Abstract
Somatic variations are a major source of genetic diversification in asexual plants, and underpin clonal evolution and the breeding of asexual crops. Sweet orange is a model species for studying somatic variation because it reproduces asexually through apomixis and is propagated asexually through grafting. To dissect the genomic basis of somatic variation, we de novo assembled a reference genome of sweet orange with an average of three gaps per chromosome and a N50 contig of 24.2 Mb, as well as six diploid genomes of somatic mutants of sweet oranges. We then sequenced 114 somatic mutants with an average genome coverage of 41×. Categorization of the somatic variations yielded insights into the single-nucleotide somatic mutations, structural variations and transposable element (TE) transpositions. We detected 877 TE insertions, and found TE insertions in the transporter or its regulatory genes associated with variation in fruit acidity. Comparative genomic analysis of sweet oranges from three diversity centres supported a dispersal from South China to the Mediterranean region and to the Americas. This study provides a global view on the somatic variations, the diversification and dispersal history of sweet orange and a set of candidate genes that will be useful for improving fruit taste and flavour.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
$119.00 per year
only $9.92 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
Genome data for di-haploid C. sinensis v.3.0 and v.4.0 have been deposited at DDBJ/ENA/GenBank under accession numbers MORK00000000 and JAFBAU000000000, respectively. The genome data for six diploid sweet oranges have been deposited at NCBI under accession PRJNA321100. All of the genome sequencing data and transcriptome sequencing data have been deposited at the Sequence Read Archive (SRA) database at NCBI. The PacBio and nanopore sequencing data for C. sinensis were deposited under the SRR accession number SRR5838837. The sequencing data that support the findings of this study have been deposited in the SRA database under accession PRJNA321100. The SRR accessions for whole-genome sequencing data and six diploid sweet oranges can be found in Supplementary Table 4. Sweet orange genome sequences are also available from our website at http://citrus.hzau.edu.cn/orange. All supporting data are included in the Supplementary Information. Source data are provided with this paper.
References
Miller, A. J. & Gross, B. L. From forest to field: perennial fruit crop domestication. Am. J. Bot. 98, 1389–1414 (2011).
Mckey, D., Elias, M., Pujol, B. & Duputié, A. The evolutionary ecology of clonally propagated domesticated plants. New Phytol. 186, 318 (2010).
Gaut, B. S., Diez, C. M. & Morrell, P. L. Genomics and the contrasting dynamics of annual and perennial domestication. Trends Genet. 31, 709–719 (2015).
Shamel, A. D. & Pomeroy, C. S. Bud mutations in horticultural crops. J. Hered. 27, 487–494 (1936).
Mendel, K. Bud mutations in Citrus and their potential commercial value. Int. Soc. Citriculture 1, 86–89 (1981).
Poduri, A., Evrony, G., Cai, X. & Walsh, C. A. Somatic mutation genomic variation and neurological disease. Science 341, 1237758 (2013).
Li, M. et al. Characterization of salt-induced epigenetic segregation by genome-wide loss of heterozygosity and its association with salt tolerance in rice (Oryza sativa L.). Front. Plant Sci. 8, 977 (2017).
Ju, Y. S. et al. Somatic mutations reveal asymmetric cellular dynamics in the early human embryo. Nature 543, 714–718 (2017).
Yao, J., Dong, Y. & Morris, B. A. Parthenocarpic apple fruit production conferred by transposon insertion mutations in a MADS-box transcription factor. Proc. Natl Acad. Sci. USA 98, 1306–1311 (2001).
Butelli, E. et al. Retrotransposons control fruit-specific, cold-dependent accumulation of anthocyanins in blood oranges. Plant Cell 24, 1242–1255 (2012).
Kobayashi, S., Goto-Yamamoto, N. & Hirochika, H. Retrotransposon-induced mutations in grape skin color. Science 304, 982 (2004).
Fernandez, L., Torregrosa, L., Segura, V., Bouquet, A. & Martinez-Zapater, J. M. Transposon-induced gene activation as a mechanism generating cluster shape somatic variation in grapevine. Plant J. 61, 545–557 (2010).
Carbonell-Bejerano, P. et al. Catastrophic unbalanced genome rearrangements cause somatic loss of berry color in grapevine. Plant Physiol. 175, 786–801 (2017).
Hiltunen, M., Grudzinska-Sterno, M., Wallerman, O., Ryberg, M. & Johannesson, H. Maintenance of high genome integrity over vegetative growth in the fairy-ring mushroom Marasmius oreades. Curr. Biol. 29, 2758–2765 (2019).
