Tree Genetics & Genomes

, 11:123 | Cite as

Genetic diversity and population structure analysis of mandarin germplasm by nuclear, chloroplastic and mitochondrial markers

  • Andres Garcia-Lor
  • François Luro
  • Patrick Ollitrault
  • Luis NavarroEmail author
Original Article
Part of the following topical collections:
  1. Population Structure


The mandarin horticultural varietal group (a basic taxon of the cultivated citrus (Citrus reticulata)) is highly polymorphic. It includes also genotypes introgressed by other species. The precise contribution of ancestral species to the mandarin group is not known. The goals of this work were (1) to characterise the mandarin germplasm using nuclear (simple sequence repeat (SSR), indel, single nucleotide polymorphism (SNP)), chloroplastic and mitochondrial markers; (2) to evaluate genetic diversity and detect redundancies; (3) to determine the possible presence of citrus ancestral genome introgressions into the mandarin genome; and (4) to determine the genetic structure within the mandarin group. Fifty microsatellites (SSRs), 24 insertion-deletions (indels), 67 SNPs, 8 chloroplastic SSRs (cpDNA) and 4 mitochondrial (mtDNA) indel markers were analysed for 191 genotypes, including the 4 main citrus ancestral species (C. reticulata, C. maxima, C. medica and C. micrantha) and Fortunella. C. maxima was the main genome introgressed in the mandarin germplasm. Seven clusters were revealed by Structure analysis at the nuclear level (N) within the mandarin germplasm. At least four of these clusters showed a clear introgression from other ancestral species. Moreover, most of the mandarins appeared to be complex mixtures of these groups. The maternal indel analysis (mtDNA and cpDNA) revealed ten cytotypes in which mandarins were represented in seven of them. This work provides new insights into the organisation of the mandarin germplasm and its structure at the nuclear and cytoplasmic levels and will be useful to design more efficient breeding programmes and management of citrus germplasm collections.


Citrus reticulata Molecular markers Diversity Germplasm Genetics Introgression 



This work was supported by a grant (AGL2011-26490) from the Ministry of Economy and Innovation-Fondo Europeo de Desarrollo Regional (FEDER), Spain.

Supplementary material

11295_2015_951_MOESM1_ESM.doc (652 kb)
ESM 1 (DOC 651 kb)
11295_2015_951_MOESM2_ESM.docx (18 kb)
ESM 2 (DOCX 17.9 kb)
11295_2015_951_MOESM3_ESM.doc (213 kb)
ESM 3 (DOC 213 kb)
11295_2015_951_MOESM4_ESM.doc (39 kb)
ESM 4 (DOC 39 kb)
11295_2015_951_MOESM5_ESM.doc (55 kb)
ESM 5 (DOC 55 kb)
11295_2015_951_MOESM6_ESM.ppt (127 kb)
ESM 6 (PPT 127 kb)
11295_2015_951_MOESM7_ESM.doc (225 kb)
ESM 7 (DOC 225 kb)


