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Journal of Integrative Agriculture  2025, Vol. 24 Issue (4): 1461-1476    DOI: 10.1016/j.jia.2024.04.031
Horticulture Advanced Online Publication | Current Issue | Archive | Adv Search |
The high quality genome of potherb mustard Xuecai (Brassica juncea var. multiceps) provides new insights into leaf shape variation

Shuangping Heng1#, Mengdi Cui1, Xiaolin Li1, Shaoheng Zhang1, Guangzhi Mao1, Feng Xing1, Zhengjie Wan2, Jing Wen3, Jinxiong Shen3, Tingdong Fu3

1 College of Life Sciences, Institute for Conservation and Utilization of Agro-Bioresources in Dabie Mountains, Xinyang Normal University, Xinyang 464000, China

2 College of Horticulture and Forestry, National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, Huazhong Agricultural University, Wuhan 430070, China

3 College of Plant Science and Technology, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China

 Highlights 
The genome of the potherb mustard was sequenced and assembled.
An increase in copy number and elevated transcript expression of the BjRCO genes resulted in the development of multipinnately lobed leaves in Brassica juncea.
Gene editing of the BjRCO genes transformed the leaf shape from multipinnately lobed to simple.
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摘要  
叶用芥菜雪里蕻(XC)是一种十字花科蔬菜,作为一种新鲜的食用和腌制蔬菜而广受欢迎。雪里蕻由于叶片裂刻的存而表现出多回羽状全裂叶片。裂叶的叶片净光合速率高于非裂叶。然而,叶形变异的分子机制仍然是不清楚的。本研究利用HiFi和Hi-C测序技术组装了雪里蕻的基因组。雪里蕻基因组长度为961.72 Mb, N50为6.565 Mb。通过与已测序的四个芥菜基因组进行了比较,鉴定了基因组共线性区域、SNPs和indels。通过芥菜比较基因组测序,在雪里蕻XC A10染色体BjRCO候选区段发现了5个BjRCO基因。大片段重复序列导致了BjRCO基因的拷贝数增加。BjRCO基因在多回羽状全裂叶中的转录表达高于锯齿状叶。BjRCO基因拷贝数增加与高表达共同调控芥菜叶形由简单变复杂。BjRCO基因编辑使芥菜叶形由多回羽状全裂叶片变为简单叶。雪里蕻的高质量基因组测序不仅为叶用芥菜基因组提供了新的见解,而且有助于进一步解析叶型变异的分子机制。我们的研究表明,通过芥菜基因组的比较分析,可以更好地解析重要性状的变异和进化。


Abstract  
The potherb mustard Xuecai (XC) cultivar is a cruciferous vegetable that is popular either fresh or pickled.  Due to the deep notches in the edges of leaves in mustard XC, this plant can be said to have multipinnately lobed leaves.  The net photosynthesis of lobed leaves is significantly greater than that of simple leaves.  However, the molecular mechanism of leaf shape variation has not been determined.  Here, we used HiFi and Hi-C data to assemble the XC genome.  The genome was 961.72 Mb in size, with a contig N50 value of 6.565 Mb.  The XC genome was compared with four previously sequenced mustard genomes, and the genomic collinearity regions, SNPs, and indels were identified.  Five BjRCO genes were found on chromosome (Chr.) A10 in potherb mustard XC when the BjRCO gene locus was compared against other sequenced Bjuncea genomes.  Segmental duplication was found to contribute to the BjRCO gene copy number.  The transcript expression of BjRCO genes was greater in multipinnately lobed leaves than in sawtooth-like leaves.  Together, these findings indicate that both the greater copy number and the expression level of BjRCO genes regulate leaf shape from simple to complex in Bjuncea.  Gene editing of the BjRCO gene from XC changed the leaf shape from multipinnately lobed to simple.  The high-quality XC genome sequence not only provides new insight into Bjuncea leaf-type genomics but also helps in deciphering leaf shape variation.  Our study provides insights into the variation and evolution of important traits in Brassica plants through a comparative analysis of the sequenced genomes.


Keywords:  Brassica juncea       genome sequence       multipinnately lobed leaves       segmental duplication       BjRCO       gene editing  
Received: 01 November 2023   Accepted: 22 March 2024
Fund: 
This study was supported by grants from the National Natural Science Foundation of China (32002056), the Science and Technology Research Key Project of Henan Province, China (242102111138), and the Nanhu Scholars Program for Young Scholars of Xinyang Normal University, China. 
About author:  #Correspondence Shuangping Heng, E-mail: shuangpingheng@126.com

Cite this article: 

Shuangping Heng, Mengdi Cui, Xiaolin Li, Shaoheng Zhang, Guangzhi Mao, Feng Xing, Zhengjie Wan, Jing Wen, Jinxiong Shen, Tingdong Fu. 2025.

