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Molecular Genetics and Genomics

, Volume 293, Issue 2, pp 417–433 | Cite as

Haplotype analysis of the germacrene A synthase gene and association with cynaropicrin content and biological activities in Cynara cardunculus

  • Ana Margarida Ferro
  • Patrícia Ramos
  • Ângela Guerra
  • Paula Parreira
  • Teresa Brás
  • Olinda Guerreiro
  • Eliana Jerónimo
  • Carmen Capel
  • Juan Capel
  • Fernando J. Yuste-Lisbona
  • Maria F. Duarte
  • Rafael Lozano
  • M. Margarida Oliveira
  • Sónia Gonçalves
Original Article

Abstract

Cynara cardunculus: L. represents a natural source of terpenic compounds, with the predominant molecule being cynaropicrin. Cynaropicrin is gaining interest since it has been correlated to anti-hyperlipidaemia, antispasmodic and cytotoxicity activity against leukocyte cancer cells. The objective of this work was to screen a collection of C. cardunculus, from different origins, for new allelic variants in germacrene A synthase (GAS) gene involved in the cynaropicrin biosynthesis and correlate them with improved cynaropicrin content and biological activities. Using high-resolution melting, nine haplotypes were identified. The putative impact of the identified allelic variants in GAS protein was evaluated by bioinformatic tools and polymorphisms that putatively lead to protein conformational changes were described. Additionally, cynaropicrin and main pentacyclic triterpenes contents, and antithrombin, antimicrobial and antiproliferative activities were also determined in C. cardunculus leaf lipophilic-derived extracts. In this work we identified allelic variants with putative impact on GAS protein, which are significantly associated with cynaropicrin content and antiproliferative activity. The results obtained suggest that the identified polymorphisms should be explored as putative genetic markers correlated with biological properties in Cynara cardunculus.

Keywords

Cynara cardunculus Germacrene A synthase Single nucleotide polymorphisms Cynaropicrin Biological activities Association analysis 

Abbreviations

a.a

Aminoacid

CDS

Coding sequence

Cc

Cynara cardunculus L.

DMSO

Dimethyl sulfoxide

EI-MS

Electron impact mass spectra

GC–MS

Gas chromatography–mass spectrometry

GAS

Germacrene A synthase

HPLC–UV

High-Performance Liquid Chromatography–Ultraviolet-Visible analysis

HRM

High-resolution melting

MRSA

Methicillin-Resistant Staphylococcus aureus

MBC

Minimum bactericidal concentration

MIC

Minimal inhibitory concentration

MAF

Minor allele frequency

MH

Mueller Hinton

PTM

Post-translational modification

PROC GLM

Procedure general linear model

SNP

Single-nucleotide polymorphism

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Notes

Acknowledgements

Plant seeds were kindly provided by Botanical Garden of University of Tuscia (Italy), Botanical Garden of University of Agronomic Sciences of Gembloux (Belgium), Botanical Garden of University of Szeged (Hungary), Botanical Garden of University of Napoli (Italy), Experimental Centre of School of Agriculture of Beja (Portugal) and Botanical Garden of Sapienza University of Rome (Italy). We are also grateful to the Horticultural Centre of School of Agriculture (ESA) of Beja, for space to grow Cc plants. Pseudomonas aeruginosa POAI was kindly donated by Professor Arsénio Fialho from Instituto Superior Técnico; Methicillin-resistant Staphylococcus aureus (clinical isolate from wound exudate) and Bacillus cereus were both kindly donated by Professor Manuela Pintado from Universidade Católica Portuguesa. We also thank Conceição Fernandes for HPLC support, Inês Pires for TASSEL software support and Anabel Usié Chimenos and Marcos Ramos for bioinformatic support. Fundação para a Ciência e a Tecnologia (FCT) is gratefully acknowledged for the PhD fellowships, refs. SFRH/BD/68847/2010; SFRH/BD/70845/2010, SFRH/BD/84406/2012, SFRH/BD/98635/2013 and SFRH/BD/110969/2015 awarded to AF, PR, OG, AG and TB, respectively. SG was supported by Programa Ciência 2008, also funded by POPH (QREN). This project was funded by European Regional Development Fund (FEDER), ValBioTecCynara (ALT20-03-0145-FEDER-000038)—Economic valorization of Cardoon (Cynara cardunculus): study of natural variability and biotechnological applications. Thanks are also due to GREEN-it “Bioresources for sustainability” (UID/Multi/04551/2013). The authors also thank Junta de Andalucía (grant P12-AGR-1482) and Campus de Excelencia Internacional Agroalimentario (CeiA3) for supporting this research collaboration.

