Molecular Biology Reports

, Volume 41, Issue 11, pp 7217–7222 | Cite as

Utility of indels for species-level identification of a biologically complex plant group: a study with intergenic spacer in Citrus

Article
First Online:
Received:
Accepted:

Abstract

The Consortium of Barcode of Life plant working group proposed to use the defined portion of plastid genes rbcL and matK either singly or in combination as the standard DNA barcode for plants. But DNA barcode based identification of biologically complex plant groups are always a challenging task due to the occurrence of natural hybridization. Here, we examined the use of indels polymorphism in trnH-psbA and trnL-trnF sequences for rapid species identification of citrus. DNA from young leaves of selected citrus species were isolated and matK gene (~800 bp) and trnH-psbA spacer (~450 bp) of Chloroplast DNA was amplified for species level identification. The sequences within the group taxa of Citrus were aligned using the ClustalX program. With few obvious misalignments were corrected manually using the similarity criterion. We identified a 54 bp inverted repeat or palindrome sequence (27–80 regions) and 6 multi residues indel coding regions. Large inverted repeats in cpDNA provided authentication at the higher taxonomic levels. These diagnostics indel marker from trnH-psbA were successful in identifying different species (5 out of 7) within the studied Citrus except Citrus limon and Citrus medica. These two closely related species are distinguished through the 6 bp deletion in trnL-trnF. This study demonstrated that the indel polymorphism based approach easily characterizes the Citrus species and the same may be applied in other complex groups. Likewise other indels occurring intergenic spacer of chloroplast regions may be tested for rapid identification of other secondary citrus species.

Keywords

Indel trnH-psbA Citrus DNA barcoding Inverted repeat Primer design 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

We acknowledge Department of Biotechnology, Govt. of India for the infrastructural support and UGC–JRF in Engineering & Technology fellowships to the author (PM).

Supplementary material

11033_2014_3606_MOESM1_ESM.doc (152 kb)
Supplementary material 1 (DOC 151 kb)

