Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication

Journal name:
Nature Biotechnology
Volume:
32,
Pages:
656–662
Year published:
DOI:
doi:10.1038/nbt.2906
Received
Accepted
Published online

Abstract

Cultivated citrus are selections from, or hybrids of, wild progenitor species whose identities and contributions to citrus domestication remain controversial. Here we sequence and compare citrus genomes—a high-quality reference haploid clementine genome and mandarin, pummelo, sweet-orange and sour-orange genomes—and show that cultivated types derive from two progenitor species. Although cultivated pummelos represent selections from one progenitor species, Citrus maxima, cultivated mandarins are introgressions of C. maxima into the ancestral mandarin species Citrus reticulata. The most widely cultivated citrus, sweet orange, is the offspring of previously admixed individuals, but sour orange is an F1 hybrid of pure C. maxima and C. reticulata parents, thus implying that wild mandarins were part of the early breeding germplasm. A Chinese wild 'mandarin' diverges substantially from C. reticulata, thus suggesting the possibility of other unrecognized wild citrus species. Understanding citrus phylogeny through genome analysis clarifies taxonomic relationships and facilitates sequence-directed genetic improvement.

At a glance

Figures

  1. A selection of mandarin, pummelo and orange fruits, including cultivars sequenced in this study.
    Figure 1: A selection of mandarin, pummelo and orange fruits, including cultivars sequenced in this study.

    Pummelos (1,2 in outline on left) are large trees that produce very large fruit with white, pink or red flesh color (2) and yellow or pink rinds. Most cultivars have large leaves with petioles with prominent wings. Apomictic reproduction is absent, and most selections are self-incompatible. Mandarins (3–7) are smaller trees bearing smaller fruit with orange flesh (9,11) and rind color. Mandarins have both apomictic and zygotic reproduction, and some are self-compatible. Oranges (8,10) are generally intermediate in tree and fruit size; the flesh (10) and rind color is commonly orange, and apomictic reproduction is always present. (The sour orange shown (12) is immature.)

  2. Nucleotide-diversity distribution in citrus.
    Figure 2: Nucleotide-diversity distribution in citrus.

    (a) Nucleotide-heterozygosity distribution computed in overlapping 100-kb windows (with 5-kb step size) across the low-acid (LAP) and Chandler (CHP) pummelo genomes and between the nonshared haplotypes of this parent-child pair (LAP/CHP). The peak at ~6 heterozygous sites/kb in all three pairwise comparisons represents the characteristic nucleotide diversity of the species C. maxima; the peak near ~1 heterozygous site/kb reflects a bottleneck in the ancestral C. maxima population after divergence from C. reticulata (Supplementary Note 10). (b) Nucleotide heterozygosity for the traditional Willowleaf mandarin (WLM) plotted along chromosome 6, computed in overlapping windows of 200 kb (with 100-kb step size). This chromosome shows an example of the clear discontinuity in single-nucleotide-variant heterozygosity levels between ~5/kb in the M/M segment (orange bar) and ~17/kb in the M/P segment (blue bar). (c) Nucleotide heterozygosity distribution computed in overlapping 500-kb windows (with 5-kb step size) in Ponkan (PKM, solid line) and Willowleaf (WLM, dashed line) mandarins. Genomic segments are designated M/M, M/P or P/P on the basis of a set of 1,537,264 SNPs that differentiate C. reticulata (M) from C. maxima (P). Both mandarins contain admixed segments from C. maxima introgression (M/P) as well as M/M segments, and these are plotted and normalized separately for easy comparison. (d) Nucleotide heterozygosity distribution computed in overlapping windows of 500 kb (5-kb offsets) for sweet orange (SWO) and sour orange (SSO). The three different genotypes of the sweet-orange genome (M/M, P/P and M/P) and the sour-orange genotype M/P are normalized and plotted separately.

  3. Admixture patterns and nucleotide diversity in cultivated citrus.
    Figure 3: Admixture patterns and nucleotide diversity in cultivated citrus.

