Skip to main content
SpringerLink
Log in

Assessment of Genetic Variability of Citrus tristeza virus by SSCP and CE-SSCP

  • Protocol
  • First Online:
Citrus Tristeza Virus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2015))

  • 626 Accesses

Abstract

Single-strand conformation polymorphism (SSCP) is a popular method used to study the genetic heterogeneity and population variability of Citrus tristeza virus (CTV) isolates. It is a simple, low-cost, and highly specific method for mutation detection of specific genes, mostly of the CTV major coat protein gene (p25). The technique is based on a comparison on polyacrylamide gel of electrophoretic profiles of single-stranded (ss) DNA sequences in terms of their spatial conformation. SSCP involves cDNA synthesis and amplification of the target gene, denaturation of single strands, and electrophoresis in non-denaturing conditions. The ssDNAs can be afterward visualized by staining the polyacrylamide gel. Alternatively, using fluorescently labeled primers, the procedure can be performed in automated sequencers equipped with an appropriate capillary (CE-SSCP), which increases the potential of high-throughput analysis, precision, and the reproducibility of results. CE-SSCP can be also directly applied to the virus particles obtained by elution from ELISA plates or tissue-print membranes.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Moreno P, Ambrós S, Albiach-Martí MR et al (2008) Citrus tristeza virus: a pathogen that changed the course of the citrus industry. Mol Plant Pathol 9:251–268

    Article  CAS  Google Scholar 

  2. Bar-Joseph M, Marcus R, Lee RF (1989) The continuous challenge of Citrus tristeza virus control. Annu Rev Phytopathol 27:291–316

    Article  Google Scholar 

  3. Dawson WO, Garnsey SM, Tatineni S et al (2013) Citrus tristeza virus-host interactions. Front Microbiol 4:88

    Article  CAS  Google Scholar 

  4. Harper SJ (2013) Citrus tristeza virus: evolution of complex and varied genotypic groups. Front Microbiol 4:93

    Article  CAS  Google Scholar 

  5. Orita M, Suzuki Y, Sekiya T et al (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5:874–879

    Article  CAS  Google Scholar 

  6. Kakavas KV, Plageras P, Vlachos AT et al (2008) PCR–SSCP: a method for the molecular analysis of genetic diseases. Mol Biotechnol 38:155–163

    Article  Google Scholar 

  7. Sunnucks P, Wilson ACC, Beheregaray LB et al (2000) SSCP is not so difficult: the application and utility of single-stranded conformation polymorphism in evolutionary biology and molecular ecology. Mol Ecol 9:1699–1710

    Article  CAS  Google Scholar 

  8. Cerni S, Skoric D, Ruscic J et al (2009) East Adriatic—a reservoir of severe Citrus tristeza virus strains. Eur J Plant Pathol 124:701–706

    Article  Google Scholar 

  9. Rubio L, Ayllón MA, Kong P et al (2001) Genetic variation of Citrus tristeza virus isolates from California and Spain: evidence for mixed infections and recombination. J Virol 75:8054–8062

    Article  CAS  Google Scholar 

  10. Kalvatchev Z, Draganov P (2005) Single-Strand Conformation Polymorphism (SSCP) analysis: a rapid and sensitive method for detection of genetic diversity among virus population. Biotechnol Biotechnol Equip 19:9–14

    Article  CAS  Google Scholar 

  11. Rubio L, Ayllón MA, Guerri J et al (1996) Differentiation of citrus tristeza closterovirus (CTV) isolates by single-strand conformation polymorphism analysis of the coat protein gene. Ann Appl Biol 129:479–489

    Article  CAS  Google Scholar 

  12. Kong P, Rubio L, Polek M et al (2000) Population structure and genetic diversity within California Citrus tristeza virus (CTV) isolates. Virus Genes 21:139–145

    Article  CAS  Google Scholar 

  13. Ayllón MA, Rubio L, Moya A et al (1999) The haplotype distribution of two genes of Citrus tristeza virus is altered after host change or aphid transmission. Virology 255:32–39

    Article  Google Scholar 

  14. D’Urso F, Sambade A, Moya A et al (2003) Variation of haplotype distributions of two genomic regions of Citrus tristeza virus populations from eastern Spain. Mol Ecol 12:517–526

    Article  Google Scholar 

  15. Morales J, Acosta O, Tamayo P et al (2013) Characterization of Citrus tristeza virus isolates from Colombia. Rev Protección Veg 28:45–53

