Population genomic insights into the domestication of Brassica juncea var. tumida

Abstract

Brassica juncea var. tumida, commonly known as Zha Cai, is a pickled stem mustard widely cultivated in southern China. Its most distinctive trait is the swollen stem, which serves as the primary economic organ for harvest. However, the origin and domestication history of tumida remain unclear, hindering genetic improvement and molecular breeding efforts. Here, we assembled a chromosome-level genome of the landrace ‘YAXY’ from Chongqing—the center of tumida diversity—totaling 909.1 Mb with a contig N50 of 4.17 Mb. We also collected and resequenced 203 tumida accessions across southern China. By integrating the ‘YAXY’ reference genome with population data, we generated the first comprehensive tumida variation dataset, comprising 1.38 million single-nucleotide polymorphisms (SNPs) and 0.27 million insertions and deletions (InDels). Joint analysis of the newly sequenced tumida population and 504 public B. juncea datasets revealed that tumida and leafy types from southern China share a common origin from local oilseed mustard. Tumida domestication was accompanied by a strong genetic bottleneck. Additionally, we conducted genome-wide association studies (GWAS) for 21 agronomic traits and identified candidate genes linked to key domestication traits in tumida. For the swollen stem trait, selective sweep and GWAS analyses jointly identified candidate genes likely involved in lignification. Transcriptome data showed consistent differential expression of BjuA05g15010, the Arabidopsis SAGL1 ortholog, across all swelling stages, suggesting a key role in stem morphogenesis. Collectively, our findings shed light on tumida evolution and provide valuable genomic resources and candidate genes to support genetic research and breeding in B. juncea.

Author summay

^‡ These authors contributed equally to this work.

Acknowledgements

This work was supported by the Fuling Academy of Southwest University Hosted Project, the Special Project of the People’s Government of Fuling District, Chongqing, the China Agriculture Research System (CARS-24-G-22), the Modern Seed Industry Enhancement Project-Tuber Mustard Industrial Cluster, the Precise Identification Project of Agricultural Germplasm Resources in Chongqing, and the Chongqing Municipal Science and Technology Bureau (CSTB2023TIAD-KPX0025).

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Acknowledgements

J.S., X.W., and Y.F. designed the experiments; H.W., Z.G., W.L., L.R., S.Y., Z.Z., and X.Z. performed experiments; H.W., X.C., J.W., Z.G., and L.P. analyzed data; H.W., X.C., J.S., and X.W. wrote the manuscript and substantively revised the manuscript; J.S., X.W., and Y.F. reviewed the manuscript. The final manuscript has been read and approved by all authors.

Data availability

The newly generated raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA025655, CRA026508, CRA026501 and CRA025656) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa. Publicly available resequencing data supporting this study include NCBI SRA PRJNA615316 and PRJNA1148674. For expression analysis of BjuA05g15010 between tumida and integrifolia, we used data from 22 integrifolia samples (PRJNA544908, PRJNA672814, PRJNA800112) and 19 tumida samples (PRJNA289188, PRJNA477240, PRJNA878553). Genome assembly files, annotated protein-coding genes, and GWAS-related file are available in the Figshare repository (https://figshare.com/s/7e8cc1eb750c4c20add7).

Conflicts of interest statement

The authors declared that they have no conflict of interest in relation to this work.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Kang L, Qian L, Zheng M. et al. Genomic insights into the origin, domestication and diversification of Brassica juncea. Nat Genet. 2021; 53:1392-402

[2] Zhang L, Li X, Chang L. et al. Expanding the genetic variation of Brassica juncea by introgression of the Brassica rapa genome. Hortic Res. 2022;9:uhab054

[3] Chang L, Liang J, Zhang L. et al. A complex locus regulates highly lobed-leaf formation in Brassica juncea. Theor Appl Genet. 2023; 136:224

[4] Vaughan JG, Hemingway JS. The utilization of mustards. Econ Bot. 1959; 13:196-204

[5] Qi X-H, Zhang M-F, Yang J-H. Molecular phylogeny of Chinese vegetable mustard (Brassica juncea) based on the internal transcribed spacers (ITS) of nuclear ribosomal DNA. Genet Resour Crop Evol. 2007; 54:1709-16

[6] Zhang L, Li Z, Garraway J. et al. The casein kinase 2 β subunit CK2B1 is required for swollen stem formation via cell cycle control in vegetable Brassica juncea. Plant J. 2020; 104:706-17

