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Pangenome analysis provides insight into the evolution of the orange subfamily and a key gene for citric acid accumulation in citrus fruits

Nature Genetics volume 55, pages 1964–1975 (2023)Cite this article

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Abstract

The orange subfamily (Aurantioideae) contains several Citrus species cultivated worldwide, such as sweet orange and lemon. The origin of Citrus species has long been debated and less is known about the Aurantioideae. Here, we compiled the genome sequences of 314 accessions, de novo assembled the genomes of 12 species and constructed a graph-based pangenome for Aurantioideae. Our analysis indicates that the ancient Indian Plate is the ancestral area for Citrus-related genera and that South Central China is the primary center of origin of the Citrus genus. We found substantial variations in the sequence and expression of the PH4 gene in Citrus relative to Citrus-related genera. Gene editing and biochemical experiments demonstrate a central role for PH4 in the accumulation of citric acid in citrus fruits. This study provides insights into the origin and evolution of the orange subfamily and a regulatory mechanism underpinning the evolution of fruit taste.

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Fig. 1: Fruit characteristics of the orange subfamily and differences in fruit citric acid levels among Citrus-related genera, wild Citrus and domesticated Citrus.
Fig. 2: Phylogenetic tree and biogeography of the orange subfamily.
Fig. 3: Pangenome analysis of the orange subfamily.
Fig. 4: Solo LTRs contribute to genome size variation and affect neighboring gene expression.
Fig. 5: Genomic variations in species from the orange subfamily.
Fig. 6: Sequence analysis and gene editing of CitPH4.
Fig. 7: Characterization of PH4, AN1 and PH5.

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Data availability

Genome assemblies have been deposited in the NCBI under accession nos. JAEVFN000000000 (M. paniculata), JASUUF000000000 (C. lansium), JASUUG000000000 (L. scandens). JASUUL000000000 (A. marmelos), JASUUH000000000 (C. gilletiana), JASUUI000000000 (A. buxifolia), JASUUJ000000000 (C. mangshanensis), JAUJEM000000000 (C. ichangensis), JAUJEF000000000 (C. linwuensis), JASUUK000000000 (C. australasica), JAUJEN000000000 (C. hongheensis), JAUJEG000000000 (C. maxima ‘Majia’). We also deposited all genome assemblies in the National Genomics Data Center (https://ngdc.cncb.ac.cn/) under accession nos. GWHDODA00000000 (M. paniculata), GWHDODB00000000 (C. lansium), GWHDODC00000000 (L. scandens). GWHDODD00000000 (A. marmelos), GWHDODE00000000 (C. gilletiana), GWHDODF00000000 (A. buxifolia), GWHDODG00000000 (C. mangshanensis), GWHDODH00000000 (C. ichangensis), GWHDODI00000000 (C. linwuensis), GWHDODJ00000000 (C. australasica), GWHDODK00000000 (C. hongheensis), GWHDODL00000000 (C. maxima ‘Majia’). The assembled genomes and annotations are also available at http://citrus.hzau.edu.cn/download.php. The detailed accession numbers of the whole-genome sequencing data are listed in Supplementary Table 4. The graph-based pangenome is available at https://figshare.com/s/a1e8071844912a7495ac. Source data are provided with this paper.

Code availability

The code used in this paper is available at GitHub (https://github.com/yilunhuangyue/citrus_pan) and Zenodo (https://doi.org/10.5281/zenodo.8108939)107.

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Acknowledgements

We thank D. Mabberley from the University of Oxford, for helpful discussions on citrus taxonomy and suggestions on how to improve the paper. We thank T. J. Siebert from Department of Botany and Plant Science, University of California, Riverside, for discussions on citrus-related genera. We also thank M. Sun for the suggestion on how to reconstruct the ancestral distribution analysis; Z.L. Ning and S.H. Zeng from the South China Botanical Garden, Chinese Academy of Sciences; and G.H. Chen from the Bureau of Agriculture and Rural Affairs, Chenzhou for providing the citrus samples. We thank J.L. Ye, D.Y. Guo and L.L. Zhong from the National Key Laboratory for Germplasm Innovation and Utilization of Horticultural Crops, Huazhong Agricultural University for support on plant growth, metabolics and the bioinformatics platform. This project was supported by the National Key Research and Development Program of China to Q.X. (nos. 2022YFF1003100 and 2018YFD1000101), the National Natural Science Foundation of China to Q.X. (no. 31925034), Major Special Projects and Key R&D Projects in Yunnan province to Q.X. (no. 202102AE090054), the Foundation of Hubei Hongshan Laboratory to Q.X. (no. 2021hszd016), Key Project of Hubei Provincial Natural Science Foundation to Q.X. (no. 2021CFA017) and the National Postdoctoral Program for Innovative Talents (no. BX20200146) to Y.X.