    CAS  Google Scholar 

  16. Abou Kubaa R, D’Onghia AM, Djelouah K et al (2008) Characterization of Citrus tristeza virus isolates recovered in Syria and Apulia (Southern Italy) using different molecular tools. Phytopathol Mediterr 51:496–504

    Google Scholar 

  17. Amin HA, Fonseca F, Santos C et al (2006) Typing of Egyptian Citrus tristeza virus (CTV) isolates based on the capsid protein gene. Phytopathol Mediterr 45:10–14

    CAS  Google Scholar 

  18. Chatzivassiliou EK, Nolasco G (2014) Detection of a new variant of Citrus tristeza virus (CTV) in Greek citrus crops. Phytopathol Mediterr 53:140–147

    CAS  Google Scholar 

  19. Davino S, Rubio L, Davino M (2005) Molecular analysis suggests that recent Citrus tristeza virus outbreaks in Italy were originated by at least two independent introductions. Eur J Plant Path 111:289–293

    Article  CAS  Google Scholar 

  20. Ferretti L, Fontana A, Sciarroni R et al (2014) Molecular and biological evidence for a severe seedling yellows strain of Citrus tristeza virus spreading in southern Italy. Phytopathol Mediterr 53:3–13

    CAS  Google Scholar 

  21. Kim DH, Shim HK, Hyeon JW et al (2006) SSCP analysis of variations in haplotypes of Citrus tristeza virus isolated from yuzu (Citrus junos) in geographically separate regions of Korea. J Plant Biol 49:88–96

    Article  CAS  Google Scholar 

  22. Lbida B, Fonseca F, Santos C et al (2004) Genomic variability of Citrus tristeza virus (CTV) isolates introduced into Morocco. Phytopathol Mediterr 43:205–210

    CAS  Google Scholar 

  23. Oliveros-Garay OA, Martinez-Salazar N, TorresRuiz Y et al (1937) CPm gene diversity in field isolates of Citrus tristeza virus from Colombia. Arch Virol 154:1933–1937

    Article  Google Scholar 

  24. Papayiannis LC, Santos C, Kyriakou A et al (2007) Molecular characterization of Citrus tristeza virus isolates from Cyprus on the basis of the coat protein gene. J Plant Path 89:291–295

    CAS  Google Scholar 

  25. Nolasco G, Fonseca F, Silva G (2008) Occurrence of genetic bottlenecks during Citrus tristeza virus acquisition by Toxoptera citricida under field conditions. Arch Virol 153:259–271

    Article  CAS  Google Scholar 

  26. Cerni S, Ruscic J, Nolasco G et al (2008) Stem pitting and seedling yellows symptoms of Citrus tristeza virus infection may be determined by minor sequence variants. Virus Genes 36:241–249

    Article  CAS  Google Scholar 

  27. Iglesias NG, Gago-Zachert SP, Robledo G et al (2008) Population structure of Citrus tristeza virus from field Argentinean isolates. Virus Genes 36:199–207

    Article  CAS  Google Scholar 

  28. Gago-Zachert S, Costa N, Semorile L (1999) Sequence variability in p27 gene of Citrus tristeza virus (CTV) revealed by SSCP analysis. Electron J Biotechnol 215:04

    Google Scholar 

  29. Costa AT, Nunes WMC, Zanutto CA et al (2010) Stability of Citrus tristeza virus protective isolates in field conditions. Pesq Agrop Brasileira 45:693–700

    Article  Google Scholar 

  30. Temporal WM, Corazza MJ, Zanutto CA et al (2011) SSCP analysis of Citrus tristeza virus protectives isolates in Pêra sweet orange clones under northern Paraná state, Brazil conditions. Citrus Res Tech 32:9–16

    Article  Google Scholar 

  31. Leonel WMS, Corazza MJ, Zanutto CA et al (2015) Stability of Citrus tristeza virus protective isolate ‘Pêra IAC’ according to SSCP analysis of old and new lines of three sweet orange varieties. Summa Phytopathol 41:8–12

    Article  Google Scholar 

  32. Sambade A, Rubio L, Garnsey SM et al (2002) Comparison of the viral RNA populations of pathogenically distinct isolates of Citrus tristeza virus. Application to monitoring cross protection. Plant Pathol 51:257–265

    Article  CAS  Google Scholar 

  33. Sambade A, Ambrós S, López C et al (2007) Preferential accumulation of severe variants of Citrus tristeza virus in plants co-inoculated with mild and severe variants. Arch Virol 152:1115–1126