[7] Hao Y, Lu F, Pyo SW. et al. PagMYB128 regulates secondary cell wall formation by direct activation of cell wall biosynthetic genes during wood formation in poplar. J Integr Plant Biol. 2024; 66:1658-74

[8] Wang G-L, Wu JQ, Chen YY. et al. More or less: recent advances in lignin accumulation and regulation in horticultural crops. Agronomy. 2023; 13:2819

[9] Yang J, Wang J, Li Z. et al. Genomic signatures of vegetable and oilseed allopolyploid Brassica juncea and genetic loci control-ling the accumulation of glucosinolates. Plant Biotechnol J. 2021; 19:2619-28

[10] Yang J, Zhang C, Zhao N. et al. Chinese root-type mustard provides phylogenomic insights into the evolution of the multi-use diversified allopolyploid Brassica juncea. Mol Plant. 2018; 11:512-4

[11] Qian L, Yang L, Liu X. et al. Natural variations in TT8 and its neighboring STK confer yellow seed with elevated oil content in Brassica juncea. Proc Natl Acad Sci USA. 2025; 122:e2417264122

[12] Yang J, Liu D, Wang X. et al. The genome sequence of allopolyploid Brassica juncea and analysis of differential homoeolog gene expression influencing selection. Nat Genet. 2016; 48:1225-32

[13] Bayer PE, Golicz AA, Scheben A. et al. Plant pan-genomes are the new reference. Nat Plants. 2020; 6:914-20

[14] Gaut BS, Seymour DK, Liu Q. et al. Demography and its effects on genomic variation in crop domestication. Nat Plants. 2018; 4:512-20

[15] Wang W, Mauleon R, Hu Z. et al. Genomic variation in 3,010 diverse accessions of Asian cultivated rice. Nature. 2018; 557: 43-9

[16] Larson G, Piperno DR, Allaby RG. et al. Current perspectives and the future of domestication studies. Proc Natl Acad Sci. 2014; 111:6139-46

[17] Liu P. Chinese Mustard. Beijing, China: China Agriculture Press, 1996, pp 15-23, 45-49, 52-55.

[18] Li E, Bhargava A, Qiang W. et al. The class II KNOX gene KNAT7 negatively regulates secondary wall formation in Ara-bidopsis and is functionally conserved in Populus. New Phytol. 2012; 194:102-15

[19] Wang S, Yang H, Mei J. et al. Rice Homeobox protein KNAT7 integrates the pathways regulating cell expansion and wall stiffness. Plant Physiol. 2019; 181:669-82

[20] Zhang Q, Xie Z, Zhang R. et al. Blue light regulates secondary cell wall thickening via MYC2/MYC4 activation of the NST1-directed transcriptional network in Arabidopsis. Plant Cell. 2018; 30:2512-28

[21] Yu S-I, Kim H, Yun D-J. et al. Post-translational and tran-scriptional regulation of phenylpropanoid biosynthesis path-way by Kelch repeat F-box protein SAGL1. Plant Mol Biol. 2019; 99:135-48

[22] Chen S. The origin and differentiation of mustard varieties in China. Cruciferae Newsletter. 1982; 7:7-10

[23] Hancock AM, Brachi B, Faure N. et al. Adaptation to climate across the Arabidopsis thaliana genome. Science. 2011; 334:83-6

[24] Lobell DB, Burke MB, Tebaldi C. et al. Prioritizing climate change adaptation needs for food security in 2030. Science. 2008; 319:607-10

[25] Hufford MB, Xu X, van Heerwaarden J. et al. Comparative pop-ulation genomics of maize domestication and improvement. Nat Genet. 2012; 44:808-11

[26] Hyten DL, Song Q, Zhu Y. et al. Impacts of genetic bottlenecks on soybean genome diversity. Proc Natl Acad Sci. 2006; 103: 16666-71

[27] Stetter MG, Gates DJ, Mei W. et al. How to make a domesticate. Curr Biol. 2017;27:R896-900

[28] Whiteley AR, Fitzpatrick SW, Funk WC. et al. Genetic rescue to the rescue. Trends Ecol Evol. 2015; 30:42-9

[29] Frankham R. Genetic rescue of small inbred populations: meta-analysis reveals large and consistent benefits of gene flow. Mol Ecol. 2015; 24:2610-8

[30] Shaw RE, Farquharson KA, Bruford MW. et al. Global meta-analysis shows action is needed to halt genetic diversity loss. Nature. 2025; 638:704-10