Author information

Author notes

  1. These authors contributed equally: Yue Huang, Jiaxian He, Yuantao Xu, Weikang Zheng.

Authors and Affiliations

  1. National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, Huazhong Agricultural University, Wuhan, People’s Republic of China

    Yue Huang, Jiaxian He, Yuantao Xu, Weikang Zheng, Xiaolin Jiang, Zishuang Liu, Lun Wang, Xia Wang, Shengjun Liu, Zhihao Lu, Ziang Liu, Huiwen Yu, Zongzhou Xie, Chunlong Li, Zhaocheng Ma, Wenbiao Jiao, Fei Zhang, Robert M. Larkin, Xiuxin Deng & Qiang Xu

  2. Hubei Hongshan Laboratory, Wuhan, People’s Republic of China

    Yue Huang, Yuantao Xu & Qiang Xu

  3. Institute of Tropical and Subtropical Cash Crops, Yunnan Academy of Agricultural Sciences, Yunnan, People’s Republic of China

    Shaohua Wang, Jianqiang Yue, Junyan Gao, Xianyan Zhou & Chunrui Long

  4. Horticulture Institute, Hunan Academy of Agricultural Sciences, Changsha, People’s Republic of China

    Peng Chen, Bin Zeng & Shuizhi Yang

  5. Qinghai-Tibet Plateau Fruit Trees Scientific Observation Test Station, Ministry of Agriculture and Rural Affairs, Lhasa, People’s Republic of China

    Xiuli Zeng

  6. Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, People’s Republic of China

    Yong-Jie Guo

  7. University of the Chinese Academy of Sciences, Beijing, People’s Republic of China

    Yong-Jie Guo

  8. Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla, People’s Republic of China

    Wen-Fu Zhang

  9. United States Department of Agriculture-Agricultural Research Service National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA, USA

    Robert R. Krueger

  10. Department of Agriculture and Fisheries, Bundaberg, Queensland, Australia

    Malcolm W. Smith

  11. Department of Plant Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Ray Ming

Authors
  1. Yue Huang
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  2. Jiaxian He
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  3. Yuantao Xu
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  4. Weikang Zheng
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  5. Shaohua Wang
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  6. Peng Chen
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  7. Bin Zeng
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  8. Shuizhi Yang
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  9. Xiaolin Jiang
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  10. Zishuang Liu
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  11. Lun Wang
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  12. Xia Wang
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  13. Shengjun Liu
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  14. Zhihao Lu
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  15. Ziang Liu
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  16. Huiwen Yu
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  17. Jianqiang Yue
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  18. Junyan Gao
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  19. Xianyan Zhou
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  20. Chunrui Long
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  21. Xiuli Zeng
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  22. Yong-Jie Guo
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  23. Wen-Fu Zhang
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  24. Zongzhou Xie
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  25. Chunlong Li
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  27. Wenbiao Jiao
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  28. Fei Zhang
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  29. Robert M. Larkin
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  30. Robert R. Krueger
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  31. Malcolm W. Smith
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  32. Ray Ming
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  33. Xiuxin Deng
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  34. Qiang Xu
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Contributions

Q.X. designed and coordinated the study. Y.H. performed the genomic and transcriptomic analyses. J.H. analyzed the function of PH4. Y.X. managed the plant material and verified the formation of the solo LTRs. W-K.Z. performed the metabolic analysis. Z-S.L., L.W., X.W., S-J.L., Z-H.L. and Z-A.L. performed the experiments and data analysis. S.W., P.C., B.Z., S.Y., X.J., H.Y., J.Y., J.G., X-Y.Z., C-R.L., X-L.Z., Y.-J.G., W-F.Z., Z.X. and Z.M. provided some of the samples. M.S. provided the images of the samples from Australia. Q.X., Y.H., J.H., Y.X. and W.Z. wrote the paper with contributions from C-L.L., R.M.L., W.J., F.Z., R.R.K., M.S., R.M. and X.D.

Corresponding author

Correspondence to Qiang Xu.

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Competing interests

The authors declare no competing interests.

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Peer review information

Nature Genetics thanks Etienne Bucher and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Characteristics of fruit and seed of Citrus-related genera (1–7), early-diverging Citrus (8–10) and three groups of Citrus species (11–25).

