Genetic Resources and Crop Evolution

, Volume 65, Issue 8, pp 2199–2214 | Cite as

New Citrus chloroplast haplotypes revealed by molecular markers using Algerian and Spanish accessions

  • Taklit Maddi
  • Estela Pérez-Román
  • Fadila Maiza-Benabdesselam
  • Bachra Khettal
  • Manuel Talon
  • Victoria Ibanez-GonzalezEmail author
Research Article


Thirty-seven chloroplast molecular markers were used to evaluate the genetic diversity and infer the phylogenetic relationship of 24 Algerian Citrus accessions from the Institut Technique de l′Arboriculture Fruitière et de la Vigne germplasm bank. The reliability and consistence of the clustering distribution was further asserted including 5 Spanish accessions from the Instituto Valenciano de Investigaciones Agrarias. The accessions were positioned on a phylogenetic tree of the genus Citrus based on previous analyses of the whole sequence of citrus chloroplast. Algerian accessions clustered into two main clades mostly differentiated by the occurrence of either mandarin or pummelo chloroplast types. All 7 mandarins analyzed were grouped in the same clade while the other cluster subdivided in 4 groups, included 1 lumia, 3 lemons, 2 grapefruits and 11 sweet oranges. Algerian grapefruit accessions were grouped together with the pummelos in a single cluster while all sweet oranges formed an independent and homogenous clade. Interestingly, the lemons studied were clustered in 3 different subclusters while Citrus lumia genotype was isolated in a different group. These results suggest that in contrast to the studied Algerian mandarins or sweet oranges, that share all the same mandarin or sweet orange chloroplast haplotype, the high diversity of current lemon accessions is at least partially correlated with the identity of different pummelo progenitors which evolved from a common ancestor. In addition, the data indicate that Citrus lumia is a new type of citrus chloroplast that appears to be phylogenetically related to the chloroplasts of the pummelo and micrantha group.


Genetic Lemon Mandarin Plant Pummelo SNP 



This work was supported by the Instituto Nacional de Investigación Agraria y Alimentaria, INIA, (Ministerio de Economía, Industria y competitividad e Innovación, Spain) under Grant [# RTA-00071-C06-01]. The authors acknowledge the access to its plant material to ITAFV (Institut Technique de l′Arboriculture Fruitière et de la Vigne, Tessala El Merdja- Algeria) and IVIA (Instituto Valenciano de Investigaciones Agrarias, Spain).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10722_2018_685_MOESM1_ESM.docx (19 kb)
Supplementary material 1 (DOCX 19 kb)
10722_2018_685_MOESM2_ESM.docx (28 kb)
Supplementary material 2 (DOCX 27 kb)


