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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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.
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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.
The authors declare no competing interests.
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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.
(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.
(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.
(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.
Source data of the citric acid content in Fig. 1b.
Source data of the pangenome analysis in Figs. 3a,b.
Statistic source data for the TEs and expression data in Fig. 4a–f.
Unprocessed gels in Fig. 4f.
Ratio of differentially expressed genes with SVs in their promoters in Fig. 5a.
pH values, citric acid and gene expression values in ph4 mutants and WT in Fig. 6d–f.
Gene expression values for citrus and citrus-related genera in Fig. 7a–c. Statistical source data of transactivation activity in Fig. 7g–i.
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