    For each of the three groups of sequenced citrus, variation in nucleotide diversity (averaged over 500-kb windows with step size 250 kb) is shown across the genome for one representative cultivar above genotype maps (horizontal bars). Green, C. maxima/C. maxima; blue, C. maxima/C. reticulata; orange, C. reticulata/C. reticulata; gray, unknown. The nine chromosomes are numbered at top. (a) Sweet orange (SWO) nucleotide diversity with genotype maps for sweet orange and sour orange (SSO), indicating the C. maxima/C. maxima genotype (green segments present on chromosomes 2 and 8) in sweet orange. (b) Willowleaf mandarin (WLM) nucleotide diversity and genotype maps for three traditional mandarins (Ponkan mandarin (PKM), Willowleaf mandarin (WLM) and Huanglingmiao (HLM)) and three recent mandarin types (Clementine (CLM), W. Murcott mandarin (WMM) and haploid Clementine reference (HCR)). For the haploid Clementine reference sequence, orange and green segments indicate C. reticulata and C. maxima haplotypes, respectively. All five mandarin types show pummelo introgressions (blue or green segments). (c) Low-acid pummelo (LAP) nucleotide diversity and genotype maps for two pummelos (low-acid pummelo and Chandler pummelo (CHP)).

  4. Mangshan mandarin is a species distinct from C. maxima and C. reticulata.
    Figure 4: Mangshan mandarin is a species distinct from C. maxima and C. reticulata.

    (a) Midpoint-rooted neighbor-joining phylogenetic tree of citrus chloroplast genomes. (b) Frequency distributions of the pairwise sequence divergences (across 100-kb windows) between Mangshan mandarin (CMS) and C. maxima (green), CMS and C. reticulata (orange), C. reticulata and C. maxima (light blue) as well as the distinctly lower CMS intrinsic nucleotide diversity (dashed blue). Ret, C. reticulata; max, C. maxima; het, heterozygous. (c) The first two coordinates of principal coordinate analysis of the citrus nuclear genomes, based on pairwise distances and metric multidimensional scaling. The C. maximaC. reticulata axis (principal coordinate 1, 47.5% variance) separates pummelos (green) from mandarins (orange), with oranges (blue) lying in between; principal coordinate 2 (19.6% of variance) separates CMS (purple) from the others.