Schmid-Siegert, E. et al. Low number of fixed somatic mutations in a long-lived oak tree. Nat. Plants 3, 926–929 (2017).
Plomion, C. et al. Oak genome reveals facets of long lifespan. Nat. Plants 4, 440–452 (2018).
Yu, L. et al. Somatic genetic drift and multilevel selection in a clonal seagrass. Nat. Ecol. Evol. 4, 952–962 (2020).
Wu, G. A. et al. Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication. Nat. Biotechnol. 32, 656–662 (2014).
Wu, G. A. et al. Genomics of the origin and evolution of Citrus. Nature 554, 311–316 (2018).
Xu, Q. et al. The draft genome of sweet orange (Citrus sinensis). Nat. Genet. 45, 59–66 (2013).
Talon, M. & Gmitter, F. G.Jr. Citrus genomics. Int. J. Plant Genomics 2008, 528361 (2008).
Zhou, K. L. & Ye, M. M. Chinese Fruit Tree: Citrus (China Forestry Publishing House, 2010).
Spiegel-Roy, P. & Goldschmidt, E. E. in The Biology of Citrus (eds Spiegel-Roy, P. & Goldschmidt, E. E.) 4–18 (Cambridge University Press, 1996).
Webber, H. J., Batchelor, L. D. & Reuther, W. in The Citrus Industry (eds Reuther, W. et al.) 1–39 (Univ. California Press, 1967).
Etienne, A., Genard, M., Lobit, P., Mbeguie, A. M. D. & Bugaud, C. What controls fleshy fruit acidity? A review of malate and citrate accumulation in fruit cells. J. Exp. Bot. 64, 1451–1469 (2013).
Jiang, T. M. Preliminary study on selection of sweet orange buds in Qianyang region. South China Fruits 2, 9–12 (1980).
Wang, L. et al. Genome of wild mandarin and domestication history of mandarin. Mol. Plant 11, 1024–1037 (2018).
Moore, G. A. Oranges and lemons: clues to the taxonomy of Citrus from molecular markers. Trends Genet. 17, 536–540 (2001).
Ramu, P. et al. Cassava haplotype map highlights fixation of deleterious mutations during clonal propagation. Nat. Genet. 49, 959–963 (2017).
Zhou, Y., Massonnet, M., Sanjak, J. S., Cantu, D. & Gaut, B. S. Evolutionary genomics of grape (Vitis vinifera ssp. vinifera) domestication. Proc. Natl Acad. Sci. USA 114, 11715–11720 (2017).
Zhang, Y. et al. F-box protein RAE1 regulates the stability of the aluminum-resistance transcription factor STOP1 in Arabidopsis. Proc. Natl Acad. Sci. USA 116, 319–327 (2019).
Liu, M. Y. et al. Two citrate transporters coordinately regulate citrate secretion from rice bean root tip under aluminum stress. Plant Cell Environ. 41, 809–822 (2018).
Fan, L. G. et al. Na+, K+/H+ antiporters regulate the pH of endoplasmic reticulum and auxin-mediated development. Plant Cell Environ. 41, 850–864 (2018).
Bassil, E. et al. Cellular ion homeostasis: emerging roles of intracellular NHX Na+/H+ antiporters in plant growth and development. J. Exp. Bot. 63, 5727–5740 (2012).
Bassil, E., Zhang, S., Gong, H., Tajima, H. & Blumwald, E. Cation specificity of vacuolar NHX-type cation/H+ antiporters. Plant Physiol. 179, 616–629 (2019).
Zhang, M. et al. A HAK family Na+ transporter confers natural variation of salt tolerance in maize. Nat. Plants 5, 1297–1308 (2019).
Terol, J. et al. Involvement of a citrus meiotic recombination TTC-repeat motif in the formation of gross deletions generated by ionizing radiation and MULE activation. BMC Genomics 16, 69 (2015).
Butelli, E. et al. Noemi controls production of flavonoid pigments and fruit acidity and illustrates the domestication routes of modern citrus varieties. Curr. Biol. 29, 158–164 (2019).
Strazzer, P. et al. Hyperacidification of Citrus fruits by a vacuolar proton-pumping P-ATPase complex. Nat. Commun. 10, 744 (2019).
Deng, X. et al. Retrospection and prospect of fruit breeding for last four decades in China (in Chinese). J. Fruit Sci. 36, 514–520 (2019).
Lijavetzky, D. et al. Molecular genetics of berry colour variation in table grape. Mol. Genet. Genomics 276, 427–435 (2006).
Vondras, A. M. et al. The genomic diversification of grapevine clones. BMC Genomics 20, 972 (2019).
