 (2014).
19. Yang, J. et al. e genome sequence of allopolyploid Brassica juncea and
analysis of dierential homoeolog gene expression inuencing selection.
Nat. Genet. 48, 1225–1232 (2016).
20. Yang, J. et al. Chinese root-type mustard provides phylogenomic insights
into the evolution of the multi-use diversied allopolyploid Brassica juncea.
Mol. Plant 11, 512–514 (2018).
21. Larson, G. et al. Current perspectives and the future of domestication
studies. Proc. Natl Acad. Sci. USA 111, 6139–6146 (2014).
22. Johnston, J. S. et al. Evolution of genome size in Brassicaceae. Ann. Bot. 95,
229–235 (2005).
23. Chin, C. S. et al. Phased diploid genome assembly with single-molecule
real-time sequencing. Nat. Methods 13, 1050–1054 (2016).
24. Durand, N. C. et al. Juicebox provides a visualization system for Hi-C
contact maps with unlimited zoom. Cell Syst. 3, 99–101 (2016).
25. Paritosh, K. et al. A chromosome-scale assembly of allotetraploid Brassica
juncea (AABB) elucidates comparative architecture of the A and B genomes.
Plant Biotechnol. J. 19, 602–614 (2021).
26. Liu, X. et al. Genome-wide identication, localization and expression
analysis of proanthocyanidin-associated genes in Brassica. Front. Plant Sci.
7, 1831 (2016).
27. Ou, S., Chen, J. & Jiang, N. Assessing genome assembly quality using the
LTR Assembly Index (LAI). Nucleic Acids Res. 46, e126 (2018).
28. He, Z. & Bancro, I. Organization of the genome sequence of the polyploid
crop species Brassica juncea. Nat. Genet. 50, 1496–1497 (2018).
29. Belser, C. et al. Chromosome-scale assemblies of plant genomes using
nanopore long reads and optical maps. Nat. Plants 4, 879–887 (2018).
30. Perumal, S. et al. A high-contiguity Brassica nigra genome localizes active
centromeres and denes the ancestral Brassica. Genome Nat. Plants 6,
929–941 (2020).
31. Song, M. J. et al. Eight high-quality genomes reveal pan-genome architecture
and ecotype dierentiation of Brassica napus. Nat. Plants 6, 34–45 (2020).
32. Zhang, L. et al. Improved Brassica rapa reference genome by
single-molecule sequencing and chromosome conformation capture
technologies. Hortic. Res. 5, 50 (2018).
33. Chalhoub, B. et al. Early allopolyploid evolution in the post-Neolithic
Brassica napus oilseed genome. Science 345, 950–953 (2014).
34. Lim, K. B. et al. Characterization of rDNAs and tandem repeats in the
heterochromatin of Brassica rapa. Mol. Cells 19, 436–444 (2005).
35. Lim, K. B. et al. Characterization of the centromere and peri-centromere
retrotransposons in Brassica rapa and their distribution in related Brassica
species. Plant J. 49, 173–183 (2007).
36. Schelout, C. J., Snowdon, R., Cowling, W. A. & Wroth, J. M. A PCR-based
B-genome-specic marker in Brassica species. eor. Appl. Genet. 109,
917–921 (2004).
37. Wang, G. et al. ChIP-cloning analysis uncovers centromere-specic
retrotransposons in Brassica nigra and reveals their rapid diversication in
Brassica allotetraploids. Chromosoma 128, 119–131 (2019).
38. Christian, D. Silk roads or steppe roads? e silk roads in world history.
J. World Hist. 11, 1–26 (2000).
39. Wu, X. M. et al. Genetic diversity in oil and vegetable mustard (Brassica
juncea) landraces revealed by SRAP markers. Genet. Resour. Crop Evol. 56,
1011–1022 (2009).
40. Pustovoit, V. S. Indian mustard in Handbook of Selection and Seed Growing
of Oil Plants (ed. Pustovoit, V. S.) 149–205 (Israel Program for Scientic
Translations, 1973).
41. Musil, A. F. Distinguishing the species of Brassica by their seed. USDA
Misc. Publ. No. 643 1–35 (1948).
42. Oram, R. N. et al. Breeding Indian mustard Brassica juncea (L.) Czern for
cold-pressed, edible oil production: a review. Aust. J. Agric. Res. 56,
581–596 (2005).
43. Hoshikawa, K. Mustard in e Origin and Propagation of Cultivated Plants
(ed. Hoshikawa, K.) 92–93 (Ninomiya Syoten Press, 1998).
44. Chauhan, J. S., Singh, K. H., Singh, V. V. & Kumar, S. Hundred years of
rapeseed-mustard breeding in India: accomplishments and future strategies.
Indian J. Agr. Sci. 81, 1093–1109 (2011).
45. Hatono, S., Nishimura, K., Murakami, Y., Tsujimura, M. & Yamagishi, H.
Complete mitochondrial genome sequences of Brassica rapa (Chinese
cabbage and mizuna), and intraspecic dierentiation of cytoplasm in
B. rapa and Brassica juncea. Breed. Sci. 67, 357–362 (2017).
46. Li, P. et al. A phylogenetic analysis of chloroplast genomes elucidates the
relationships of the six economically important Brassica species comprising
the triangle of U. Front. Plant Sci. 8, 111 (2017).
47. Chang, S. et al. Mitochondrial genome sequencing helps show the
evolutionary mechanism of mitochondrial genome formation in Brassica.
BMC Genomics 12, 497 (2011).
48. Tsunoda, S. Eco-physiology of wild and cultivated forms in Brassica and
allied genera in Brassica Crops and Wild Allies (eds. Tsunoda, S. et al.)
109–120 (Japan Scientic Societies Press, 1980).
49. Olsson, G. Species crosses within the genus Brassica I. Articial Brassica
juncea Coss. Hereditas 46, 171–222 (1960).