References

  1. 1.
    Laskar BA, Bhattacharjee MJ, Dhar B, Mahadani P, Kundu S, Ghosh SK (2013) The species dilemma of Northeast Indian Mahseer (Actinopterygii: Cyprinidae): DNA barcoding in clarifying the riddle. PLoS ONE 8(1):e53704PubMedCrossRefPubMedCentralGoogle Scholar
  2. 2.
    Hollingsworth MP, Forrest LL, Spouge LJ, Hajibabaei M, Ratnasingham S, van der Bank M, Chase WM, Cowan SR et al (2009) A DNA barcode for land plants. Proc Natl Acad Sci U S A 106(31):12794–12797CrossRefPubMedCentralGoogle Scholar
  3. 3.
    Luo K, Chen S, Chen K, Song J, Yao H, Ma X, Zhu Y, Pang X, Yu H, Li X, Liu Z (2010) Assessment of candidate plant DNA barcodes using the Rutaceae family. Sci China Life Sci 53(6):701–708PubMedCrossRefGoogle Scholar
  4. 4.
    Maia VH, Mata CS, Franco LO, Cardoso MA, Cardoso SR, Hemerly AS, Ferreira PC (2012) DNA barcoding Bromeliaceae: achievements and pitfalls. PLoS ONE 7(1):e29877PubMedCrossRefPubMedCentralGoogle Scholar
  5. 5.
    Roy S, Tyagi A, Shukla V, Kumar A, Singh UM, Chaudhary LB, Datt B, Bag SK, Singh PK, Nair NK, Husain T, Tuli R (2010) Universal plant DNA barcode loci may not work in complex groups: a case study with Indian berberis species. PLoS ONE 5(10):e13674PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Spooner DM (2009) DNA barcoding will frequently fail in complicated groups: an example in wild potatoes. Am J Bot 96(6):1177–1189PubMedCrossRefGoogle Scholar
  7. 7.
    Britten RJ, Rowen L, Williams J, Cameron RA (2003) Majority of divergence between closely related DNA samples is due to indels. Proc Natl Acad Sci U S A 100(8):4661–4665PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Tian D, Wang Q, Zhang P, Araki H, Yang S, Kreitman M, Nagylaki T, Hudson R, Bergelson J, Chen JQ (2008) Single-nucleotide mutation rate increases close to insertions/deletions in eukaryotes. Nature 455(7209):105–108PubMedCrossRefGoogle Scholar
  9. 9.
    Golenberg EM, Clegg MT, Durbin ML, Doebley J, Ma DP (1993) Evolution of a noncoding region of the chloroplast genome. Mol Phy Evol 2(1):52–64CrossRefGoogle Scholar
  10. 10.
    Ingvarsson PK, Ribstein S, Taylor DR (2003) Molecular evolution of insertions and deletion in the chloroplast genome of silene. Mol Bio Evol 20(11):1737–1740CrossRefGoogle Scholar
  11. 11.
    Blair C, Murphy RW (2011) Recent trends in molecular phylogenetic analysis: where to next? J Hered 102(1):130–138PubMedCrossRefGoogle Scholar
  12. 12.
    Hebert PD, Cywinska A, Ball SL, deWaard JR (2003) Biological identifications through DNA barcodes. Proc Biol Sci 270(1512):313–321PubMedCrossRefPubMedCentralGoogle Scholar
  13. 13.
    Liu J, Provan J, Gao L, Li D (2012) Sampling strategy and potential utility of indels for DNA barcoding of closely related plant species: a case study in Taxus. Int J Mol Sci 13(7):8740–8751PubMedCrossRefPubMedCentralGoogle Scholar
  14. 14.
    Bayer RJ, Mabberley DJ, Morton C, Miller CH, Sharma IK, Pfeil BE, Rich S, Hitchcock R, Sykes S (2009) A molecular phylogeny of the orange subfamily (Rutaceae: Aurantioideae) using nine cpDNA sequences. Am J Bot 96(3):668–685PubMedCrossRefGoogle Scholar
  15. 15.
    Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24:4876–4882CrossRefGoogle Scholar
  16. 16.
    Ragupathy S, Newmaster SG, Murugesan M, Balasubramaniam V (2009) DNA barcoding discriminates a new cryptic grass species revealed in an ethnobotany study by the hill tribes of the Western Ghats in southern India. Mol Ecol Resour 9(Suppl s1):164-171Google Scholar
  17. 17.
    Simmons MP, Ochoterena H (2000) Gaps as characters in sequence-based phylogenetic analyses. Syst Biol 49(2):369–381PubMedCrossRefGoogle Scholar
  18. 18.
    Fazekas AJ, Kesanakurti PR, Burgess KS, Percy DM, Graham SW, Barrett SC, Newmaster SG, Hajibabaei M, Husband BC (2009) Are plant species inherently harder to discriminate than animal species using DNA barcoding markers? Mol Ecol Resour 9: 130–139Google Scholar
  19. 19.
    Jena SN, Kumar S, Nair NK (2009) Molecular phylogeny in IndianCitrus L. (Rutaceae) inferred through PCR-RFLP and trnL-trnF sequence data of chloroplast DNA. Sci Hortic 119(4):403–416CrossRefGoogle Scholar
  20. 20.
    Strochova H, Olson MS (2007) The architecture of the chloroplast psbA-trnH non coding region in angiosperms. Plant Syst Evol 268:235–256CrossRefGoogle Scholar
  21. 21.
    Mahadani P, Sharma GD, Ghosh SK (2013) Identification of ethnomedicinal plants (Rauvolfioideae: Apocynaceae) through DNA barcoding from northeast India. Pharmacogn Mag 9(35):255–263PubMedCrossRefPubMedCentralGoogle Scholar
  22. 22.
    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):1–19PubMedCrossRefPubMedCentralGoogle Scholar
  23. 23.
    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 Genet Genomics 287(1):77–94PubMedCrossRefGoogle Scholar
  24. 24.
    Hamilton MB, Braverman JM, Soria-Hernanz DF (2003) Patterns and relative rates of nucleotide and insertion/deletion evolution at six chloroplast intergenic regions in new world species of the Lecythidaceae. Mol Biol Evol 20(10):1710–1721PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  1. 1.DNA Barcode and Genomics Laboratory, Department of BiotechnologyAssam UniversitySilcharIndia

Personalised recommendations