Wang, L. et al. The architecture of intra-organism mutation rate variation in plants. PLoS Biol. 17, e3000191 (2019).
Lovell, J. T., Williamson, R. J., Wright, S. I., McKay, J. K. & Sharbel, T. F. Mutation accumulation in an asexual relative of Arabidopsis. PLoS Genet. 13, e1006550 (2017).
Ming, R. et al. The pineapple genome and the evolution of CAM photosynthesis. Nat. Genet. 47, 1435–1442 (2015).
Yang, S. et al. Parent–progeny sequencing indicates higher mutation rates in heterozygotes. Nature 523, 463–467 (2015).
Pelsy, F., Dumas, V., Bevilacqua, L., Hocquigny, S. & Merdinoglu, D. Chromosome replacement and deletion lead to clonal polymorphism of berry color in grapevine. PLoS Genet. 11, e1005081 (2015).
Hu, J. et al. Genetically diverse long-lived clonal lineages of Phytophthora capsici from pepper in Gansu, China. Phytopathology 103, 920–926 (2013).
Calabrese, F. in Citrus: The Genus Citrus (eds Dugo, G. & Di Giacomo, A) 1–15 (Taylor & Francis, 2002).
Wang, X. et al. Genomic analyses of primitive, wild and cultivated citrus provide insights into asexual reproduction. Nat. Genet. 49, 765–772 (2017).
Chin, C. S. et al. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat. Methods 10, 563–569 (2013).
Walker, B. J. et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE 9, e112963 (2014).
Boetzer, M., Henkel, C. V., Jansen, H. J., Butler, D. & Pirovano, W. Scaffolding pre-assembled contigs using SSPACE. Bioinformatics 27, 578–579 (2010).
Luo, R. et al. SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. Gigascience 1, 18 (2012).
Kajitani, R. et al. Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads. Genome Res. 24, 1384–1395 (2014).
Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
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).
Chen, Y. et al. Efficient assembly of nanopore reads via highly accurate and intact error correction. Nat Commun. 12, 60 (2021).
Vaser, R., Sović, I., Nagarajan, N. & Šikić, M. Fast and accurate de novo genome assembly from long uncorrected reads. Genome Res. 737–746 (2017).
Hu, J., Fan, J., Sun, Z. & Liu, S. NextPolish: a fast and efficient genome polishing tool for long-read assembly. Bioinformatics 36, 2253–2255 (2020).
Dudchenko, O. et al. De novo assembly of the Aedes aegypti genome using Hi-C yields chromosome-length scaffolds. Science 356, 92–95 (2017).
Durand, N. C. et al. Juicer provides a one-click system for analyzing loop-resolution Hi-C experiments. Cell Syst. 3, 95–98 (2016).
Stanke, M. et al. AUGUSTUS: ab initio prediction of alternative transcripts. Nucleic Acids Res. 34, W435–W439 (2006).
Majoros, W. H., Pertea, M. & Salzberg, S. L. TigrScan and GlimmerHMM: two open source ab initio eukaryotic gene-finders. Bioinformatics 20, 2878–2879 (2004).
Slater, G. S. C. & Birney, E. Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics 6, 31 (2005).
Huang, X. Q., Adams, M. D., Zhou, H. & Kerlavage, A. R. A tool for analyzing and annotating genomic sequences. Genomics 46, 37–45 (1997).
Grabherr, M. G. et al. Full-length transcriptome assembly from RNA-seq data without a reference genome. Nat. Biotechnol. 29, 644–652 (2011).
Haas, B. J. et al. Automated eukaryotic gene structure annotation using EVidenceModeler and the program to assemble spliced alignments. Genome Biol. 9, R7 (2008).
Sedlazeck, F. J. et al. Accurate detection of complex structural variations using single-molecule sequencing. Nat. Methods 15, 461–468 (2018).
Edge, P., Bafna, V. & Bansal, V. HapCUT2: robust and accurate haplotype assembly for diverse sequencing technologies. Genome Res. 27, 801–812 (2016).
Roach, M. J., Schmidt, S. A. & Borneman, A. R. Purge Haplotigs: allelic contig reassignment for third-gen diploid genome assemblies. BMC Bioinformatics 19, 460 (2018).
Alonge, M. et al. RaGOO: fast and accurate reference-guided scaffolding of draft genomes. Genome Biol. 20, 224 (2019).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).
Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014).
Price, A. L. et al. Principal components analysis corrects for stratification in genome-wide association studies. Nat. Genet. 38, 904–909 (2006).
Danecek, P. et al. The variant call format and VCFtools. Bioinformatics 27, 2156–2158 (2011).
Liang, P., Saqib, H. S. A., Zhang, X., Zhang, L. & Tang, H. Single-base resolution map of evolutionary constraints and annotation of conserved elements across major grass genomes. Genome Biol. Evol. 10, 473–488 (2018).
Hubisz, M. J., Pollard, K. S. & Siepel, A. PHAST and RPHAST: phylogenetic analysis with space/time models. Brief. Bioinform. 12, 41–51 (2011).
Xie, C. & Tammi, M. T. CNV-seq, a new method to detect copy number variation using high-throughput sequencing. BMC Bioinformatics 10, 80 (2009).
Boeva, V. et al. Control-FREEC: a tool for assessing copy number and allelic content using next-generation sequencing data. Bioinformatics 28, 423–425 (2011).
Li, S. J. et al. Citrus CitNAC62 cooperates with CitWRKY1 to participate in citric acid degradation via up-regulation of CitAco3. J. Exp. Bot. 68, 3419–3426 (2017).
Liu, Q. et al. A novel bud mutation that confers abnormal patterns of lycopene accumulation in sweet orange fruit (Citrus sinensis L. Osbeck). J. Exp. Bot. 58, 4161–4171 (2007).
Liu, B. et al. Estimation of genomic characteristics by analyzing k-mer frequency in de novo genome projects. Preprint at http://arxiv.org/abs/1308.2012 (2012).
Acknowledgements
We thank Y. Zhang from Chongqing Academy of Agricultural Sciences and W. Song from Zigui Agricultural Bureau, Yichang for sampling support. We also thank L. Chen for suggestions on the bioinformatics analysis. This project was financially supported by the National Key Research and Development Program of China granted to Q.X. (number 2018YFD1000101), the National Natural Science Foundation of China granted to Q.X. (numbers 31925034 and 31872052), the Fundamental Research Funds for the Central Universities granted to Q.X. (number 2662015PY109) and the support from Agricultural Research Service, US Department of Agriculture (number 8062-21000-043-02S to E.S.B.). L.W. was supported by the China Postdoctoral Science Foundation (number 2020M672375).
Author information
Authors and Affiliations
Contributions
Q.X. conceived and designed the project. L.W. developed the method for the bioinformatics analyses of the somatic mutant, designed primers for experiments, prepared the figures and coordinated teamwork. Y.H. assembled the sweet orange genomes and performed gene annotation. Z. Liu carried out the somatic variant validation experiments (with contribution by J.H.). Z. Liu and J.H. performed gene expression. Z. Liu, Z. Lu and J.H. performed the transient overexpression experiments. F.H., X.J., S.Y., P.C., B.Z., L.K. and Z.X. collected and evaluated the samples. Z. Liu, F.H. and J.H. measured the fruit quality. Z. Liu, H.Y. and L.K. performed the DNA and RNA extraction experiments. D.J. provided partial sweet-orange samples. E.S.B. and R.M. supervised the bioinformatics analyses. Q.X., L.W., Y.H. and R.M.L. wrote the manuscript with contributions from X.D. and R.M.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Peer review information Nature Plants thanks Olivier Panaud, Dacheng Tian and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 Citrus bud mutation and asexual propagation.
The mutation occurred somatically on a bud of one branch of the tree. If this mutation was observed by human, the mutated branch will be grafted on rootstock. Then this mutant was further propagated if developed as cultivars. The whole process is on somatic level.
Extended Data Fig. 2 Validation of 12 TE insertions in low acid sweet orange (BTC) by PCR experiments.
DH2, TCPS1, MORO, NHE, AJTC, SO3, ZAOJ are control sweet oranges; BTC, BT2, REN4, REN5, JH (accession name: JHBTC), HYJH (accession name: HYJHBTC), RRJH (accession name: RRJHBTC) are Bingtangcheng. The accession name was provided in the Supplementary Table 4 and the primers and reproducibility of gel validation experiments was provided in Supplementary Table 9.
Extended Data Fig. 3 Feature of the large duplication at 0..7.4M on chromosome 7.
a. The allele frequency in the mutants FW95-1 and the control (T1) Statistical source data was provided. b Copy number ratios between FW95-1 and the control (T1). Windows in increasing red color tones with significance P values correspond to the signal of CNV. c. the copy number profile results of FREEC. Window with red represent the signal of copy number increase.
Extended Data Fig. 4 Validation the TE insertion in CsRAE1 gene in the blood orange, transient gene transformation assay, and gene expression analysis of CsRAE1 gene.
a. DH2 and DHWH2 are high acid oranges; SO3 and MIDNIT are Valencia oranges; MoroN2, TaroWC, TaroROS, TaroUn and QXC are blood oranges. All the accession name was provided in the Supplementary Table 4. Nine independent experiments were repeated with similar results. Primer design information and experiments reproducibility was provided in Supplementary Table 9. b. Expression of RAE1 in blood orange (XC), a moderate sweet orange and high acid sweet orange (DH, Dahong). Values are means ± S.E.M (n = 3 biological independent samples), c. the pH value in the fruit development of Newhall navel (NHE) and late maturing orange (NW). Values are means ± S.E.M (n = 3 biological independent samples), d-e Gene expression of the RAE1 in the NHE (d) and NW (e). Values are means ± S.E.M (n = 3 biological independent samples), f. The expression of the CsRAE1 gene in the overexpression (OE) lines and the control, g. the citric acid content in the OE lines and the control (EV), Values are means ± S.E.M (n = 4 biological independent samples), h. pH value in the OE lines of CsRAE1 and EV, Values are means ± S.E.M (n = 4 biological independent samples). Asterisks indicate significant difference (*p ≤ 0.05, P = 0.025, one-sided t-test,). All primer pairs were listed in Supplementary Tables 9 and 16.
Extended Data Fig. 5 Validation the TE insertion in promoter of NHX gene in the low acid orange (Bingtangcheng), transient gene transformation assay, and gene expression analysis of CsNHX gene.
a. The structure of Mule transposon sequence and the CsNHX (Na+/H+ transporter) gene. b. PCR confirmation of the TE insertion. BTC, BT2, REN4, REN5 are low acid mutants (Bingtangcheng). DH2, TCPS1 are high acid oranges; Valencia (SO3) and blood orange (MORO) are moderate acid; AJTC is the acidless mutant. All the accession name was provided in the Supplementary Table 4. Seven independent experiments were repeated with similar results. Primer design information and experiments reproducibility was provided in Supplementary Table 9. c. Expression of CsNHX in different citrus varieties. Values are means ± S.E.M (n = 3 biological independent samples). XC means blood orange, a moderate sweet orange; DH (Dahong) is a high acid sweet orange. DAF, days after flowering. d-e Gene expression of the NHX in the Newhall navel orange (d) and Lanlate late-maturing orange (e). Values are means ± S.E.M (n = 3 biological independent samples). f. The expression of the CsNHX gene in the overexpression (OE) lines and the control (EV), Values are means ± S.E.M (n = 4 biological independent samples). g. the citric acid content in the OE lines and the EV, Values are means ± S.E.M (n = 3 biological independent samples). h. the pH value in the overexpression line of CsNHX and the EV, Values are means ± S.E.M (n = 4 biological independent samples). Asterisks indicate significant difference (**p ≤ 0.01, P = 0.0093, one-sided t-test). All primer pairs were listed in Supplementary Tables 9 and 16.
Supplementary information
Supplementary Information
Supplementary Figs. 1–32 and unprocessed DNA gels.
Supplementary Data
Supplementary Tables 1–16.
Source data
Source Data Fig. 1
Statistical source data.
Source Data Fig. 2
Statistical source data.
Source Data Fig. 3
Statistical source data.
Source Data Fig. 4
Statistical source data.
Source Data Fig. 5
Statistical source data.
Source Data Extended Data Fig. 2
Unprocessed DNA gels.
Source Data Extended Data Fig. 3
Statistical source data.
Source Data Extended Data Fig. 4
Statistical source data.
Source Data Extended Data Fig. 5
Statistical source data.
Rights and permissions
About this article
Cite this article
Wang, L., Huang, Y., Liu, Z. et al. Somatic variations led to the selection of acidic and acidless orange cultivars. Nat. Plants 7, 954–965 (2021). https://doi.org/10.1038/s41477-021-00941-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41477-021-00941-x
This article is cited by
-
A tetraploid-dominated cytochimera developed from a natural bud mutant of the nonapomictic mandarin variety ‘Orah’
Molecular Breeding (2024)
-
Weighted gene coexpression correlation network analysis reveals the potential molecular regulatory mechanism of citrate and anthocyanin accumulation between postharvest ‘Bingtangcheng’ and ‘Tarocco’ blood orange fruit
BMC Plant Biology (2023)
-
Chromosome-level genome assembly and population genomics of Robinia pseudoacacia reveal the genetic basis for its wide cultivation
Communications Biology (2023)
-
Chromosome-level assemblies of cultivated water chestnut Trapa bicornis and its wild relative Trapa incisa
Scientific Data (2023)
-
Centromeric repeats in Citrus sinensis provide new insights into centromeric evolution and the distribution of G-quadruplex structures
Horticulture Advances (2023)