50. Tsunoda, S. & Nishi, S. Origin, dierentiation and breeding of cultivated
Brassica. Proc. XII Int. Congr. Genet. 2, 77–88 (1968).
51. Kayaçetin, F. Morphological characterization and relationships among some
important wild and domestic Turkish mustard genotypes (Brassica spp.).
Turk. J. Bot. 43, 499–515 (2019).
52. Dönmeza, A. A., Aydına, Z. U. & Wang, X. W. Wild Brassica and its close
relatives in Turkey, the genetic treasures. Hort. Plant J. 7, 97–107 (2021).
53. Wang, S. M. & Shu, G. G. in Explanations of Cucurbits and Vegetable Crops
11, 1576–1588 (e Commercial Press, 1937)..
54. Willcox, G. Charred plant remains from a 10th millenium B.P. kitchen at
Jerf el Ahmar (Syria). Veget. Hist. Archaeobot. 11, 55–60 (2002).
55. Institute of Archaeology of Chinese Academy of Sciences. Xian Banpo
country. 223 (Special issue of Archaeology, Archaeology Press, 1963).
56. Liu, X. et al. Inheritance, mapping, and origin of yellow-seeded trait in
Brassica juncea. Acta Agron. Sin. 35, 839–847 (2009).
57. Liu, Z. et al. Domestication and molecular mechanism underlying yellow
seed in Brassica juncea Czern & Coss. 131 (14th International Rapeseed
Congress, Saskatoon, Canada, 2015).
58. Vavilov, N. I. Origin and Geography of Cultivated Plants (translated by Love,
D.) (Cambridge Univ. Press, 1992).
59. Hutchinson, J. B. India: local and introduced crops. Philos. Trans. R. Soc.
Lond. B Biol. Sci. 275, 129–141 (1976).
60. Pokharia, A. K. et al. Neolithic Early historic (2500–200 BC) plant use: the
archaeobotany of Ganga Plain, India. Quatern. Int. 443, 223–237 (2017).
61. Rahman, M., Castillo, C. C., Murphy, C., Rahman, S. M. & Fuller, D. Q.
Agricultural systems in Bangladesh: the rst archaeobotanical results from
Early Historic Wari-Bateshwar and Early Medieval Vikrampura. Archaeol.
Anthropol. Sci. 12, 37 (2020).
62. Prakash, S., Wu, X. & Bhat, S. R. History, evolution and domestication of
Brassica crops. Plant Breed. Rev. 35, 19–84 (2012).
63. Jia, S. X. & Shu, Q. M. Y. Important Arts for the Peoples’ Welfare (Shanghai
Classics Publishing House, 2009).
64. Liu, Z. M. e origin and development of cultivated rice in China. Acta
Genet. Sin. 2, 23–29 (1975).
65. Staiger, D. et al. e Arabidopsis SRR1 gene mediates phyB signaling
and is required for normal circadian clock function. Genes Dev. 17,
256–268 (2003).
66. Sung, S. & Amasino, R. M. Vernalization in Arabidopsis thaliana is
mediated by the PHD nger protein VIN3. Nature 427, 159–164 (2004).
67. Shi, L. et al. A CACTA‐like transposable element in the upstream region of
BnaA9.CYP78A9 acts as an enhancer to increase silique length and seed
weight in rapeseed. Plant J. 98, 524–539 (2019).
68. Cheng, Y. et al. GhCaM7-like, a calcium sensor gene, inuences cotton
ber elongation and biomass production. Plant Physiol. Biochem. 109,
128–136 (2016).
69. Rancour, D. M., Park, S., Knight, S. D. & Bednarek, S. Y. Plant UBX
domain-containing protein 1, PUX1, regulates the oligomeric structure and
activity of Arabidopsis CDC48. J. Biol. Chem. 279, 54264–54274 (2004).
70. Xie, Y. et al. Comparative proteomic analysis provides insight into a
complex regulatory network of taproot formation in radish (Raphanus
sativus L.). Hortic. Res. 5, 51 (2018).
71. Noh, S. A. et al. Down-regulation of the IbEXPB1 gene enhanced storage
root development in sweet potato. J. Exp. Bot. 64, 129–142 (2013).
72. Shi, H. et al. Cell division and endoreduplication play important roles in
stem swelling of tuber mustard (Brassica juncea Coss. var. tumida Tsen et
Lee). Plant Biol. 14, 956–963 (2012).
73. Cheng, F. et al. Subgenome parallel selection is associated with morphotype
diversication and convergent crop domestication in Brassica rapa and
Brassica oleracea. Nat. Genet. 48, 1218–1224 (2016).
74. Wang, F. et al. Genome-wide identication and analysis of the
growth-regulating factor family in Chinese cabbage (Brassica rapa L. ssp.
pekinensis). BMC Genomics 15, 807 (2014).
75. Liu, M. et al. What makes turnips: anatomy, physiology and transcriptome
during early stages of its hypocotyl-tuber development. Hortic. Res. 6,
38 (2019).
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
NATURE GENETICS | VOL 53 | SEPTEMBER 2021 | 1392–1402 | www.nature.com/naturegenetics 1401
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles NATuRE GENETICS
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing, adap-
tation, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The images or other
third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons license and your intended use is not permitted by statu-
tory regulation or exceeds the permitted use, you will need to obtain permission directly
from the copyright holder. To view a copy of this license, visit http://creativecommons.
org/licenses/by/4.0/.
© The Author(s) 2021
NATURE GENETICS | VOL 53 | SEPTEMBER 2021 | 1392–1402 | www.nature.com/naturegenetics
1402
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles
NATuRE GENETICS
Methods
Genome sequencing and assembly. Genome sequencing. High-molecular-weight
DNA was isolated from fresh young leaves of B. juncea ssp. juncea var. SY. A
SMRTbell library constructed with Sequel 1.0 reagents was sequenced on the
PacBio Sequel. Illumina paired-end libraries of 350 bp in length were prepared
following the manufacturer’s protocol. Hi-C libraries were performed as previously
described76. Hi-C libraries were controlled for quality and sequenced on the
Illumina HiSeq X Ten platform. Total RNA samples were extracted from root,
stem, leaf, ower bud, siliques (7 and 15 d post-anthesis (DPA)), pod wall
(20 DPA) and seed (20 DPA). RNA-sequencing (RNA-seq) libraries were
made using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB)
following the manufacturer’s recommendations and also sequenced on the
Illumina X Ten platform.
