Research Article |
Corresponding author: Debora Giorgi ( debora.giorgi@enea.it ) Academic editor: Marcelo Guerra
© 2016 Debora Giorgi, Gianmarco Pandozy, Anna Farina, Valentina Grosso, Sergio Lucretti, Andrea Gennaro, Paola Crinò, Francesco Saccardo.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Giorgi D, Pandozy G, Farina A, Grosso V, Lucretti S, Gennaro A, Crinò P, Saccardo F (2016) First detailed karyo-morphological analysis and molecular cytological study of leafy cardoon and globe artichoke, two multi-use Asteraceae crops. Comparative Cytogenetics 10(3): 447-463. https://doi.org/10.3897/CompCytogen.v10i3.9469
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Traditionally globe artichoke and leafy cardoon have been cultivated for use as vegetables but these crops are now finding multiple new roles in applications ranging from paper production to cheese preparation and biofuel use, with interest in their functional food potential. So far, their chromosome complements have been poorly investigated and a well-defined karyotype was not available. In this paper, a detailed karyo-morphological analysis and molecular cytogenetic studies were conducted on globe artichoke (Cynara cardunculus var. scolymus Fiori, 1904) and leafy cardoon (C. cardunculus var. altilis De Candolle, 1838). Fluorescent
Cynara , SSR simple sequence repeats, repetitive sequences, flow cytometry, FISHIS, FISH
The globe artichoke (Cynara cardunculus var. scolymus (L.) Fiori, 1904) and the cultivated leafy cardoon (C. cardunculus var. altilis De Candolle, 1838) are dicotyledonous angiosperms belonging to the family Asteraceae and originate from the Mediterranean area (
In spite of the agronomic, nutritional and industrial importance of globe artichoke and leafy cardoon for the Mediterranean basin, their genetics and cytogenetics is relatively poorly characterized, as recently stated by
In addition to standard chromosome morphological analysis, cytogenetics can take advantage of a molecular approach based on fluorescence
Globe artichoke cultivar (cv) Istar and cardoon cv Bianco Avorio seeds were kindly provided by the Seed Company Topseed (Sarno, Salerno, Italy) while Pisum sativum (Linnaeus, 1573) cv Citrad seeds were generously provided by Dr. J. Doležel (Centre of Plant Structural and Functional Genomics, Institute of Experimental Botany, Olomouc, Ceck Republic). For both DNA content determination and cytogenetic analysis, Cynara spp. seeds were germinated in the dark on moist filter paper at 24±1 °C for 5-10 days, after a hot treatment at 50 °C for 10 min (for P. sativum, no hot treatment was performed). Actively growing roots and young leaves were excised for further treatment.
Nuclei were extracted from 50 mg of young leaves using two different protocols. The first was performed according to
Flow cytometric estimation of nuclear DNA content stained with PI (λ ext max: 293 nm and 514 nm; λ ems max 625 nm) was performed using a FACS Vantage SE flow cytometer (BD Bioscience, San Jose, CA) with a solid state laser (Genesis CX STM, Coherent, Santa Clara, CA), UV emission at 200 mW, and a 70 µm flow tip running at 27 psi with a solution of 50 mM NaCl as sheath fluid. P. sativumcv “Citrad” was used as internal standard (2C = 9.09 pg) (
unknown 2C DNA content = [(unknown G1 peak mean)/ (standard G1 peak mean)] × standard 2C DNA content.
2C DNA content (pg) was converted to base pairs (bp) following the factor: 1 pg DNA = 0.978 × 109bp (
Actively growing roots were excised and pre-treated in 2 mM 8-Hydroxyquinoline (8HQ) for 3-5 h at room temperature (r.t.) or in 30 µM Oryzalin for 20 h at 4 °C, and then fixed in Carnoy solution (ethanol : glacial acetic acid = 3:1) at -20 °C for at least 18 h. Chromosome spreads were prepared according to the “drop spreading method” developed by
In order to better discriminate each chromosome and pairing homologous chromosomes, single and double-target fluorescence
The 18S-5.8S-26S rDNA clone pTa71 (
Metaphases were denatured at r.t for 5 min in 70% ethanol (pH13 using 4 N NaOH). Preparations were dehydrated at r.t. through an ice-cold ethanol series (70%, 85% and 100%) for 2 min each, and air dried. The denatured probe was applied after chromosomes alkaline denaturation and plastic cover slips were placed over the specimens and the slides were incubated in a humid chamber at 37 °C for 16 h. After hybridization, the coverslips were removed and the preparations were subjected to a single stringency wash in 50% (v/v) formamide in 1 x SSC, followed by 2 additional washes in 2 x SSC, for 5 min at 45 °C each. Finally, samples were counterstained with DAPI and mounted in a Vectashield antifade solution (Vector Laboratories, Burlingame, CA, USA).