    Article  CAS  Google Scholar 

  34. Waldecy Matos da Silva L, Corazza MJ, Zanutto CA et al (2015) Stability of Citrus tristeza virus protective isolate ‘Pêra IAC’ according to SSCP analysis of old and new lines of three sweet orange varieties. Summa Phytopathol 41:8–12

    Article  Google Scholar 

  35. Jespersgaard C, Larsen LA, Baba S et al (2006) Optimization of capillary array electrophoresis single-strand conformation polymorphism analysis for routine molecular diagnostics. Electrophoresis 27:3816–3822

    Article  CAS  Google Scholar 

  36. Larsen LA, Jespersgaard C, Andersen PS (2007) Single-strand conformation polymorphism analysis using capillary electrophoresis for large-scale mutation detection. Nat Protoc 2:1458–1466

    Article  CAS  Google Scholar 

  37. Quinto JD, Wang CK (2004) A high-throughput single-stranded conformation polymorphism assay for detection, subtyping and genotyping of influenza viruses. Int Congr Ser 1263:653–657

    Article  CAS  Google Scholar 

  38. Licciardello G, Raspagliesi D, Bar Joseph M et al (2012) Characterization of isolates of Citrus tristeza virus by sequential analyses of enzyme immunoassays and capillary electrophoresis-single-strand conformation polymorphisms. J Virol Methods 181:139–147

    Article  CAS  Google Scholar 

  39. Dallot S, Boeglin M, Labonne G (2008) Spatial pattern and genetic structure of PPV-M in a delimited area of stone fruit orchards in southern France. Acta Hort 781:235–242

    Article  Google Scholar 

  40. Delaunay A, Dallot S, Filloux D et al (2015) SNaPshot and CE-SSCP: two simple and cost-effective methods to reveal genetic variability within a virus species. Method Mol Biol 1302:187–206

    Article  Google Scholar 

  41. OEPP/EPPO (2004) Diagnostic protocols for regulated pests/Protocoles de diagnostic pour les organismes réglementés - Citrus tristeza closterovirus. OEPP/EPPO Bull 34:155–157

    Article  Google Scholar 

Download references

Acknowledgments

The CE-SSCP protocol was developed thanks to the project IT-Citrus Genomics PON 01_1623, funded by MIUR and MISE and co-funded by the EU, and the “Lotta al virus della tristezza degli agrumi” project funded by Assessorato delle Risorse Agricole ed Agroalimentari, Regione Siciliana granted to Parco Scientifico e Tecnologico della Sicilia. The method was patented in Italy, EU: “Metodo per la discriminazione di ceppi virali Citrus tristeza virus ” N° 1405881, 30/01/2014, by Licciardello G., Raspagliesi D., Lombardo A., Catara A (Parco Scientifico e Tecnologico della Sicilia).

Author information

Authors and Affiliations

  1. Plant Pathology Laboratory, Faculty of Crop Science, Agricultural University of Athens, Athens, Greece

    Elisavet K. Chatzivassiliou

  2. Consiglio per la Ricerca in Agricoltura e l’analisi dell’Economia Agraria (CREA), Centro di Olivicoltura, Frutticoltura e Agrumicoltura (CREA-OFA), Acireale (Catania), Italy

    Grazia Licciardello

Authors
  1. Elisavet K. Chatzivassiliou
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Grazia Licciardello
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Grazia Licciardello .

Editor information

Editors and Affiliations

  1. Department of Phytosanitary Sciences, University of Catania, Science and Technology Park of Sicily, Catania, Italy

    Antonino F. Catara

  2. The S. Talkowski Laboratory, Department of Plant Pathology and Weed Research, The Volcani Center, Agricultural Research Organization, Bet Dagan, Israel

    Moshe Bar-Joseph

  3. Consiglio per la Ricerca in agricoltura e l’analisi dell’Economia Agraria, Centro di Olivicoltura, Frutticoltura e Agrumicoltura (CREA-OFA), Acireale (Catania), Italy

    Grazia Licciardello

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Chatzivassiliou, E.K., Licciardello, G. (2019). Assessment of Genetic Variability of Citrus tristeza virus by SSCP and CE-SSCP. In: Catara, A., Bar-Joseph, M., Licciardello, G. (eds) Citrus Tristeza Virus. Methods in Molecular Biology, vol 2015. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9558-5_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9558-5_7

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9557-8

  • Online ISBN: 978-1-4939-9558-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation