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Somatic variations led to the selection of acidic and acidless orange cultivars

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.

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Fig. 1: Distribution of sweet oranges sampled in South China and phenotypes of sweet orange somatic mutants.
Fig. 2: PCA and phylogenetic relationships of sweet oranges and other sexual/asexual citrus species.
Fig. 3: Genomic location of somatic variations in 114 sweet oranges.
Fig. 4: Categorization of somatic TE transposition events in sweet oranges.
Fig. 5: The diversification and dispersal history of sweet orange.

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.

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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

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Authors

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

Correspondence to Qiang Xu.

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The authors declare no competing interests.

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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.

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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.

Source data

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.

Source data

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.

Source data

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.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–32 and unprocessed DNA gels.

Reporting Summary

Supplementary Data

Supplementary Tables 1–16.

Source data

Source Data Fig. 1

Statistical source data.

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Source Data Extended Data Fig. 2

Unprocessed DNA gels.

Source Data Extended Data Fig. 3

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Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 5

Statistical source data.

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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

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