[31] Breed MF, Harrison PA, Blyth C. et al. The potential of genomics for restoring ecosystems and biodiversity. Nat Rev Genet. 2019; 20:615-28

[32] Lim K-B, Yang TJ, Hwang YJ. et al. Characterization of the cen-tromere and peri-centromere retrotransposons in Brassica rapa and their distribution in related Brassica species. Plant J. 2007; 49:173-83

[33] Koo D-H, Hong CP, Batley J. et al. Rapid divergence of repeti-tive DNAs in Brassica relatives. Genomics. 2011; 97:173-85

[34] Ou S, Su W, Liao Y. et al. Benchmarking transposable element annotation methods for creation of a streamlined, compre-hensive pipeline. Genome Biol. 2019; 20:275

[35] Tarailo-Graovac M, Chen N. Using RepeatMasker to identify repetitive elements in genomic sequences. Curr Protoc Bioin-formatics. 2009; Chapter 25:4.10.1-4.10.14

[36] Besemer J, Borodovsky M. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Res. 2005;33:W451-4

[37] Birney E, Clamp M, Durbin R. GeneWise and Genomewise. Genome Res. 2004; 14:988-95

[38] Grabherr MG, Haas BJ, Yassour M. et al. Full-length tran-scriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011; 29:644-52

[39] Haas BJ, Delcher AL, Mount SM. et al. Improving the Arabidop-sis genome annotation using maximal transcript alignment assemblies. Nucleic Acids Res. 2003; 31:5654-66

[40] Haas BJ, Salzberg SL, Zhu W. et al. Automated eukaryotic gene structure annotation using EVidenceModeler and the pro-gram to assemble spliced alignments. Genome Biol. 2008;9:R7

[41] Hunter S, Apweiler R, Attwood TK. et al. InterPro: the integrative protein signature database. Nucleic Acids Res. 2008;37:D211-5

[42] Emms DM, Kelly S. OrthoFinder: phylogenetic orthology infer-ence for comparative genomics. Genome Biol. 2019; 20:238

[43] Chen C, Chen H, Zhang Y. et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020; 13:1194-202

[44] Goel M, Sun H, Jiao W-B. et al. SyRI: finding genomic rear-rangements and local sequence differences from whole-genome assemblies. Genome Biol. 2019; 20:277

[45] Wingett SW, Andrews S. FastQ screen: a tool for multi-genome mapping and quality control. F1000Res. 2018; 7:1338

[46] Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25: 1754-60

[47] Li H, Handsaker B, Wysoker A. et al. The sequence alignmen-t/map format and SAMtools. Bioinformatics. 2009; 25:2078-9

[48] McKenna A, Hanna M, Banks E. et al. The genome anal-ysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010; 20: 1297-303

[49] Purcell S, Neale B, Todd-Brown K. et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet. 2007; 81:559-75

[50] Browning BL, Browning SR. Genotype imputation with mil-lions of reference samples. Am J Hum Genet. 2016; 98: 116-26

[51] Cingolani P, Platts A, Wang LL. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin). 2012; 6:80-92

[52] Kumar S, Stecher G, Tamura K. MEGA7: molecular evolution-ary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016; 33:1870-4

[53] Bradbury PJ, Zhang Z, Kroon DE. et al. TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics. 2007; 23:2633-5

[54] Tang H, Peng J, Wang P. et al. Estimation of individual admix-ture: analytical and study design considerations. Genet Epi-demiol. 2005; 28:289-301

[55] Malekpour SA, Pezeshk H, Sadeghi M. MSeq-CNV: accurate detection of copy number variation from sequencing of mul-tiple samples. Sci Rep. 2018; 8:4009

[56] Danecek P, Auton A, Abecasis G. et al. The variant call format and VCFtools. Bioinformatics. 2011; 27:2156-8

[57] Zhou X, Stephens M. Genome-wide efficient mixed-model analysis for association studies. Nat Genet. 2012; 44:821-4

[58] Li MX, Yeung JM, Cherny SS. et al. Evaluating the effective numbers of independent tests and significant p-value thresh-olds in commercial genotyping arrays and public imputation reference datasets. Hum Genet. 2012; 131:747-56

[59] Chen S, Zhou Y, Chen Y. et al. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34:i884-90

[60] Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015; 12:357-60

[61] Kovaka S, Zimin AV, Pertea GM. et al. Transcriptome assembly from long-read RNA-seq alignments with StringTie2. Genome Biol. 2019; 20:278