  1. Aleza P, Froelicher Y, Schwarz S, Hernández M, Juárez J, Morillon R, Navarro L, Ollitrault P (2011) Tetraploidization events by chromosome doubling of nucellar cells are frequent in apomictic citrus and are dependent on genotype and environment. Ann Bot 108:37–50PubMedCentralCrossRefPubMedGoogle Scholar
  2. Barkley NA, Roose ML, Krueger RR, Federici CT (2006) Assessing genetic diversity and population structure in a citrus germplasm collection utilizing simple sequence repeat markers (SSRs). TAG Theor Appl Genet 112:1519–1531CrossRefPubMedGoogle Scholar
  3. Barkley NA, Krueger RR, Federici CT, Roose ML (2009) What phylogeny and gene genealogy analyses reveal about homoplasy in citrus microsatellite alleles. Plant Syst Evol 282:71–86CrossRefGoogle Scholar
  4. Barrett H, Rhodes A (1976) A numerical taxonomic study of affinity relationships in cultivated citrus and its close relatives. Syst Bot 1:105–136CrossRefGoogle Scholar
  5. Barry GH, Gmitter FG Jr, Chen C, Roose ML, Federici CT, McCollum GT (2015) Investigating the parentage of ‘Orri’ and ‘Fortune’ mandarin hybrids. Acta Hortic 1065:449–456. doi: 10.17660/ActaHortic.2015.1065.55 CrossRefGoogle Scholar
  6. Bayer RJ, Mabberley DJ, Morton C et al (2009) A molecular phylogeny of the orange subfamily (Rutaceae: Aurantioideae) using nine cpDNA sequences. Am J Bot 96:668–685CrossRefPubMedGoogle Scholar
  7. Belkhir K, Borsa P, Goudet J, Chikhi L, Bonhomme F (2002) GENETIX v. 4.03, logiciel sous windows pour la génétique des populations. In Laboratoire Génome, Populations, Interactions, CNRS UMR 5000, Université de Montpellier II, Montpellier (France). Available at [].
  8. Cheng Y, De Vicente MC, Meng H, Guo W, Tao N et al (2005) A set of primers for analyzing chloroplast DNA diversity in citrus and related genera. Tree Physiol 25:661–672CrossRefPubMedGoogle Scholar
  9. Coletta-Filho HD, Machado MA, Targon MLPN, Moreira MCPQDG, Pompeu Júnior J (1998) Analysis of the genetic diversity among mandarins (citrus spp.) using RAPD markers. Euphytica 102:133–139CrossRefGoogle Scholar
  10. Cuenca J, Froelicher Y, Aleza P, Juarez J, Navarro L, Ollitrault P (2011) Multilocus half-tetrad analysis and centromere mapping in citrus: evidence of SDR mechanism for 2n megagametophyte production and partial chiasma interference in mandarin cv ‘Fortune’. Heredity 107:462–470PubMedCentralCrossRefPubMedGoogle Scholar
  11. Curk et al (2014) Next generation haplotyping to decipher nuclear genomic interspecific admixture in Citrus species: analysis of chromosome 2. BMC Genet 15:1272CrossRefGoogle Scholar
  12. Curk F, Ancillo G, Ollitrault F, Perrier X, Jacquemoud-Collet JP, Garcia-Lor A, Navarro L, Ollitrault P (2015) Nuclear species-diagnostic SNP markers mined from 454 amplicon sequencing reveal admixture genomic structure of modern Citrus varieties. PLoS ONE 10:e0125628. doi: 10.1371/journal.pone.0125628 PubMedCentralCrossRefPubMedGoogle Scholar
  13. Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software structure: a simulation study. Mol Ecol 14:2611–2620CrossRefPubMedGoogle Scholar
  14. Falush D, Stephens M, Pritchard JK (2003) Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics 164:1567–1587PubMedCentralPubMedGoogle Scholar
  15. Fanciullino AL, Tomi F, Luro F, Desjobert JM, Casanova J (2006) Chemical variability of peel and leaf oils of mandarins. Flavour Fragrance J 21:359–367CrossRefGoogle Scholar
  16. Federici CT, Fang DQ, Scora RW, Roose ML (1998) Phylogenetic relationships within the genus citrus (rutaceae) and related genera as revealed by RFLP and RAPD analysis. Theor Appl Genet 96:812–822CrossRefGoogle Scholar
  17. Food and Agriculture Organization of the United Nations, FAOSTAT database (2013) The state of the world’s plant genetic resources for food and agriculture. FAO, Rome, Italy. Available at
  18. Froelicher Y, Dambier D, Bassene JB, Costantino G, Lotfy S, Didout C, Beaumont V, Brottier P, Risterucci AM, Luro F, Ollitrault P (2008) Characterization of microsatellite markers in mandarin orange (Citrus reticulata Blanco). Mol Ecol Resour 8:119–122CrossRefPubMedGoogle Scholar