Optical mapping. High-molecular-weight DNA extracted using the BioNano Plant
Tissue DNA isolation kit (BioNano Genomics) was digested by Nt.BspQI and
labeled with IrysPrep Labeling mix. The labeled DNA sample was loaded on the
IrysChip and imaged using the BioNano Irys System.
Construction of a high-density Brassica juncea genetic map. A set of 172
recombinant inbred lines were derived from the cross SY × Purple Leaf Mustard
(PM). Genomic DNA extracted from recombinant inbred line individual plants
were digested with MseI. The fragments between 330 and 550 bp were gel excised
and eluted. The pooled libraries were amplified and sequenced on a HiSeq 2000
platform. After stringent filtering, a total of 51,018 SNPs were identified in 21,210
genotyping-by-sequencing tags using the UNEAK pipeline77. To map the reads,
the published B. juncea ‘T84-66’ genome (http://brassicadb.cn/#/SearchJBrowse/
?Genome=Bju15/) was used as the reference. Genotyping of recombinant inbred
lines was performed using a hidden Markov mode78 and the genetic map was
constructed using MSTMap79.
De novo genome assembly. The genome size of SY was estimated by Jellyfish
(v.2.2.9)80 using the k-mer of 17. After low-quality PacBio subreads shorter than
500 bp or with a quality score lower than 0.8 were filtered out, clean PacBio
subreads were error-corrected and assembled into contigs by FALCON23 with the
parameters --max_diff 100, --max_cov 100 and --min_cov 3, and then connected
to scaffolds using Sspace-longread (v.1.1)81. After filling gaps using PacBio reads
with PBJelly (v.1.9.1)82, gap-closed scaffolds were polished by Quiver83 and Pilon84
software with PacBio reads and Illumina data, respectively.
Scaffolding by integrating BioNano optical map. High-quality labeled molecules
were pairwise aligned, clustered and assembled into contigs following the
BioNano Genomics assembly pipeline. The BioNano Solve (V3.1) pipeline
module ‘HybridScaffold’ was used to perform the hybrid assembly between
the initial scaffold sequences and BioNano-assembled genome maps with
the one-enzyme method. Using 202-fold coverage of BioNano data, we then
generated an optical consensus map, which was implemented to assemble 1,897
super-scaffolds with an N50 of 5.87 Mb (assembly v2). Visualization of alignments
between genome sequences and BioNano optical maps was performed by
BioNano Access software (v1.5.1).
Pseudo-chromosomes assembly using the high-density genetic map. For
pseudo-chromosomes assembly, markers of the high-density B. juncea genetic
map were aligned to SY assembly V.2 by BWA (v. 0.7.8)85 mem. We set a threshold
of at least three linked markers to order and orientate the contigs. Contigs
which showed conflicts to the genetic map were called as potential mis-joins
and checked based on marker continuity. A total of 35 mis-joins were found in
2,329 contigs and split to give 2,364 contigs after correction (Supplementary
Table2). Subsequently, the software Chromonomer (v.1.07, http://catchenlab.
life.illinois.edu/chromonomer/manual/) was used to construct the initial
pseudo-chromosomes of SY, with default parameters, following the internationally
agreed nomenclature for Brassica chromosomes (http://www.brassica.info/
resource/maps/lg-assignments.php).
Pseudo-chromosomes validation using Hi-C. To avoid artificial bias, the following
type of reads were removed: (1) reads with ≥10% unidentified nucleotides (N);
(2) reads with >10 bp aligned to the adaptor, allowing ≤10% mismatches; (3) reads
with >50% bases having phred quality < 5. The filtered Hi-C reads were aligned to
the initial pseudo-chromosome genome by BWA (v0.7.8)85 with default parameters.
Reads were excluded from subsequent analysis if they did not align within 500 bp
of a restriction site. Only uniquely mapped reads and valid paired-end ditags were
used to validate the pseudo-chromosome sequences. The scaffolds of assembly V3
were used to make the Hi-C map by HiCPlotter86, and the interaction matrix of
each chromosome was visualized with heat maps at the 25-kb resolution. A total
of 165 mis-joined contigs were identified and manually broken using Juicebox24
according to the discrete chromatin interaction pattern. Of these, 150 mis-joined
contigs, which lacked sufficient linked markers (three or more per contig or
subcontig), were corrected and ordered by Hi-C contact map. Next, 13 mis-joins
showing conflicts between the results of Hi-C data and the high-density map were
broken, then re-clustered and ordered according to the Hi-C contact signal. Two
remaining unanchored contigs that could not be anchored by the genetic map were
repositioned to their pseudo-chromosome based on the Hi-C data.
Assessment of SY genome quality. The 1,440 conserved protein models in the
BUSCO embryophyta_odb9 dataset (https://busco.ezlab.org/frame_wget.html) and
the 248 conserved protein models in the CEGMA dataset (http://korflab.ucdavis.
edu/dataseda/cegma/) were searched against the SY genome by using the BUSCO
(v2)87 and the CEGMA (v. 2.5)88 programs with default parameters. Eighty-one
BAC sequences and 2,567 BAC-end sequences from the PM BAC library were
aligned to the SY genome by LASTZ89 with parameters (M = 254, K = 4,500,
L = 3,000, Y = 15,000; --seed = match12 --step = 20 --identity = 85). LTRharvest90
(with parameters --similar 85.00 --vic 10 --seed 30 --seqids yes --motif TGCA
--motifmis 1 --minlenltr 100 --maxlenltr 3,500 --mindistltr 1,000 --maxdistltr
20,000 --mintsd 4 --maxtsd 20) and LTR_FINDER91 (with parameters: --w 2 --l 100
--L 3,500 --d 1,000 --D 20,000 --M 0.3) were used to de novo predict the candidate
LTR-RTs (full-length LTRs retrotransposon) in the SY assembly sequences.