  1. Aldeguer M, López-Andreo M, Gabaldón JA, Puyet A (2014) Detection of mandarin in orange juice by single-nucleotide polymorphism qPCR assay. Food Chem 145:1086–1091. CrossRefPubMedGoogle 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). Theor Appl Genet 112:1519–1531. CrossRefPubMedGoogle Scholar
  3. Barrett HC, Rhodes AM (1976) A numerical taxonomic study of affinity relationships in cultivated Citrus and its close relatives. Syst Bot 1:105–136. CrossRefGoogle Scholar
  4. Bausher MG, Singh ND, Lee SB, Jansen RK, Daniell H (2006) 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. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bielsa B, Jiwan D, Fernández i Martí A, Dhingra A, Rubio-Cabetas MJ (2014) Detection of SNP and validation of a SFP Indel (deletion) in inverted repeat region of the Prunus species chloroplast genome. Sci Hortic Amst 168:108–112. CrossRefGoogle Scholar
  6. Cameron J, Soost R (1961) Chandler: an early-ripening hybrid pummelo derived from a low-acid parent. Hilgardia 30:359–364. CrossRefGoogle Scholar
  7. Carbonell-Caballero J, Alonso R, Ibanez V, Terol J, Talon M, Dopazo J (2015) A phylogenetic analysis of 34 chloroplast genomes elucidates the relationships between wild and domestic species within the genus Citrus. Mol Biol Evol 32:2015–2036. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Chen C, Gmitter FG Jr (2013) Mining of haplotype-based expressed sequence tag single nucleotide polymorphisms in Citrus. BMC Genomics 14:746. CrossRefPubMedPubMedCentralGoogle Scholar
  9. Coletta Filho H, Machado M, Targon M (1998) Analysis of the genetic diversity among mandarins (Citrus spp.) using RAPD markers. Euphytica 102:133–139. CrossRefGoogle Scholar
  10. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  11. Fujii H, Ohta S, Nonaka K, Katya’s Y, Matsumoto T, Endo T, Yoshioka T, Omura M, Shimada T (2016) Parental diagnosis of satsuma mandarin (Citrus unshiu Marc.) revealed by nuclear and cytoplasmic markers. Breed Sci 66:683–691. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Garcia-Lor A, Curk F, Snoussi-Trifa H, Morillon R, Ancillo G, Luro F, Navarro L, Ollitrault P (2013) 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 111:1–19. CrossRefPubMedGoogle Scholar
  13. Hodgson RW (1967) Horticultural varieties of Citrus. The Citrus industry 1: 431–591, University of California Press, BerkeleyGoogle Scholar
  14. Jena SN, Kumar S, Nair NK (2009) Molecular phylogeny in Indian Citrus L. (Rutaceae) inferred through PCR-RFLP and trnL-trnF sequence data of chloroplast DNA. Sci Hortic Amst 199:403–416. CrossRefGoogle Scholar
  15. Jukes TH, Cantor CR (1969) Evolution of protein molecules. In: Munro HN (ed) Mammalian protein metabolism. Academic Press, New York, pp 21–132CrossRefGoogle Scholar
  16. Koressaar T, Remm M (2007) Enhancements and modifications of primer design program Primer3. Bioinformatics 23:1289–1291. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874. CrossRefGoogle Scholar
  18. Ling P, Ducan LW, Deng Z, Dunn D, Hu X, Huang S, Gmitter FG (2000) Inheritance of Citrus nematode resistance and its linkage with molecular markers. Theor Appl Genet 100:1010–1017. CrossRefGoogle Scholar
  19. Liu W, Wu H, Luo Y, Xi W, Zhou Z (2017) Comparative analysis of chloroplast genomes of the genus Citrus and its close relatives. Mitochondrial DNA 28:33–36. CrossRefPubMedGoogle Scholar
  20. Luro F, Laigret F, Bove JM, Ollitrault P (1995) DNA amplified fingerprinting, a useful tool for determination of genetic origin and diversity analysis in Citrus. HortScience 30:1063–1067Google Scholar
  21. Mahadani P, Ghosh Sankar K (2014) Utility of indels for species-level identification of a biologically complex plant group: a study with intergenic spacer in Citrus. Mol Biol Rep 41:7217–7222. CrossRefPubMedGoogle Scholar
  22. Mammadov J, Aggarwal R, Buyyarapu R, Kumpatla S (2012) SNP markers and their impact on plant breeding. Int J Plant Genomics 2012:728398. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Nicolosi E, Deng ZN, Gentile A, La Malfa S, Continella G, Tribulato E (2000) Citrus phylogeny and genetic origin of important species as investigated by molecular markers. Theor Appl Genet 100:1155–1166. CrossRefGoogle Scholar
  24. Oueslati A, Ollitrault F, Baraket G, Salhi-Hannachi A, Navarro L, Ollitrault P (2016) Towards a molecular taxonomic key of the Aurantioideae subfamily using chloroplastic SNP diagnostic markers of the main clades genotyped competitive allele-specific PCR. BMC Genet 17(1):118. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Pardo MA (2015) Evaluation of a dual-probe real time PCR system for detection of mandarin in commercial orange juice. Food Chem 172:377–384. CrossRefPubMedGoogle Scholar
  26. 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:e62574. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Pessoa-Filho M, Magalhães MA, Elias-Ferreira M (2017) Molecular dating of phylogenetic divergence between Urochloa species based on complete chloroplast genomes. BMC Genomics 18:516. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Primmer CR, Borge T, Lindell J, Saetre GP (2002) Single-nucleotide polymorphism characterization in species with limited available sequence information: high nucleotide diversity revealed in the avian genome. Mol Ecol 11:603–612. CrossRefPubMedGoogle Scholar