The high quality genome of potherb mustard Xuecai (Brassica juncea var. multiceps) provides new insights into leaf shape variation
. Journal of Integrative Agriculture, 24(4): 1461-1476.

Alvim Kamei C L, Pieper B, Laurent S, Tsiantis M, Huijser P. 2020. CRISPR/Cas9-mediated mutagenesis of RCO in Cardamine hirsutaPlants9, 268.

Beier S, Thiel T, Münch T, Scholz U, Mascher M. 2017. MISA-web: A web server for microsatellite prediction. Bioinformatics33, 2583–2585.

Benson G. 1999. Tandem repeats finder A program to analyze DNA sequences. Nucleic Acids Research27, 573–580.

Burton J N, Adey A, Patwardhan R P, Qiu R, Kitzman J O, Shendure J. 2013. Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions. Nature Biotechnology31, 1119–1125.

Chang L, Liang J, Zhang L, Zhang Z, Cai X, Wu J, Wang X. 2023. A complex locus regulates highly lobed-leaf formation in Brassica junceaTheoretical and Applied Genetics136, 224.

Chen C, Chen H, Zhang Y, Thomas H R, Frank M H, He Y, Xia R. 2020. TBtools: An integrative Toolkit developed for interactive analyses of big biological data. Molecular Plant13, 1194–1202.

Chen C, Wu Y, Xia R. 2022. A painless way to customize Circos plot from data preparation to visualization using TBtools. iMeta1, e35.

Chen S, Zhou Y, Chen Y, Gu J. 2018. fastp: An ultra-fast all-in-one FASTQ preprocessor. Bioinformatics34, i884-i890.

Cheng H, Concepcion G T. 2021. Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nature Methods18, 170–175.

Danecek P, McCarthy S A, Birol I. 2017. BCFtools/csq Haplotype-aware variant consequences. Bioinformatics33, 2037–2039.

Feng X, Yang X, Zhong M, Li X, Zhu P. 2022. BoALG10, an α-1,2 glycosyltransferase, plays an essential role in maintaining leaf margin shape in ornamental kale. Horticulture Research9, uhac137.

Flynn J M, Hubley R. 2020. RepeatModeler2 for automated genomic discovery of transposable element families. Proceedings of the National Academy of Sciences of the United States of America117, 9451–9457.

Haas B J, Salzberg S L, Zhu W, Pertea M, Allen J E, Orvis J, White O, Buell C R, Wortman J R. 2008. Automated eukaryotic gene structure annotation using EVidenceModeler and the Program to Assemble Spliced Alignments. Genome Biology9, R7.

Hajheidari M. 2019. Autoregulation of RCO by low-affinity binding modulates cytokinin action and shapes leaf diversity. Current Biology29, 4183–4192.e4186.

Heng S, Gao J, Wei C, Chen F, Li X, Wen J, Yi B, Ma C, Tu J, Fu T, Shen J. 2018. Transcript levels of orf288 are associated with the hau cytoplasmic male sterility system and altered nuclear gene expression in Brassica junceaJournal of Experimental Botany69, 455–466.

Heng S, Huang H, Cui M, Liu M, Lv Q, Hu P, Ren S, Li X, Fu T, Wan Z. 2020. Rapid identification of the BjRCO gene associated with lobed leaves in Brassica juncea via bulked segregant RNA-seq. Molecular Breeding40, 42.

Hu L, Zhang H, Sun Y, Shen X, Amoo O, Wang Y, Fan C, Zhou Y. 2020. BnA10.RCO, a homeobox gene, positively regulates leaf lobe formation in Brassica napus L. Theoretical and Applied Genetics133, 3333–3343.

Ji G, Liang C, Cai Y, Pan Z, Meng Z, Li Y, Jia Y, Miao Y, Pei X, Gong W, Wang X, Gao Q, Peng Z, Wang L, Sun J, Geng X, Wang P, Chen B, Wang P, Zhu T, et al. 2021. A copy number variant at the HPDA-D12 locus confers compact plant architecture in cotton. New Phytologist229, 2091–2103.