LTR_retriever92 was then used to combine and refactor all the candidates to get the
final full-length LTR-RTs. LAI27 was calculated based on the formula: LAI = (intact
LTR-RTs length/total LTR-RTs length) × 100%. As recommended by the steering
group of the Multinational Brassica Genome Project (https://www.brassica.info/),
the consistency of syntenic gene ordering was evaluated by exploiting the linkage
mapping information depicted by the genome-ordered graphical genotypes28.
Protein sequences of annotated HC genes from B. juncea vars. SY, T84-66 (ref. 19)
and Varuna25, both progenitors B. rapa29 and B. nigra30, and previously reported
B. napus cv. ZS11 (ref. 31) were reciprocal aligned using BLASTP with an E-value
cutoff of 1e−5. The reciprocal best hit for each alignment was used to build
whole-genome synteny between SY and the other five Brassica subgenomes by
MCScanX93.
Detailed procedures for the SY genome annotation are provided in
theSupplementary Note.
Genome blocks and centromere detection. We first constructed the three
subgenomes (LF, MF1 and MF2) following methods described previously94. Then,
we defined the genomic blocks in SY based on the syntenic relationship of the
B. juncea and A. thaliana genomes95. We aligned the A subgenome centromeric
repeat sequences (CentBrs, CRB and TR805)34,35 and the B subgenome centromeric
repeat sequences (CRB, pBNBH35 and CLs)30,35–37 to the SY assembly using BLAST
(E-value 1e−5). The pericentromeric regions of A subgenome were detected using
peri-centromere-specific retrotransposons and the tandem repeat sequence TR238
(ref. 35), whereas the pericentromeric regions of B subgenome contained more
LTR/gypsy elements30. Then, the densities of centromeric repeat sequences were
calculated to detect the centromere locations.
Re-sequencing, reads mapping and SNP calling. A panel of 480 mustard
accessions (Supplementary Table18) were self-pollinated over multiple generations
before re-sequencing. Genomic DNA extracted from fresh leaves was used for
350-bp Illumina libraries preparation. Sequencing protocols were the same as
mentioned above. A total of 7.01 Tb (~14.48 Gb per sample) of clean data was
generated after removing reads with ≥10% unidentified nucleotides (N), >10
nucleotides aligned to the adaptor or of which >50% bases had Phred quality
scores less than 5. The paired-end reads were mapped to the SY genome using
BWA (v0.7.8)85 with the command ‘mem --t 4 --k 32 --M’. Duplicated reads were
removed with SAMtools (v.0.1.19)96. The genomic variants for each accession
were then identified with the HaplotypeCaller module and the GVCF model by
Genome Analysis Toolkit97 (GATK) software. All the GVCF files were merged. The
high-quality SNPs and InDels were created in the HaplotypeCaller module filtered
with the following four parameters: depth for individual ≥ 4, genotype quality for
individual ≥ 5, minor allele frequency (MAF) ≥ 0.05, with missing rate ≤ 0.1 and
heterozygous rate < 0.1. The identified SNPs and InDels were further annotated
with ANNOVARtool (v2013-05-20)98, and divided into the following groups:
variations occurring in intergenic regions, within 1 kb upstream (downstream) of
transcription start (stop) sites, in coding sequences and in introns.
Population structure and phylogenetic analyses. The population genetic structure
was examined using the program ADMIXTURE (v1.23)99 with K values (the
putative number of populations) from 2 to 10. The K = 6 value was chosen because
clusters maximized the marginal likelihood. To better clarify the relationships of
B. juncea accessions, 390 accessions with the genetic components of larger than
0.6 were retained for the further analysis. To construct maximum-likelihood
phylogeny, we screened 30,609 synonymous SNPs to reduce influences of natural
or artificial selection. Phylogenetic tree analysis was performed using IQ-TREE
(v1.6.6)100, based on the best model (GTR + F + ASC + R7) determined by the
Bayesian information criterion. Bootstrap support values were calculated using
the ultrafast bootstrap approach (UFboot) with 1,000 replicates. Five known
closely related species A. thaliana, Crambe hispanica, Cardamine hirsuta, Eutrema
halophilum and Eutrema salsugineum were used as outgroups. The phylogenetic
tree was visualized by the online tool EvolView (https://www.evolgenius.info//
evolview/). PCAs were done by GCTA101. The population relatedness and
NATURE GENETICS | www.nature.com/naturegenetics
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles NATuRE GENETICS
migration events were inferred using TreeMix102. We ran the tree with the group
1 as the root group and made this the base tree topology. Then we ran TreeMix
using introducing migration events from 1 to 6. To detect admixture, we computed
D-statistics103 based on ABBA and BABA SNP frequency differences. For a triplet
of taxa P1, P2 and P3, and an outgroup O, that follows the phylogeny of (((P1,
P2), P3), O), a D statistic significantly different from zero indicates P3 exchanged
gene with P1 (D value 0) or P2 (D value >0). Then, the f-branch statistic calculated
introgressions among the six groups by the software package Dsuite104. The fd
statistic105 was used to calculate the fraction of introgression in G4 from G2 in
100-SNP windows, which signifies gene flow when 0 < fd < 1.
Nucleotide diversity (π) and fixation index (FST) were calculated by vcftools106
and pairwise genetic distance was calculated by Arlequin (v.3.5.2.2)107. To estimate
and compare the pattern of LD among different groups, the squared correlation
coefficient (r2) between pairwise SNPs was computed using the PopLDdecay
(v.3.40)108 software. Parameters in the program were --MaxDist 500 --MAF 0.05
--Miss 0.1. The average r2 value was calculated for pairwise markers in a 500-kb
window and averaged across the whole genome.