  29. Ramadugu C, Keremane ML, Hu X, Karp D, Federici CT, Kahn T, Roose ML, Lee RF (2015) Genetic analysis of citron (Citrus medica L.) using simple sequence repeats and single nucleotide polymorphisms. Sci Hortic Amst 195:124–137. CrossRefGoogle Scholar
  30. Robinson JT, Thorvaldskóttir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat Biotechnol 29:24–26. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Scora RW (1975) On the history and origin of Citrus. B Torrey Bot Club 102:369–375. CrossRefGoogle Scholar
  32. Scora RW (1988) Biochemistry, taxonomy and evolution of modern cultivated Citrus. Proc Int Soc Citric 1:277–289Google Scholar
  33. Shimizu T, Kitajima A, Nonaka K, Yoshioka T, Ohta S, Goto S, Toyoda A, Fujiyama A, Mochizuki T, Nagasaki H, Kaminuma E, Nakamura Y (2016) Hybrid origins of Citrus varieties inferred from DNA marker analysis of nuclear and organelle genomes. PLoS ONE 11:e0166969. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Singh IP (2002) Micropropagation in Citrus—A review. Agri Rev 23:1–13Google Scholar
  35. Swingle WT (1943) The botany of Citrus and its wild relatives of the orange subfamily. The Citrus Industry 1: 129–474. In: Webber HJ, Batchelor LD (eds) University of California Press, CaliforniaGoogle Scholar
  36. Tanaka T (1954) Species problem in Citrus: a critical study of wild and cultivated units of Citrus, based upon field studies in their native homes (Revisio Aurantiacearum IX), Japanese Society for the Promotion of Science, p 152Google Scholar
  37. Terol J, Ibañez V, Carbonell J, Alonso R, Estornell L, Licciardello C, Gut IG, Dopazo J, Talon M (2015) Involvement of a Citrus meiotic recombination TTC-repeat motif in the formation of gross deletions generated by ionizing radiation and MULE activation. BMC Genom 16:69. CrossRefGoogle Scholar
  38. Thorvaldsdóttir H, Robinson JT, Mesirov JP (2013) Integrative genomics viewer (IGV): performance genomics data visualization and exploration. Brief Bioinform 14(2):178–192. CrossRefPubMedGoogle Scholar
  39. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3—new capabilities and interfaces. Nucleic Acids Res 40(15):e115. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Uzun A, Yesiloglu T, Aka-Kacar Y, Tuzcu O, Gulsen O (2009) Genetic diversity and relationships within Citrus and related genera based on sequence related amplified polymorphism markers (SRAPs). Sci Hortic Amst 121:306–312. CrossRefGoogle Scholar
  41. Weising K, Nybom H, Wolff K, Kahl G (2005) DNA fingerprinting in plants: principles, methods and applications, 2nd edn. Taylor and Francis group, Boca RatonCrossRefGoogle Scholar
  42. Whitkus R, Doebley J, Wendel JF (1994) Nuclear DNA markers in systematics and evolution. In: Philips RL, Vasil IK (eds) DNA-based markers in plants. Advances in cellular and molecular biology of plants. Kluwer Academic Publishers, DordrechtGoogle Scholar
  43. Wu GA, Prochnik S, Jenkins J, Salse J, Hellsten U, Murat F, Perrier S, Ruiz M, Scalabrin S, Terol J, Takita MA, Labadie K, Poulain J, Coulous A, Jabbari K, Cattonaro F, Del Fabbro C, Pinosio S, Zuccolo A, Chapman J, Grimwood J, Tadeo F, Estornell L, Muñoz-Sanz JV, Ibanez V, Herrero-Ortega A, Aleza P, Pérez-Pérez J, Ramón D, Brunel D, Luro F, Chen C, Farmerie W, Desany B, Kodira C, Mohiuddin M, Harkins T, Fredrikson K, Burns P, Lomsadze A, Borodovsky M, Reforgiato G, Freitas-Astúa J, Quetier F, Navarro L, Roose M, Wincker P, Schmutz J, Morgante M, Machado MA, Talon M, Jaillon O, Ollitrault P, Gmitter F, Rokhsar D (2014) Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during Citrus domestication. Nat Biotechnol 2:656–662. CrossRefGoogle Scholar
  44. Wu GA, Terol J, Ibanez V, López-García A, Pérez-Román E, Borredá C, Domingo C, Tadeo FR, Carbonell-Caballero J, Alonso R, Curk F, Du D, Ollitrault P, Roose M, Dopazo J, Gmitter F, Rokshar D, Talon M (2018) Genomics of the origin and evolution of Citrus. Nature 554(7692):311–316. CrossRefPubMedGoogle Scholar
  45. Xing C, Schumacher F, Xing G, Lu Q, Wang T, Elston R (2005) Comparison of microsatellites, single-nucleotide polymorphisms (SNPs) and composite markers derived from SNPs in linkage analysis. BMC Genet 6:S29. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Yu H, Yang X, Guo F, Jiang X, Deng X, Xu Q (2017) Genetic diversity and population structure of pummelo (Citrus maxima) germplasm in China. Tree Genet Genomes 13:58. CrossRefGoogle Scholar
  47. Zaragozá S, Pina Lorca JA, Forner MA, Navarro L, Medina A, Soler G, Chomé PM (2011) Las variedades de Cítricos. El material vegetal y el registro de variedades comerciales de España. Spain. Ministerio de Medio Ambiente, Medio Rural y MarinoGoogle Scholar
  48. Zhen-hua L, Zhi-qin Z, Rang-jin X (2011) Molecular phylogeny of the “True Citrus Fruit Trees” group (Aurantioideae, Rutaceae) as inferred from chloroplast DNA sequence. Agric Sci China 10:49–57. CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Laboratoire de Biotechnologie végétale et EthnobotaniqueFaculté des Sciences de la Nature et de la Vie, Université de BejaiaBejaiaAlgeria
  2. 2.Centro de GenómicaInstituto Valenciano de Investigaciones AgrariasMoncadaSpain
  3. 3.Escuela de Empresarios (EDEM)ValenciaSpain
  4. 4.Universidad de Valencia (UV)ValenciaSpain

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