Accession codes

Primary accessions

BioProject

NCBI Reference Sequence

References

  1. Bové, J. Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. J. Plant Pathol. 88, 737 (2006).
  2. The Citrus Industry 1st edn, Vol. 1 (eds. Reuther, W., Webber, H.J. & Batchelor, L.D.). (University of California, Division of Agricultural Sciences, Berkeley, California, USA, 1967).
  3. Spiegel-Roy, P. & Goldschmidt, E.E. Biology of citrus (Cambridge University Press, Cambridge and New York, 1996).
  4. Scora, R.W. On the history and origin of citrus. Bull. Torrey Bot. Club 102, 369375 (1975).
  5. Barrett, H.C. & Rhodes, A.M. A numerical taxonomic study of affinity relationships in cultivated citrus and its close relatives. Syst. Bot. 1, 105136 (1976).
  6. Nicolosi, E. et al. Citrus phylogeny and genetic origin of important species as investigated by molecular markers. Theor. Appl. Genet. 100, 11551166 (2000).
  7. Swingle, W.T. & Reece, H.C. in The Citrus Industry 2nd edn, Vol. 1 (eds. Reuther, W., Webber, H.J. & Batchelor, L.D.) 190430 (University of California Press, Berkeley, California, USA, 1967).
  8. Tanaka, T. Fundamental discussion of Citrus classification. Studia Citrologica 14, 16 (1977).
  9. Moore, G.A. Oranges and lemons: clues to the taxonomy of Citrus from molecular markers. Trends Genet. 17, 536540 (2001).
  10. Cornille, A. et al. New insight into the history of domesticated apple: secondary contribution of the European wild apple to the genome of cultivated varieties. PLoS Genet. 8, e1002703 (2012).
  11. Myles, S. et al. Genetic structure and domestication history of the grape. Proc. Natl. Acad. Sci. USA 108, 35303535 (2011).
  12. Huang, X. et al. A map of rice genome variation reveals the origin of cultivated rice. Nature 490, 497501 (2012).
  13. Hufford, M.B. et al. Comparative population genomics of maize domestication and improvement. Nat. Genet. 44, 808811 (2012).
  14. Morrell, P.L., Buckler, E.S. & Ross-Ibarra, J. Crop genomics: advances and applications. Nat. Rev. Genet. 13, 8596 (2011).
  15. Germana, M.A. et al. Cytological and molecular characterization of three gametoclones of Citrus clementina. BMC Plant Biol. 13, 129 (2013).
  16. Aleza, P. et al. Recovery and characterization of a Citrus clementina Hort. ex Tan. 'Clemenules' haploid plant selected to establish the reference whole Citrus genome sequence. BMC Plant Biol. 9, 110 (2009).
  17. Xu, Q. et al. The draft genome of sweet orange (Citrus sinensis). Nat. Genet. 45, 5966 (2013).
  18. Ollitrault, P. et al. A reference genetic map of C. clementina hort. ex Tan.: citrus evolution inferences from comparative mapping. BMC Genomics 13, 593 (2012).
  19. Salse, J. In silico archeogenomics unveils modern plant genome organisation, regulation and evolution. Curr. Opin. Plant Biol. 15, 122130 (2012).
  20. Froelicher, Y. et al. New universal mitochondrial PCR markers reveal new information on maternal citrus phylogeny. Tree Genet. Genomes 7, 4961 (2011).
  21. Cameron, J.W.S. R K Chandler: an early-ripening hybrid pummelo derived from a low-acid parent. Hilgardia 30, 359364 (1961).
  22. Barkley, N.A., Roose, M.L., Krueger, R.R. & Federici, C.T. Assessing genetic diversity and population structure in a citrus germplasm collection utilizing simple sequence repeat markers (SSRs). Theor. Appl. Genet. 112, 15191531 (2006).
  23. Johnson, N.A. et al. Ancestral components of admixed genomes in a Mexican cohort. PLoS Genet. 7, e1002410 (2011).
  24. Bustamante, C.D., Burchard, E.G. & De la Vega, F.M. Genomics for the world. Nature 475, 163165 (2011).
  25. Tuskan, G.A. et al. The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313, 15961604 (2006).
  26. Pfeil, B.E. & Crisp, M.D. The age and biogeography of Citrus and the orange subfamily (Rutaceae: Aurantioideae) in Australasia and New Caledonia. Am. J. Bot. 95, 16211631 (2008).
  27. Trabut, J.L. L'hybridation des Citrus: une nouvelle tangérine 'la Clémentine'. Revue Horticole 10, 232234 (1902).
  28. Samaan, L.G. Studies on the origin of Clementine tangerine (Citrus reticulata Blanco). Euphytica 31, 167173 (1982).
  29. Novelli, V.M., Cristofani, M., Souza, A.A. & Machado, M.A. Development and characterization of polymorphic microsatellite markers for the sweet orange (Citrus sinensis L. Osbeck). Genet. Mol. Biol. 29, 9096 (2006).
  30. Garcia-Lor, A. et al. A nuclear phylogenetic analysis: SNPs, indels and SSRs deliver new insights into the relationships in the 'true citrus fruit trees' group (Citrinae, Rutaceae) and the origin of cultivated species. Ann. Bot. 111, 119 (2013).
  31. Liu, G.F., He, S.W. & Li, W.B. Two new species of citrus in China. Acta Botanica Yunnanica 12, 287289 (1990).
  32. Gmitter, F.G. & Hu, X. The possible role of Yunnan, China, in the origin of contemporary citrus species (Rutaceae). Econ. Bot. 44, 267277 (1990).
  33. Morton, J.F. Fruits of Warm Climates (Florida Flair Books, Miami, 1987).
  34. Gottwald, T.R. Current epidemiological understanding of citrus Huanglongbing. Annu. Rev. Phytopathol. 48, 119139 (2010).
  35. Talon, M. & Gmitter, F.G. Jr. Citrus genomics. Int. J. Plant Genomics 2008, 528361 (2008).
  36. Gmitter, F.G. et al. Citrus genomics. Tree Genet. Genomes 8, 611626 (2012).
  37. Bausher, M.G., Singh, N.D., Lee, S.B., Jansen, R.K. & Daniell, H. The complete chloroplast genome sequence of Citrus sinensis (L.) Osbeck var 'Ridge Pineapple': organization and phylogenetic relationships to other angiosperms. BMC Plant Biol. 6, 21 (2006).