To construct subgenome trees, we selected 390 B. juncea accessions with
genetic components greater than 60% in each group and 68 B. rapa and 11
B. nigra samples (Supplementary Table24). We selected 14,264 and 10,629
synonymous SNPs for the A and B subgenomes, respectively, filtered with the
following processes: depth for individual ≥ 4, missing rate ≤ 0.1, MAF > 5%. The
maximum-likelihood phylogeny for each subgenome was constructed using
IQ-TREE (v1.6.6)100 based on the optimal models (TVM + F + ASC + R6) following
the same pipeline implemented as that for the B. juncea phylogeny.
Pairwise identity-by-descent detection. To investigate genome-wide introgression
between G4 and G2, we identified haplotypes in the G4 accessions that were
identical by descent (IBD) with individuals from both the original source of
diversification, the G5 leaf mustard, and the source of introgression, the G2
yellow-seeded mustard, following an approach described previously109. To
estimate the frequency of shared haplotypes along individual chromosomes, each
chromosome was divided into bins of 10 kb with a sliding window of 5 kb, and the
number of recorded IBD tracts between G4 and the two groups (G2 and G5) was
computed per bin. As the total number of pairwise comparisons differed between
the groups, these numbers were normalized from 0 (no IBD detected) to 1 (IBD
shared by all individuals within the group). The normalized IBD between G4
and the G2 (nIBDG2) and the normalized IBD between G4 and the G5 (nIBDG5)
were then used to calculate the rIBD (nIBDG2 − nIBDG5). Finally, the putative
introgression segments from the G2 to each of the G4 accessions were identified.
Estimation of divergence time and demographic history. With genome-scale
characterization of the divergence of orthologous genes, we managed to date the
divergence between B. rapa A genome and B. juncea A subgenome, between B.
nigra B genome and B. juncea B subgenome, and between Brassica and Arabidopsis.
The synonymous divergence (KS) values for A. thaliana, B. rapa, B. nigra, and A
and B subgenomes of B. juncea were calculated using the KA/KS Calculator (v2.0)110.
The divergence time between species was calculated as KS/2 µ, where µ is the
mutation rate (1.5 × 10−8 ~ 9 × 10−9 per synonymous site111).
SMC++ (v1.13)112 was used to estimate the divergence time and historical
Ne among different groups of B. juncea. For normalizing the population size, we
selected seven different samples from each group. Generations were calculated by
the upper and lower mutation rates of 1.5 × 10−8 and 9 × 10−9 per synonymous site
for each generation111, and the generation time was 1 year.
Organellar genome analysis. The CP genomes were assembled by NOVOPlasty113
using genome re-sequencing data. After manually correcting the orientation
of the two inverted repeats, the assembled CP genomes were annotated by
GeSeq114. The InDel variants in CP genomes of B. juncea were identified
through sequence alignment and confirmed by PCR (Extended Data Fig.2a).
The maximum-likelihood phylogeny of CP genomes was constructed based
on high-quality variants (variants with >20% missing calls and MAF < 0.01)
using RAxML (v8.0.17)115 with the GTRGAMMAI model. A bootstrap of 1,000
repetitions was used to assess the reliability of the phylogeny reconstructed. The
MT genomes were assembled by Celera Assembler116 with default parameters
using PacBio reads of ten B. juncea accessions. For the mitotype analysis, an InDel
and a reported SNP locus45 were identified by sequence alignment and confirmed
by PCR (Extended Data Fig.2b). Phylogenetic tree analysis of MT genomes
was performed through IQ-TREE (v1.6.6)100 using the best model (HKY + F)
determined by the Bayesian information criterion with 1,000 bootstrap replicates.
Measurement and statistical analysis of agronomic traits. The 390 B. juncea
accessions were grown in four locations: Guiyang (Guizhou, E106.72/N026.58,
short-day, mild-winter), Xiangtan (Hunan, E112.90/N027.86, short-day,
mild-winter), Kunming (Yunnan, E102.72/N025.04, long-day, subtropical) and
Urumqi (Xinjiang, E087.60/N043.80, long-day, continental steppe with large
diurnal temperature differences) in 2018 (designated G18, X18, K18 and U18,
respectively). The field trials were conducted with two replications. The flowering
time was recorded as days to flowering by 25% plants. Open pollinated seeds
were harvested and dried. The mean weight of a thousand seeds from the three
replications was used for further analysis. Statistical analyses of phenotypic data
were performed with the R packages Hmisc (v4.1.1)117 and Psych (v1.8.4)118.
GWAS analysis. Only SNPs with MAF ≥ 0.05 and missing rate ≤ 0.1 in a
population were used to carry out GWAS. This resulted in 4,423,439 SNPs that
were used in GWAS for 390 B. juncea accessions. We performed GWAS using
GEMMA (the genome-wide efficient mixed-model association) program119 under
the mixed-linear model. The top three PCs were used for population-structure
correction. The genetic relationship between individuals was modeled as a random
effect using the kinship (K) matrix. Significant P-value thresholds (P < 10−6 and
10−5 for flowering time and TSW, respectively) were set to control the genome-wide
type I error rate.
Selective-sweep analysis. The XP-CLR score were calculated using the XP-CLR120
package with sliding windows of 10 kb that had a 5-kb overlap between adjacent
windows. The top 5% regions were assigned to candidate selective regions, and
genes in these regions were considered as candidate genes.
Transcriptome analysis. Total RNA was isolated from a sampled organ with two
biological replicates at a specific developmental stage to investigate expression
of the genes associated with formation of special organs for enlarged roots and
tuber stems. As above, RNA-seq libraries were constructed and sequenced on
an Illumina X Ten. The clean reads were mapped against the SY genome using
TopHat (v2.0.12)121 software. The number of reads mapped was counted using
HTSeq (v0.6.1)122 and then FPKM values were calculated for each gene. Transcripts
of less than one per million mapped reads were ignored. Analysis of differential
gene expression between two samples was performed using the DESeq R package
(v1.18.0)123. Genes with an adjusted P value < 0.05 found by DESeq were assigned
as differentially expressed. Procedures for the RT–qPCR analysis are provided in
theSupplementary Note.