Author contribution

AMF performed the experimental work and carried out the bioinformatic data analysis. PR contributed for GC-MS and GC-FID planning. AG contributed for antithrombin and antiproliferative activities and PP contributed for the antimicrobial activity. TB helped in the preparation of leaf lipophilic-derived extracts and cynaropicrin quantification by HPLC. OG and EJ contributed for statistical analysis. CC, JC and FJY-L contributed for HRM planning and execution. MFD, MMO, SG and RL contributed for the research planning, funding, discussions and paper writing. All authors read and approved the final manuscript

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Human and animal studies

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

438_2017_1388_MOESM1_ESM.pdf (58 kb)
Cynara cardunculus accessions used in this study (PDF 57 KB)
438_2017_1388_MOESM2_ESM.pdf (99 kb)
GAS gene structure and primer pairs (triangle arrows) designed for HRM analysis. (*)SNPs position; (#) a.a. alteration. The introns are indicated in gray (PDF 98 KB)
438_2017_1388_MOESM3_ESM.pdf (87 kb)
Primer pairs used in HRM analysis (PDF 86 KB)
438_2017_1388_MOESM4_ESM.pdf (113 kb)
Haplotypes identified for GAS gene. Individuals A1, A2, A4, B3, C1, D2, G3, J3, K3, U1, Y1, AB1 and AC2 were selected for phenotypic trait analysis and are indicated in gray. Some of these individuals present two haplotypes for GAS gene and are denominated X_1 and X_2. For instance, the denomination A2_1 and A2_2 correspond to the two haplotypes of the same individual A2, which was selected for phenotypic analysis (results presented in table 3) (PDF 113 KB)
438_2017_1388_MOESM5_ESM.pdf (53 kb)
SNPs identified on GAS gene (PDF 53 KB)

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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Ana Margarida Ferro
    • 1
    • 2
  • Patrícia Ramos
    • 1
    • 3
  • Ângela Guerra
    • 1
    • 3
  • Paula Parreira
    • 1
  • Teresa Brás
    • 1
    • 4
  • Olinda Guerreiro
    • 1
    • 5
  • Eliana Jerónimo
    • 1
  • Carmen Capel
    • 6
  • Juan Capel
    • 6
  • Fernando J. Yuste-Lisbona
    • 6
  • Maria F. Duarte
    • 1
  • Rafael Lozano
    • 6
  • M. Margarida Oliveira
    • 2
  • Sónia Gonçalves
    • 1
    • 7
  1. 1.Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo (CEBAL)Instituto Politécnico de Beja (IPBeja)BejaPortugal
  2. 2.Instituto de Tecnologia Química e Biológica António Xavier (ITQB)Universidade Nova de LisboaOeirasPortugal
  3. 3.CICECO, Departamento de QuímicaUniversidade de AveiroAveiroPortugal
  4. 4.LAQV, REQUIMTE, FCTUniversidade Nova de LisboaCaparicaPortugal
  5. 5.Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, ULisboaAvenida da Universidade TécnicaLisboaPortugal
  6. 6.Centro de Investigación en Biotecnología Agroalimentaria (BITAL)Universidad de AlmeríaAlmeriaSpain
  7. 7.Wellcome Trust Sanger InstituteCambridgeUK

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