Before FISH analysis on metaphase spreads, a selection of SSR oligonucleotides was carried out on nuclei using FISHIS. Nuclei were isolated from roots following the same procedure previously described for fixed leaves. Hybridization was performed according to
A fast FISH method was developed and carried out on metaphase spreads of artichoke and cardoon using selected SSR oligonucleotides as probes. Chromosome DNA was denatured in an alkaline 70% ethanol solution, as previously described, and the preparations were dehydrated at r.t. through an ice-cold ethanol series (70%, 85% and 100%) for 2 min each, and air dried. A mix containing 1.5 - 3 ng µl-1 of labelled oligonucleotide in 2X SSC (300 mM sodium chloride, 0.3 mM trisodium citrate) was applied on the slide (final volume 60 µl per slide) and hybridization was carried on at r.t. for 1 h After washing in 4X SSC, 0.2% Tween20 for 10 min, samples were counterstained with DAPI and mounted in antifade solution.
Microscope slides with chromosomes or nuclei were examined with a Nikon Eclipse TE2000-S inverted microscope equipped with an HB0100 W lamp and a CFI Plan Apo oil objective 100X and appropriate filter sets for DAPI, FITC and Cy3 fluorescence. Separate images from each filter set were digitalized and analysed using a DXM1200F Nikon camera and the NIS AR 3.1 software (Nikon Instruments S.p.A, Florence, Italy), respectively. Image analysis and measurements were performed using ImageJ v1.46 (
After optimization of the isolation procedure, flow cytometry estimation of nuclear DNA content was performed analysing nuclei isolated from globe artichoke and cardoon using Pisum sativumcv. Citrad as internal standard, When using the most common isolation buffers and the classical method of chopping of fresh tissues (
Flow Cytometry DNA content histogram. Flow cytometry analysis of DNA fluorescence peaks (FL1A) from G0/G1 (2C) PI stained leaf nuclei from cultivated cardoon (M1), globe artichoke (M2) and P. sativum (pea) (M3). Pea was used as an internal standard and the peak was set at channel 400; M4 shows pea G2 nuclei (4C).
In order to perform a good morphological analysis, the quantity and quality of metaphase spreads, in terms of absence of cytoplasm and low percentages of overlapping chromosomes, is of critical relevance. Here a pre-treatment with oryzalin, as antimitotic agent, was tested for the first time in Cynara and compared to the 8-hydroxyquinoline used in previous studies; a further slight increase in methaphases number (about 5%) was observed.
The morphometric analysis of cardoon and globe artichoke chromosomes was carried out by measuring the total length (tl), the arm length and the arm ratio of all 34 individual chromosomes (Table
Morphometric analysis of C. cardunculus chromosomes. Morphological analysis of the 2n = 34 chromosomes of cultivated cardoon (A) and globe artichoke (B) based on Fig.
C.p. | T.l. (µm) | p (µm) | q (µm) | AR | Class | |||||
---|---|---|---|---|---|---|---|---|---|---|
A | B | A | B | A | B | A | B | A | B | |
1 | 3.16 | 3.19 | 1.23 | 1.27 | 1.93 | 1.92 | 1.57 | 1.51 | s | s |
2 | 2.97 | 3.16 | 1.07 | 1.24 | 1.78 | 1.93 | 1.78 | 1.55 | sm | sm |
3 | 2.58 | 2.81 | 1.05 | 1.21 | 1.55 | 1.60 | 1.46 | 1.32 | m | m |
4 | 2.35 | 2.65 | 0.97 | 1.16 | 1.38 | 1.49 | 1.42 | 1.28 | m | m |
5 | 2.35 | 2.45 | 0.78 | 0.95 | 1.57 | 1.50 | 2.01 | 1.57 | sm | sm |
6 | 2.12 | 2.27 | 0.85 | 0.95 | 1.27 | 1.32 | 1.49 | 1.39 | m | m |
7 | 2.10 | 2.25 | 0.87 | 0.93 | 1.23 | 1.32 | 1.41 | 1.42 | m | m |
8 | 2.03 | 2.00 | 0.79 | 0.78 | 1.24 | 1.22 | 1.56 | 1.56 | sm | sm |
9 | 2.00 | 1.98 | 0.92 | 0.92 | 1.08 | 1.06 | 1.17 | 1.15 | m | m |
10 | 2.00 | 1.98 | – | – | 2.00 | 1.98 | >3 | >3 | a | a |
11 | 1.95 | 1.95 | – | – | 1.95 | 1.95 | >3 | >3 | a | a |
12 | 1.79 | 1.95 | 0.81 | 0.71 | 0.98 | 1.24 | 1.20 | 1.74 | m | sm |
13 | 1.68 | 1.70 | 0.62 | 0.79 | 1.06 | 0.91 | 1.70 | 1.15 | sm | m |
14 | 1.40 | 1.41 | – | 0.65 | 1.40 | 0.76 | >3 | 1.17 | a | m |
15 | 1.30 | 1.38 | – | – | 1.30 | 1.38 | >3 | >3 | a | a |
16 | 1.25 | 1.27 | – | – | 1.25 | 1.27 | >3 | >3 | a | a |
17 | 1.22 | 1.22 | – | – | 1.22 | 1.22 | >3 | >3 | a | a |
The smallness and variable sizing of Cynara chromosomes is shown; in both panel A and B, leafy cardoon chromosome 1 has been circled as an example of the different condensation level of the same metaphase chromosomes. In A chromosome 1 is 3.2 µm, in B it is 4.3 µm (a 36% size variation). Scale bars: 5 µm.