Reporting Summary. Further information on research design is available in
theNature Research Reporting Summary linked to this article.
Data availability
The genome sequence and annotation data for B. juncea var. SY, the re-sequencing
data for 480 B. juncea accessions and transcriptome data are accessible under
NCBI BioProject no. PRJNA615316. For Functional annotation of the SY genome,
the SwissProt (https://ftp.uniprot.org/pub/databases/uniprot/current_release/
knowledgebase/complete/uniprot_sprot.fasta.gz/), NR (https://ftp.ncbi.nlm.nih.
gov/blast/db/FASTA/nr.gz/) and KEGG (release 53; https://www.genome.jp/
kegg/brite.html) databases were used. Seeds of accessions used, phenotype data
and sequences of the CP and MT genomes reported here are available from the
corresponding authors upon request.Source data are provided with this paper.
References
76. Lieberman-Aiden, E. et al. Comprehensive mapping of long-range
interactions reveals folding principles of the human genome. Science 326,
289–293 (2009).
77. Wu, Z. K. et al. Evaluation of linkage disequilibrium pattern and association
study on seed oil content in Brassica napus using ddRAD sequencing. PLoS
ONE 11, e0146383 (2016).
78. Xie, W. B. et al. Parent-independent genotyping for constructing an
ultrahigh-density linkage map based on population sequencing. Proc. Natl
Acad. Sci. USA 107, 10578–10583 (2010).
79. Wu, Y., Bhat, P. R., Close, T. J. & Lonardi, S. Ecient and accurate
construction of genetic linkage maps from the minimum spanning tree of a
graph. PLoS Genet. 4, e1000212 (2008).
80. Marçais, G. & Kingsford, C. A fast, lock-free approach for ecient
parallel counting of occurrences of k-mers. Bioinformatics 27,
764–770 (2011).
81. Boetzer, M. & Pirovano, W. SSPACE-LongRead: scaolding bacterial dra
genomes using long read sequence information. BMC Bioinformatics 15,
211 (2014).
82. English, A. C. et al. Mind the gap: upgrading genomes with Pacic
Biosciences RS long-read sequencing technology. PLoS ONE 7,
e47768 (2012).
83. Chin, C. S. et al. Nonhybrid, nished microbial genome assemblies
from long-read SMRT sequencing data. Nat. Methods 10,
563–569 (2013).
84. Walker, B. J. et al. Pilon: an integrated tool for comprehensive microbial
variant detection and genome assembly improvement. PLoS ONE 9,
e112963 (2014).
85. Li, H. & Durbin, R. Fast and accurate short read alignment with
Burrows–Wheeler Transform. Bioinformatics 25, 1754–1760 (2009).
86. Akdemir, K. C. & Chin, L. HiCPlotter integrates genomic data with
interaction matrices. Genome Biol. 16, 198 (2015).
NATURE GENETICS | www.nature.com/naturegenetics
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles
NATuRE GENETICS
87. Simão, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V. & Zdobnov,
E. M. BUSCO: assessing genome assembly and annotation completeness
with single-copy orthologs. Bioinformatics 31, 3210–3212 (2015).
88. Parra, G., Bradnam, K. & Korf, I. CEGMA: a pipeline to accurately
annotate core genes in eukaryotic genomes. Bioinformatics 23,
1061–1067 (2007).
89. Kent, W. J. BLAT—e BLAST-like alignment tool. Genome Res. 12,
656–664 (2002).
90. Ellinghaus, D., Kurtz, S. & Willhoe, U. LTRharvest, an ecient and
exible soware for de novo detection of LTR retrotransposons. BMC
Bioinformatics 9, 18 (2008).
91. Xu, Z. & Wang, H. LTR_FINDER: an ecient tool for the
prediction of full-length LTR retrotransposons. Nucleic Acids Res. 35,
W265–W268 (2007).
92. Ou, S. & Jiang, N. LTR_retriever: a highly accurate and sensitive program
for identication of long terminal repeat retrotransposons. Plant Physiol.
176, 1410–1422 (2018).
93. Wang, Y. et al. MCScanX: a toolkit for detection and evolutionary analysis
of gene synteny and collinearity. Nucleic Acids Res. 40, e49 (2012).
94. Cheng, F. et al. Deciphering the diploid ancestral genome of the
mesohexaploid Brassica rapa. Plant Cell 25, 1541–1554 (2013).
95. Schranz, M. E., Lysak, M. A. & Mitchell-Olds, T. e ABC’s of comparative
genomics in the Brassicaceae: building blocks of crucifer genomes. Trends
Plant Sci. 11, 535–542 (2006).
96. Li, H. et al. e Sequence Alignment/Map format and SAMtools.
Bioinformatics 25, 2078–2079 (2009).
97. McKenna, A. et al. e genome analysis toolkit: a MapReduce framework
for analyzing next-generation DNA sequencing data. Genome Res. 20,
1297–1303 (2010).
98. Wang, K., Li, M. & Hakonarson, H. ANNOVAR: functional annotation of
genetic variants from high-throughput sequencing data. Nucleic Acids Res.
38, e164 (2010).
99. Alexander, D. H., Novembre, J. & Lange, K. Fast model-based estimation of
ancestry in unrelated individuals. Genome Res. 19, 1655–1664 (2009).
100. Nguyen, L. T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a
fast and eective stochastic algorithm for estimating maximum-likelihood
phylogenies. Mol. Biol. Evol. 32, 268–274 (2015).