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Author information

  1. These authors contributed equally to this work.

    • G Albert Wu &
    • Simon Prochnik

Affiliations

  1. US Department of Energy Joint Genome Institute, Walnut Creek, California, USA.

    • G Albert Wu,
    • Simon Prochnik,
    • Uffe Hellsten,
    • Jarrod Chapman &
    • Daniel Rokhsar
  2. HudsonAlpha Biotechnology Institute, Huntsville, Alabama, USA.

    • Jerry Jenkins,
    • Jane Grimwood &
    • Jeremy Schmutz
  3. Institut National de la Recherche Agronomique (INRA), Université Blaise Pascal (UBP) UMR 1095 Génétique, Diversité, Ecophysiologie des Céréales (GDEC), Clermont Ferrand, France.

    • Jerome Salse &
    • Florent Murat
  4. Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), UMR Amélioration Génétique et Adaptation des Plantes Méditerranéennes et Tropicales (AGAP), Montpellier, France.

    • Xavier Perrier,
    • Manuel Ruiz &
    • Patrick Ollitrault
  5. Istituto di Genomica Applicata, Udine, Italy.

    • Simone Scalabrin,
    • Federica Cattonaro,
    • Cristian Del Fabbro,
    • Sara Pinosio,
    • Andrea Zuccolo &
    • Michele Morgante
  6. Centro de Genomica, Instituto Valenciano de Investigaciones Agrarias (IVIA), Valencia, Spain.

    • Javier Terol,
    • Francisco R Tadeo,
    • Leandro H Estornell,
    • Juan V Muñoz-Sanz,
    • Victoria Ibanez,
    • Amparo Herrero-Ortega &
    • Manuel Talon
  7. Centro de Citricultura Sylvio Moreira, Instituto Agronômico (IAC), Cordeirópolis, Brazil.

    • Marco Aurélio Takita,
    • Juliana Freitas-Astúa &
    • Marcos Antonio Machado
  8. Commissariat à l'Energie Atomique (CEA), Institut de Génomique (IG), Genoscope, Evry, France.

    • Karine Labadie,
    • Julie Poulain,
    • Arnaud Couloux,
    • Kamel Jabbari,
    • Dominique Brunel,
    • Francis Quetier,
    • Patrick Wincker &
    • Olivier Jaillon
  9. Institute of Life Sciences, Scuola Superiore Sant'Anna, Pisa, Italy.

    • Andrea Zuccolo
  10. Centro de Protección Vegetal y Biotecnología–Instituto Valenciano de Investigaciones Agrarias, Moncada, Spain.

    • Pablo Aleza &
    • Luis Navarro
  11. Lifesequencing, Valencia, Spain.

    • Julián Pérez-Pérez &
    • Daniel Ramón
  12. Secugen, Madrid, Spain.

    • Julián Pérez-Pérez
  13. INRA, US 1279 Etude du Polymorphisme des Génomes Végétaux (EPGV), Evry, France.

    • Dominique Brunel
  14. INRA Génétique et Écophysiologie de la Qualité des Agrumes (GEQA), San Giuliano, France.

    • François Luro
  15. Citrus Research and Education Center (CREC), Institute of Food and Agricultural Sciences (IFAS), University of Florida, Lake Alfred, Florida, USA.

    • Chunxian Chen &
    • Frederick Gmitter
  16. Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, Florida, USA.

    • William G Farmerie
  17. 454 Life Sciences, Roche, Branford, Connecticut, USA.