  19. Froelicher Y, Mouhaya W, Bassene JB, Costantino G, Kamiri M et al (2011) New universal mitochondrial PCR markers reveal new information on maternal citrus phylogeny. Tree Genet Genomes 7:49–61CrossRefGoogle Scholar
  20. Furr JR (1964) New tangerines for the desert. Calif Citrog 49:266–276Google Scholar
  21. Garcia-Lor A, Luro F, Navarro L, Ollitrault P (2012) Comparative use of InDel and SSR markers in deciphering the interspecific structure of cultivated citrus genetic diversity: a perspective for genetic association studies. Mol Gen Genomics 287:77–94CrossRefGoogle Scholar
  22. Garcia-Lor A, Curk F, Snoussi-Trifa H, Morillon R, Ancillo G et al (2013a) 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 111:1–19PubMedCentralCrossRefPubMedGoogle Scholar
  23. Garcia-Lor A, Ancillo G, Navarro L, Ollitrault P (2013b) Citrus (Rutaceae) SNP markers based on Competitive Allele­Specific PCR; transferability across the Aurantioideae subfamily. Applications in Plant Sciences 1: doi: 10.3732/apps.1200406.
  24. Green RM, Vardi A, Galun E (1986) The plastome of Citrus. Physical map, variation among citrus cultivars and species and comparison with related genera. Theor Appl Genet 72:170–177CrossRefPubMedGoogle Scholar
  25. Hirai M, Mitsue S, Kita K, Kajiura I (1990) A survey and isozyme analysis of wild mandarin, tachibana (Citrus tachibana (Mak.) Tanaka) growing in Japan. J Japan Soc Hort Sci 59:1–7CrossRefGoogle Scholar
  26. Hodgson RW (1967) Horticultural varieties of citrus. In: Reuther W, Webber HJ, Batchelor LD (eds) The Citrus industry. University of California, Berkeley, pp 431–591Google Scholar
  27. Kaçar YA, Uzun A, Polat I, Yesiloglu T, Yilmaz B et al (2013) Molecular characterization and genetic diversity analysis of mandarin genotypes by SSR and SRAP markers. J Food, Agric Environ 11:516–521Google Scholar
  28. Kamiri M, Stift M, Srairi I, Costantino G, Moussadik AE, Hmyene A, Bakry F, Ollitrault P, Froelicher Y (2011) Evidence for non-disomic inheritance in a Citrus interspecific tetraploid somatic hybrid between C. reticulata and C. limon using SSR markers and cytogenetic analysis. Plant Cell Rep 30:1415–1425CrossRefPubMedGoogle Scholar
  29. Kijas JMH, Thomas MR, Fowler JCS, Roose ML (1997) Integration of trinucleotide microsatellites into a linkage map of Citrus. Theor Appl Genet 94:701–706CrossRefGoogle Scholar
  30. Koehler-Santos P, Dornelles ALC, Freitas LB (2003) Characterization of mandarin citrus germplasm from Southern Brazil by morphological and molecular analyses. Pesq Agrop Brasileira 38:797–806CrossRefGoogle Scholar
  31. Krueger RR, Roose ML (2003) Use of molecular markers in the management of citrus germplasm resources. J Am Soc Hortic Sci 128:827–837Google Scholar
  32. Krueger RR, Navarro L (2007) Citrus germplasm resources. In: Khan I (ed) Citrus genetics, breeding and biotechnology. CABI Publishing, Wallington, pp 45–140CrossRefGoogle Scholar
  33. Li WB, Liu GF, He SW (1992) Leaf isozymes of mandarin. Proc Int Soc Citricult 1:217–220Google Scholar
  34. Liu K, Muse S (2005) PowerMarker: an integrated analysis environment for genetic marker analysis. Bioinformatics 21:2128–2129CrossRefPubMedGoogle Scholar
  35. Lota ML, Serra DR, Tomi F, Casanova J (2000) Chemical variability of peel and leaf essential oils of mandarins from citrus reticulata blanco. Biochem Syst Ecol 28:61–78CrossRefGoogle Scholar
  36. Luro FL, Costantino G, Terol J, Argout X, Allario T, Wincker P, Talon M, Ollitrault P, Morillon R (2008) Transferability of the EST-SSRs developed on Nules clementine (Citrus clementina Hort ex Tan) to other Citrus species and their effectiveness for genetic mapping. BMC Genomics 9:287PubMedCentralCrossRefPubMedGoogle Scholar
  37. Mabberley D (1997) A classification for edible citrus (rutaceae). Telopea 7:167–172CrossRefGoogle Scholar
  38. Morton CM (2009) Phylogenetic relationships of the Aurantioideae (Rutaceae) based on the nuclear ribosomal DNAITS region and three noncoding chloroplast DNA regions, atpB-rbcL spacer, rps16, and trnL-trnF. Org Divers Evol 9:52–68CrossRefGoogle Scholar