101. Yang, J., Lee, S. H., Goddard, M. E. & Visscher, P. M. GCTA: a tool for
genome-wide complex trait analysis. Am. J. Hum. Genet. 88, 76–82 (2011).
102. Pickrell, J. K. & Pritchard, J. K. Inference of population splits and mixtures
from genome-wide allele frequency data. PLoS Genet. 8, e1002967 (2012).
103. Durand, E. Y., Patterson, N., Reich, D. & Slatkin, M. Testing for ancient
admixture between closely related populations. Mol. Biol. Evol. 28,
2239–2252 (2011).
104. Malinsky, M., Matschiner, M. & Svardal, H. Dsuite—Fast D-statistics and
related admixture evidence from VCF les. Mol. Ecol. Resour. 21,
584–595 (2021).
105. Martin, S. H., Davey, J. W. & Jiggins, C. D. Evaluating the use of ABBA-BABA
statistics to locate introgressed loci. Mol. Biol. Evol. 32, 244–257 (2015).
106. Danecek, P. et al. e variant call format and VCFtools. Bioinformatics 27,
2156–2158 (2011).
107. Excoer, L. & Lischer, H. E. L. Arlequin suite ver 3.5: a new series of
programs to perform population genetics analyses under Linux and
Windows. Mol. Ecol. Resour. 10, 564–567 (2010).
108. Zhang, C. et al. PopLDdecay: a fast and eective tool for linkage
disequilibrium decay analysis based on variant call format les.
Bioinformatics 35, 1786–1788 (2019).
109. Bosse, M. et al. Genomic analysis reveals selection for Asian genes in
European pigs following human-mediated introgression. Nat. Commun. 5,
4392 (2014).
110. Zhang, Z. et al. KaKs_Calculator: calculating Ka and Ks through model
selection and model averaging. Genomics Proteomics Bioinformatics 4,
259–263 (2006).
111. Qi, X. et al. Genomic inferences of domestication events are corroborated
by written records in Brassica rapa. Mol. Ecol. 26, 3373–3388 (2017).
112. Terhorst, J., Kamm, J. A. & Song, Y. S. Robust and scalable inference of
population history from hundreds of unphased whole genomes. Nat. Genet.
49, 303–309 (2017).
113. Dierckxsens, N., Mardulyn, P. & Smits, G. NOVOPlasty: de novo assembly
of organelle genomes from whole genome data. Nucleic Acids Res. 45,
e8 (2016).
114. Tillich, M. et al. GeSeq – versatile and accurate annotation of organelle
genomes. Nucleic Acids Res. 45, W6–W11 (2017).
115. Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and
post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014).
116. Gennady, D. et al. Consensus generation and variant detection by Celera
Assembler. Bioinformatics 24, 1035–1040 (2008).
117. Harrell, F. Hmisc: Harrell Miscellaneous. https://CRAN.R-project.org/
package=Hmisc (2018).
118. Revelle, W. Psych: procedures for personality and psychological Research.
https://CRAN.R-project.org/package=psych (2018).
119. Zhou, X. & Stephens, M. Genome-wide ecient mixed-model analysis for
association studies. Nat. Genet. 44, 821–824 (2012).
120. Chen, H., Patterson, N. & Reich, D. Population dierentiation as a test for
selective sweeps. Genome Res. 20, 393–402 (2010).
121. Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence
of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).
122. Mortazavi, A., Williams, B. A., McCue, K., Schaeer, L. & Wold, B.
Mapping and quantifying mammalian transcriptomes by RNA-seq. Nat.
Methods 5, 621–628 (2008).
123. Wang, L., Feng, Z., Wang, X., Wang, X. & Zhang, X. DEGseq: an R package
for identifying dierentially expressed genes from RNA-seq data.
Bioinformatics 26, 136–138 (2010).
Acknowledgements
This research was supported by Nat ional Natural Science Foundation of China (U20A2029),
National Key Research and Developmental Program of China (2017YFD0101702) and
Key Projects of Hunan Provincial Education Department (18A086). H.A. and J.C.P. were
supported by the US Department of Energy (DOE HDTRA 1-16-1-0048) and the US
National Science Foundation (NSF IOS 1339156). X. Wu at O CRI, CAAS, W. Qian at Xi’nan
University, J. Zhang and D. Liu at Sichuan Academy of Agricultural Science and Y. Yuan at
Tibet Academy of Agricultural Science kindly provided a part of the accessions used in this
study. We thank Y. Liu at Johns Hopkins University and Q. Yang at Huazhong Agricultural
University for constructive discussion and feedback on this manuscript.
Author contributions
Z.L. and W.H. conceived and designed the study. L.K., L.Q., M.Z., L.C., H.C. and H.L.
performed data analysis. L. Yang, L. You, B.Y., X.L. and X.X. managed the fieldwork
and prepared the samples. B.Y., M.Y., Y.G., D. Zhang, Y.R., D.J., D. Zhou, H.X. and Y.W.
measured the agronomic traits. L.Q. and T.W. performed GWAS analysis. L.K., M.Z.,
L.C. and H.C. performed RNA-seq data analysis. H.A. and P.B. carried out the f-branch
analysis. L.K., L.Q., M.Z. and Z.L. wrote the manuscript. S.-V.S., H.A., P.B., A.S.M., J.C.P.
and R.J.S. revised the manuscript and gave suggestions and comments. All authors read
and approved the final manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Extended data is available for this paper at https://doi.org/10.1038/s41588-021-00922-y.
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41588-021-00922-y.
Correspondence and requests for materials should be addressed to
Wei Hua or Zhongsong Liu.
Peer review information Nature Genetics thanks Caroline Belser and Xiaowu Wang for
their contribution to the peer review of this work.
Reprints and permissions information is available at www.nature.com/reprints.