Kang L, Qian L, Zheng M, Chen L, Chen H, Yang L, You L, Yang B, Yan M, Gu Y, Wang T, Schiessl S V, An H, Blischak P, Liu X, Lu H, Zhang D, Rao Y, Jia D, Zhou D, et al. 2021. Genomic insights into the origin, domestication and diversification of Brassica junceaNature Genetics53, 1392–1402.

Kawakatsu Y, Sakamoto T, Nakayama H, Kaminoyama K, Igarashi K, Yasugi M, Kudoh H, Nagano A J, Yano K, Kubo N, Notaguchi M, Kimura S. 2021. Combination of genetic analysis and ancient literature survey reveals the divergence of traditional Brassica rapa varieties from Kyoto, Japan. Horticulture Research8, 132.

Keilwagen J, Wenk M, Erickson J L, Schattat M H, Grau J, Hartung F. 2016. Using intron position conservation for homology-based gene prediction. Nucleic Acids Research44, e89.

Kim D, Langmead B, Salzberg S L. 2015. HISAT: A fast spliced aligner with low memory requirements. Nature Methods12, 357–360.

Korf I. 2004. Gene finding in novel genomes. BMC Bioinformatics5, 59.

Lee S-M, Song H, Yi H, Hur Y. 2019. Transcriptomic analysis of contrasting inbred lines and F2 segregant of Chinese cabbage provides valuable information on leaf morphology. Genes & Genomics41, 811–829.

Li H, Birol I. 2018. Minimap2: Pairwise alignment for nucleotide sequences. Bioinformatics34, 3094–3100.

Li P, Su T, Li H, Wu Y, Wang L, Zhang F, Wang Z, Yu S. 2023. Promoter variations in a homeobox gene, BrLMI1, contribute to leaf lobe formation in Brassica rapa ssp. chinensis Makino. Theoretical and Applied Genetics136, 188.

Liu D, Zhang C, Zhang J, Xin X, Liao X. 2021. Metagenomics reveals the formation mechanism of flavor metabolites during the spontaneous fermentation of potherb mustard (Brassica juncea var. multiceps). Food Research International148, 110622.

Liu Q, Xu J, Zhu Y, Mo Y, Yao X F, Wang R, Ku W, Huang Z, Xia S, Tong J, Huang C, Su Y, Lin W, Peng K, Liu C M, Xiao L. 2020. The copy number variation of OsMTD1 regulates rice plant architecture. Frontiers in Plant Science11, 620282.

Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2–∆∆CT method. Methods25, 402–408.

Marçais G, Delcher A L, Phillippy A M, Coston R, Salzberg S L, Zimin A. 2018. MUMmer4: A fast and versatile genome alignment system. PLoS Computational Biology14, e1005944.

Marçais G, Kingsford C. 2011. A fast, lock-free approach for efficient parallel counting of occurrences of k-mers. Bioinformatics27, 764–770.

Ou S, Chen J, Jiang N. 2018. Assessing genome assembly quality using the LTR Assembly Index (LAI). Nucleic Acids Research46, e126.

Ou S, Jiang N. 2017. LTR_retriever: A highly accurate and sensitive program for identification of long terminal repeat retrotransposons. Plant Physiology176, 1410–1422.

Paritosh K, Yadava S K, Singh P, Bhayana L, Mukhopadhyay A, Gupta V, Bisht N C, Zhang J, Kudrna D A, Copetti D, Wing R A, Reddy V B, Pradhan A K, Pental D. 2020. A chromosome-scale assembly of allotetraploid Brassica juncea (AABB) elucidates comparative architecture of the A and B genomes. Plant Biotechnology Journal19, 602–614.

Parker T A, Cetz J, de Sousa L L, Kuzay S, Lo S, Floriani T O, Njau S, Arunga E, Duitama J, Jernstedt J, Myers J R, Llaca V, Herrera-Estrella A, Gepts P. 2022. Loss of pod strings in common bean is associated with gene duplication, retrotransposon insertion and overexpression of PvINDNew Phytologist235, 2454–2465.

Parra G, Bradnam K, Korf I. 2007. CEGMA: A pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics23, 1061–1067.

Pertea M, Pertea G M, Antonescu C M, Chang T C, Mendell J T, Salzberg S L. 2015. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nature Biotechnology33, 290–295.