1, Murraya paniculata; 2, Atalantia buxifolia; 3, Glycosmis pentaphylla; 4, Bergera koenigii; 5, Clausena lansium; 6, Aegle marmelos; 7, Citropsis gilletiana; 8, Citrus trifoliata; 9, Citrus mangshanensis; 10, Citrus ichangensis; 11, Citrus linwuensis; 12, Citrus reticulata; 13, Citrus aurantium; 14, Citrus sinensis; 15, Citrus hystrix; 16, Citrus hongheensis; 17, Citrus maxima; 18, Citrus medica; 19, Citrus indica; 20, Citrus paradisi; 21, Citrus limon; 22, Citrus polyandra; 23, Citrus australasica; 24, Citrus hindsii; 25, Citrus glauca. Individual pieces of fruit from different pictures were collected and are shown together. Scale bars, 1 cm. Generally, a large number of seeds and the emergence of juice vesicles in the fruits of the early-diverging Citrus species were observed, showing an intermediate form between Citrus-related genera and Citrus species. Scale bars = 1 cm.

Extended Data Fig. 2 Leaf characteristics of Citrus-related genera (1–7), early diverging Citrus (8–10) and three groups of Citrus species (11–21).

1, Murraya paniculata; 2, Glycosmis pentaphylla; 3, Bergera koenigii; 4, Clausena lansium; 5, Aegle marmelos; 6, Atalantia buxifolia; 7, Citropsis gilletiana; 8, Citrus trifoliata; 9, Citrus ichangensis; 10, Citrus mangshanensis; 11, Citrus linwuensis; 12, Citrus reticulata; 13, Citrus aurantium; 14, Citrus hystrix; 15, Citrus medica; 16, Citrus hongheensis; 17, Citrus indica; 18, Citrus sinensis; 19, Citrus hindsii; 20, Citrus australasica; 21, Citrus maxima. The pinnately compound leaf frequently occurs in Citrus-related genera, while simple leaf is popular in Citrus species. The leaves of the early-diverging Citrus (8, 9, 10) represent an intermediate state in the range from the Citrus-related genera to Citrus species. Scale bars = 1 cm.

Extended Data Fig. 3 Characteristics of Citrus linwuensis.

(a) Soft and dasyphyllous leaves and purple young leaves. (b-c) Flowers. (d) Pistils. (e) Longitudinal sections of pistils. (f) Fruit shape (top row), longitudinal section (bottom left) and equatorial cross section (bottom right) of mature fruit. Scale bars = 1 cm.

Extended Data Fig. 4 Characterization of Citrus mangshanensis.

(a) Flowering branches. (b-c) Flower. (d) Pistil. (e) Longitudinal section of a pistil. (f, g) Fruits. (h) Longitudinal and equatorial cross sections of fruits. (i) Morphological characteristics of young seedlings from Citrus mangshanensis, Citrus maxima and their F2 progeny. The germination rate of the seeds from the F1 hybrid progeny is indicated. Scale bars = 1 cm.

Extended Data Fig. 5 Newly found accession of Citrus trifoliata in Yongshun, Hunan Province.

(a) Wild stand of the newly found Citrus trifoliata ‘Yongshun’. (b-c) Longitudinal and equatorial cross sections of fruits and leaves from the newly found Citrus trifoliata ‘Yongshun’. (d) Leaves from common Citrus trifoliata from SCC. (e) Leaves from Citrus trifoliata ‘Fumin’ from Yunnan province. (f) Phylogenetic analysis of the newly found Citrus trifoliata ‘Yongshun’, common Citrus trifoliata from SCC, Citrus trifoliata ‘Fumin’ from Yunnan province and other Citrus species. Bootstrap support values higher than 80 are denoted above the branch. Divergence times are indicated as millions of years ago (MA). Red color indicates deciduous trifoliate orange. Green color indicates evergreen trifoliate orange. In a, scale bars= 1 m. In b-e, scale bars = 1 cm.

Extended Data Table 1 Statistics for assembly and annotation of 12 genomes
Full size table

Supplementary information

Supplementary Information

Supplementary Notes 1–9 and Figs. 1–36.

Reporting Summary

Peer Review File

Supplementary Table 1

Supplementary Tables 1–21.

Source data

Source Data Fig. 1

Source data of the citric acid content in Fig. 1b.

Source Data Fig. 3

Source data of the pangenome analysis in Figs. 3a,b.

Source Data Fig. 4

Statistic source data for the TEs and expression data in Fig. 4a–f.

Source Data Fig. 4

Unprocessed gels in Fig. 4f.

Source Data Fig. 5

Ratio of differentially expressed genes with SVs in their promoters in Fig. 5a.

Source Data Fig. 6

pH values, citric acid and gene expression values in ph4 mutants and WT in Fig. 6d–f.

Source Data Fig. 7

Gene expression values for citrus and citrus-related genera in Fig. 7a–c. Statistical source data of transactivation activity in Fig. 7g–i.

Source Data Fig. 7

Unprocessed blots in Fig. 7e.

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Huang, Y., He, J., Xu, Y. et al. Pangenome analysis provides insight into the evolution of the orange subfamily and a key gene for citric acid accumulation in citrus fruits. Nat Genet 55, 1964–1975 (2023). https://doi.org/10.1038/s41588-023-01516-6

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