  39. Motohashi T, Matsuyama T, Akihama T (1992) DNA fingerprinting in citrus cultivars. Proc Int Soc Citriculture: 221–224.Google Scholar
  40. Nicolosi E, Deng ZN, Gentile A, Malfa S, Continella G et al (2000) Citrus phylogeny and genetic origin of important species as investigated by molecular markers. Theor Appl Genet 100:1155–1166CrossRefGoogle Scholar
  41. Ollitrault P, Terol J, Garcia-Lor A, Berard A, Chauveau A et al (2012a) SNP mining in C. clementina BAC end sequences; transferability in the Citrus genus (rutaceae), phylogenetic inferences and perspectives for genetic mapping. BMC Genomics 13:13PubMedCentralCrossRefPubMedGoogle Scholar
  42. Ollitrault P, Terol J, Chen C, Federici C, Lotfy S et al (2012b) A reference genetic map of C. clementina hort. ex Tan.; citrus evolution inferences from comparative mapping. BMC Genomics 13:593PubMedCentralCrossRefPubMedGoogle Scholar
  43. Penjor T, Yamamoto M, Uehara M, Ide M, Matsumoto N, Matsumoto R, Nagano Y (2013) Phylogenetic relationships of citrus and its relatives based on matK gene sequences. PLoS ONE 8:e62574PubMedCentralCrossRefPubMedGoogle Scholar
  44. Perrier X, Jacquemoud-Collet J (2006) DARwin software. Available at 5.0.158.
  45. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959PubMedCentralPubMedGoogle Scholar
  46. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  47. Swingle W, Reece P (1967) The botany of citrus and its wild relatives. In: Reuther W, Webber HJ, Batchelor LD (eds) The Citrus industry. University of California, Berkeley, pp 190–430Google Scholar
  48. Tanaka T (1954) Species problem in Citrus (Revisio aurantia cearum IX). Japanese Society for Promotion of Science, Tokyo, Japan, pp 1–157Google Scholar
  49. Tanaka T (1977) Fundamental discussion of citrus classification. Studia Citrologia 14:1–6Google Scholar
  50. Tapia Campos E, Gutiérrez Espinosa MA, Warburton ML, Santacruz Varela A, Villegas Monter Á (2005) Characterization of mandarin (Citrus spp.) using morphological and AFLP markers. Interciencia 30:687–693, Available at: ISSN 0378–1844Google Scholar
  51. Webber HJ (1943) Cultivated varieties of citrus. In: Anonymous the Citrus industry. History, World Distribution, Botany and Varieties, California, pp 475–668Google Scholar
  52. Weir BS, Cockerham CC (1984) Estimating F-statistics for the analysis of population structure. Evolution 38:1358–1370CrossRefGoogle Scholar
  53. Wu GA, Prochnik S, Jenkins J, Salse J, Hellsten U, Murat F et al (2014) Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication. Nat Biotechnol 32:656–663PubMedCentralCrossRefPubMedGoogle Scholar
  54. Wright S (1969) The theory of gene frequencies. In: Anonymous evolution and the genetics of populations. University of Chicago Press, ChicagoGoogle Scholar
  55. Yamamoto M, Tominaga S (2003) High chromosomal variability of mandarins (citrus spp.) revealed by CMA banding. Euphytica 129:267–274CrossRefGoogle Scholar
  56. Yamamoto M, Kobayashi S, Nakamura Y, Yamada Y (1993) Phylogenic relationships of Citrus revealed by diversity of cytoplasmic genomes. In: Hayashi T, Omura M, Scott NS (eds) Techniques on gene diagnosis and breeding in fruit trees. Res Sta, Okitsu, Japan, pp 39–46Google Scholar
  57. Yamamoto M, Tsuchimochi Y, Ninomiya T, Koga T, Kitajima A, Yamasaki A, Inafuku-Teramoto S, Yang X, Yang X, Zhong G, Nasir N, Kubo T and Tominaga S (2013) Diversity of chloroplast DNA in various mandarins (Citrus spp.) and other citrus demonstrated by CAPS analysis. J. Japan. Soc. Hort. Sci. 82: 106-113Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Andres Garcia-Lor
    • 1
  • François Luro
    • 2
  • Patrick Ollitrault
    • 1
    • 3
  • Luis Navarro
    • 1
    Email author
  1. 1.Centro de Protección Vegetal y BiotecnologíaInstituto Valenciano de Investigaciones Agrarias (IVIA)MoncadaSpain
  2. 2.UMR AGAP Institut National de la Recherche Agronomique (INRA Corse)San GiulianoFrance
  3. 3.UMR AGAP, Centre de coopération Internationale en Recherche Agronomique pour le Développement (CIRAD)Petit-BourgGuadeloupe

Personalised recommendations