Perumal S, Koh C S, Jin L, Buchwaldt M, Higgins E E, Zheng C, Sankoff D, Robinson S J, Kagale S, Navabi Z K, Tang L, Horner K N, He Z, Bancroft I, Chalhoub B, Sharpe A G, Parkin I A P. 2020. A high-contiguity Brassica nigra genome localizes active centromeres and defines the ancestral Brassica genome. Nature Plants6, 929–941.

Price M N, Dehal P S, Arkin A P. 2010. FastTree 2 - approximately maximum-likelihood trees for large alignments. PLoS ONE5, e9490.

Servant N, Varoquaux N, Lajoie B R, Viara E, Chen C J, Vert J P, Heard E, Dekker J, Barillot E. 2015. HiC-Pro: An optimized and flexible pipeline for Hi-C data processing. Genome Biology16, 259.

Simão F A, Waterhouse R M, Ioannidis P, Kriventseva E V, Zdobnov E M. 2015. BUSCO: Assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics31, 3210–3212.

Stanke M, Diekhans M, Baertsch R, Haussler D. 2008. Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics24, 637–644.

Streubel S, Fritz M A, Teltow M, Kappel C, Sicard A. 2018. Successive duplication-divergence mechanisms at the RCO locus contributed to leaf shape diversity in the Brassicaceae. Development145, dev164301.

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6 molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution30, 2725–2729.

Tang S, Lomsadze A, Borodovsky M. 2015. Identification of protein coding regions in RNA transcripts. Nucleic Acids Research43, e78.

Tang T, Yu X, Yang H, Gao Q, Ji H, Wang Y, Yan G, Peng Y, Luo H, Liu K, Li X, Ma C, Kang C, Dai C. 2018. Development and validation of an effective CRISPR/Cas9 vector for efficiently isolating positive transformants and transgene-free mutants in a wide range of plant species. Frontiers in Plant Science9, 1533.

Tarailo-Graovac M, Chen N. 2009. Using RepeatMasker to identify repetitive elements in genomic sequences. Current Protocols in Bioinformatics, doi: 10.1002/0471250953.bi0410s25.

Vlad D, Kierzkowski D, Rast M I, Vuolo F, Dello Ioio R, Galinha C, Gan X, Hajheidari M, Hay A, Smith R S, Huijser P, Bailey C D, Tsiantis M. 2014. Leaf shape evolution through duplication, regulatory diversification, and loss of a homeobox gene. Science343, 780–783.

Wang X, Wang H, Wang J, Sun R, Wu J, Liu S, Bai Y, Mun J H, Bancroft I, Cheng F, Huang S, Li X, Hua W, Wang J, Wang X, Freeling M, Pires J C, Paterson A H, Chalhoub B, Wang B, et al. 2011. The genome of the mesopolyploid crop species Brassica rapaNature Genetics43, 1035–1039.

Wang X, Zheng Z, Cai Y, Chen T, Li C, Fu W, Jiang Y. 2017. CNVcaller: highly efficient and widely applicable software for detecting copy number variations in large populations. Gigascience6, 1–12.

Wang Y, Liu X, Ji X, Zhang L, Liu Y, Lv X, Feng H. 2015. Identification and validation of a major QTL controlling the presence/absence of leaf lobes in Brassica rapa L. Euphytica205, 761–771.

Wang Y, Strauss S, Liu S, Pieper B, Lymbouridou R, Runions A, Tsiantis M. 2022. The cellular basis for synergy between RCO and KNOX1 homeobox genes in leaf shape diversity. Current Biology32, 3773–3784.e3775.

Wang Y, Tang H, DeBarry J D, Tan X, Li J, Wang X, Lee T H, Jin H, Marler B, Guo H, Kissinger J C, Paterson A H. 2012. MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Research40, e49.

Yang J, Liu D, Wang X, Ji C, Cheng F. 2016. The genome sequence of allopolyploid Brassica juncea and analysis of differential homoeolog gene expression influencing selection. Nature Genetics48, 1225–1232.

Yang J, Wang J, Li Z, Li X. 2021. Genomic signatures of vegetable and oilseed allopolyploid Brassica juncea and genetic loci controlling the accumulation of glucosinolates. Plant Biotechnology Journal19, 2619–2628.

Zhang L, Jia S, Yang M, Xu Y, Li C, Sun J, Huang Y, Lan X, Lei C, Zhou Y, Zhang C, Zhao X, Chen H. 2014. Detection of copy number variations and their effects in Chinese bulls. BMC Genomics15, 480.

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