    • Brian Desany,
    • Chinnappa Kodira,
    • Mohammed Mohiuddin,
    • Tim Harkins &
    • Karin Fredrikson
  18. Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA.

    • Paul Burns,
    • Alexandre Lomsadze &
    • Mark Borodovsky
  19. School of Computational Science and Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA.

    • Paul Burns,
    • Alexandre Lomsadze &
    • Mark Borodovsky
  20. Department of Biological and Medical Physics, Moscow Institute of Physics and Technology, Dolgoprudny, Russia.

    • Mark Borodovsky
  21. Consiglio per la Ricerca e la Sperimentazione in Agricoltura (CRA-ACM), Acireale, Italy.

    • Giuseppe Reforgiato
  22. Embrapa Cassava and Fruits, Cruz das Almas, Brazil.

    • Juliana Freitas-Astúa
  23. Département de Biologie, Université d'Evry, Evry, France.

    • Francis Quetier,
    • Patrick Wincker &
    • Olivier Jaillon
  24. Department of Botany and Plant Sciences, University of California, Riverside, Riverside, California, USA.

    • Mikeal Roose
  25. Centre National de Recherche Scientifique (CNRS), Evry, France.

    • Patrick Wincker &
    • Olivier Jaillon
  26. Department of Agriculture and Environmental Sciences, University of Udine, Udine, Italy.

    • Michele Morgante
  27. Division of Genetics, Genomics and Development, University of California, Berkeley, Berkeley, California, USA.

    • Daniel Rokhsar
  28. Present addresses: Life Technologies, Grand Island, New York, USA (T.H.) and US Department of Agriculture, Agricultural Research Service, Southeastern Fruit and Tree Nut Research Laboratory, Byron, Georgia, USA (C.C.).

    • Chunxian Chen &
    • Tim Harkins

Contributions

G.A.W., development and application of methods to analyze citrus genetic diversity, population history and ancestry; S. Prochnik, genome annotation and initial analysis of genetic diversity; J.J., J.G. and J.C., sequence assembly and map integration of haploid Clementine reference; J. Salse and F.M., analysis of synteny and genome evolution.; U.H., analysis of population history and ancestry; K.L., J.P.-P., A.C., J.P., D.B. and K.J., dideoxy shotgun sequencing and analysis of haploid Clementine reference; S.S., S. Pinosio, A.Z., C.D.F., X.P. and M. Ruiz, analysis of sequencing and resequencing data, and repetitive sequence annotation and analysis; F.C., Sanger and Illumina sequencing; A.L., P.B. and M.B., sweet-orange gene model predictions; C.C. and W.G.F., 454 sequencing of sweet orange and Illumina sequencing of Siamese Sweet pummelo; C.C., contributions to sweet-orange transcriptome, annotation and strategic rationale for comparative analyses; P.A., J.P.-P. and L.N., haploid Clementine DNA; J.P.-P. and D. Ramón, haploid Clementine transcriptome; J.T., F.R.T., L.H.E., J.V.M.-S., V.I., A.H.-O. and M.T., generation of BAC clones of the haploid Clementine and contribution of genome sequences of sweet orange, Ponkan, diploid Clementine and Willowleaf mandarins; B.D., C.K., M. Mohiuddin, T.H. and K.F., sweet-orange 454 transcriptome and genome sequencing and assembly; M.A.M. and M.A.T., Ponkan shotgun sequence; M. Roose, W. Murcott shotgun sequence; M. Morgante, Chandler pummelo and Seville sour-orange shotgun sequence; G.R., J.F.-A., F.Q., L.N., F.L. and M. Roose, project coordination; D. Rokhsar, F.G., G.A.W. and S. Prochnik, writing of the paper with substantial input from M.T., P.O., M. Mohiuddin, O.J. and M. Roose; F.G., D. Rokhsar, O.J., P.O., M.A.M., M. Morgante, M.T., J. Schmutz and P.W., project coordination and scientific leadership.

Competing financial interests

The authors declare no competing financial interests.

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