NATURE GENETICS | www.nature.com/naturegenetics
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles NATuRE GENETICS
Extended Data Fig. 1 | Distribution of genomic blocks along the eighteen chromosomes of the Brassica juncea var. Sichuan Yellow genome. Genome
blocks on eighteen chromosomes were assigned to the subgenomes LF (orange), MF1 (dark cyan), and MF2 (deep sky blue). The 24 conserved genomic
blocks are defined and labelled from A to X (colored) based on the syntenic relationship of the B. juncea and A. thaliana genomes. The centromeres in the
SY genome are shown as black. The orientation of chromosomes is according to international standards such that the centromeres are toward the top of
the chromosome.
NATURE GENETICS | www.nature.com/naturegenetics
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles
NATuRE GENETICS
Extended Data Fig. 2 | Three types of Brassica juncea chloroplast and mitochondrial genomes. a, Three B. juncea chloroplast genome types were identified
by sequence alignment. PCR validation of the two InDels in the chloroplast genomes of B. juncea accessions. b, Three B. juncea mitotypes were shown by
sequence alignment. PCR validation of the InDel and the SNP in the mitochondrial genomes of B. juncea accessions. The amplified DNA was treated with
the restriction enzyme EarI. All the PCR experiments were repeated independently for three times with similar results. The primers used for PCR were
listed in Supplementary table42. Source data for the gels were provided as a Source Data file.
NATURE GENETICS | www.nature.com/naturegenetics
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles NATuRE GENETICS
Extended Data Fig. 3 | See next page for caption.
NATURE GENETICS | www.nature.com/naturegenetics
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles
NATuRE GENETICS
Extended Data Fig. 3 | Estimation of introgressions among the six groups of Brassica juncea. a, Treemix analysis. Migration arrows are colored according
to their weight. Horizontal branch length is proportional to the amount of genetic drift that has occurred on the branch. Scale bar shows ten times the
average standard error of the entries in the sample covariance matrix. b, f-branch values. c, fd values from G2 to G4. The center lines in box plots indicate
the median values, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. Whiskers extend to data no more than 1.5
times the interquartile range. p-value was calculated using two-sided t-test.
NATURE GENETICS | www.nature.com/naturegenetics
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles NATuRE GENETICS
Extended Data Fig. 4 | Co-evolution analysis of the flowering time genes SRR1 (BjuA10g14550S) and VIN3 (BjuB05g31990S) in Brassica juncea. a, LD
analysis between SRR1 and VIN3 genes. b, The combinations of both SRR1 and VIN3 haplotypes (SRR1-A10-Hap1 + VIN3-B05-Hap1, SRR1-A10-Hap2 +
VIN3-B05-Hap2, and SRR1-A10-Hap3 + VIN3-B05-Hap3). c, Boxplot showing comparison between these three haplotypes corresponding to accessions
across four environments. Box edges represent the 0.25 and 0.75 quantiles with the median values shown by bold lines. Whiskers extend to data no more
than 1.5 times the interquartile range, and remaining data are indicated by dots. p-value was calculated with two-sided t-test. na, data missing (G1 group
did not flower in Kunming).
NATURE GENETICS | www.nature.com/naturegenetics
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles
NATuRE GENETICS
Extended Data Fig. 5 | Genome-wide selective sweep scan and GWAS for seed weight in Brassica juncea. a, Genome-wide distribution of selective-sweep
signals identified through comparisons between G5 or G6 with G2 using XP-CLR values (sliding window = 10 kb, step = 1 kb). The thousand seed
weight candidate genes in the selection regions are labeled. b and e, Local Manhattan plot showing the 0.60 - 0.65 Mb and 41.48 - 41.50 Mb
region on chromosomes A04 and B05, respectively. The green plots represent the position of these SNPs in CYP78A9 (BjuA04g00760S) and CaM7
(BjuB05g28000S). Three and one SNPs in CYP78A9 and CaM7 are significantly associated with thousand seed weight, respectively. The heatmaps
span the SNP markers that show linkage disequilibrium (LD) with the most strongly associated SNPs. The grey dashed lines indicate the significance
threshold (-log10p = 5.0). c and f, Comparison of conserved SNPs specific to six groups in CYP78A9 and CaM7 gene region, respectively. Two haplotypes
with frequency greater than 0.01 were identified in CYP78A9 and CaM7 gene region, respectively. d and g, Comparison in thousand seed weight between
accessions of three haplotypes in CYP78A9 and CaM7 gene region, respectively. Box edges represent the 0.25 quantile and 0.75 quantile with the median
values shown by bold lines. Whiskers extend to data no more than 1.5 times the interquartile range, and remaining data are indicated by dots. p-value was
calculated with two-sided t-test.
NATURE GENETICS | www.nature.com/naturegenetics
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Articles NATuRE GENETICS
Extended Data Fig. 6 | Identification of candidate genes for stem tuber formation in stem mustard. a, Genome-wide distribution of selective sweeps
in stem mustard for stem tuber formation. b, Haplotypes for the candidate gene GRF7 (BjuA05g02460S) in stem mustard (T) and leaf mustard (I). c,
Haplotypes for the candidate gene IAA33 (BjuA10g12920S) in stem mustard (T) and leaf mustard (I). d, The expression levels of GRF7 and IAA33 in
non-stem mustard, stem mustard (one week after the stem swelled, three weeks after the stem swelled) (from left to right) were estimated based on
FPKM values. In box plots, the center lines indicate the median values and the bottom and top edges of the box indicate the 25th and 75th percentiles,
respectively. Whiskers extend to data no more than 1.5 times the interquartile range, and remaining data are indicated by dots. p-value was calculated
using two-sided t-test. Scale bars, 2 cm.
NATURE GENETICS | www.nature.com/naturegenetics
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Content courtesy of Springer Nature, terms of use apply. Rights reserved
1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-scale
personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By accessing,
sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these purposes, Springer
Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription (to
the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue, royalties,
rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal content cannot be
used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any other, institutional
repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or content on
this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature may revoke this
licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied with
respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law, including
merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed from
third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com
Available via license: CC BY 4.0
Content may be subject to copyright.