FISH localization of rDNA was investigated using the pTa71 sequence as a probe. For both crops there were eight hybridization signals (Figures
FISH molecular cytogenetic analysis with rDNA. FISH on metaphase spreads of cultivated cardoon (A) and globe artichoke (B) using the rDNA probe pTa71-Cy3 (red fluorescence). In Fig.
Screening SSR by FISHIS analysis revealed that only the telomeric sequence (TTTAGGG)5 and the oligonucleotide (GAA)7 showed clear and discrete hybridization signals on nuclei of both crops (Figure
Fast screening of labelling probes was performed by FISHIS on cultivated cardoon (panel A and C) and globe artichoke (panel B and D) nuclei suspensions. The nuclei with the clearest and discrete telomeric (TTTAGGG)5 and SSR (GAA)7 signals are shown. All oligonucleotides were fluorescently labelled by Cy3 (red fluorescence). Nuclear DNA was counterstained with DAPI (blue fluorescence). Scale bar: 5 µm.
All four oligonucleotides (TTTAGGG)5, (GA)10, (CA)10, and (GAA)7 were used for fast standard FISH on chromosome spreads. As expected, (TTTAGGG)5 hybridized at the telomeres, facilitating identification of the ends of the chromosome and more accurate measurements (Figures
FISH molecular cytogenetic analysis with SSR probes. FISH on metaphase spreads of cardoon (left hand side) and of globe artichoke (right had side). The oligonucleotides sequence is indicated in each panel. Oligonucleotides were labelled with Cy3 (red fluorescence) or FITC (green fluorescence) fluorochromes. In (E) and (F) chromosomes 3, 5 and 8 of cardoon and globe artichoke, respectively, are indicated by arrows and can be discriminated by the widespread (GA)10 hybridization pattern on the long arms. Scale bars: 5 µm.
The (CA)10 and (GA)10 di-nucleotides showed very similar hybridization patterns on the chromosomes of the two crops (Figures
An example of globe artichoke pairing of homologous chromosomes based on (GA)10 labelling pattern. Scale bar: 5 µm.
A less clear hybridization pattern was obtained using the (GAA)7 probe which showed a variable and sometimes asymmetric distribution of the signals on sister chromatids, mainly on the large chromosome of globe artichoke and cardoon. For at least two large, one medium and one small chromosome pair a hybridization signal was visible in all the observed metaphases for both crops (Figures
Ideograms summarising chromosome morphology and molecular karyotyping with the (CA)10 /(GA)10 and pTa71 DNA probes of the two crops are shown in Figure
Ideogram with molecular characterization of cardoon and globe artichoke complement. Ideogram showing chromosome morphology (in black) and the (CA)10, (GA)10 di-nucleotides (red) and pTa71 sequence (green) distribution on cardoon (A) and globe artichoke (B) complement. The two di-nucleotides localize at similar chromosome regions. Scale bar: 5 µm.
In spite of the recent release of globe artichoke genome sequence (
The genomes size measured in this study for leafy cardoon (2C = 2.20 pg) and globe artichoke (2C = 2.40 pg) are slightly different to those previously reported by
For cytogenetic studies in globe artichoke we have previously tested several microtubule assembling inhibitors, that is, 8-hydroxyquinoline, amiprophos-methyl, colchicine and a-bromonaphtalene, to increase the number of metaphases. 8-hydroxyquinoline was identified as the most effective inhibitor but even so, the mitotic index of Cynara remained as low as 10% (
To enhance metaphase spread quality, a drop spreading method recommended for plants with small chromosomes was used (
Chromosome characterization by FISH labelling was preceded by FISHIS on nuclei suspensions. This procedure was initially developed to label chromosomes in suspension for flow karyotyping and sorting, as an effective method to discriminate, purify and isolate specific plant chromosomes (
The rDNA genes sites identified using traditional FISH analysis agrees, as number, with that reported in the recent work of
The publication of the globe artichoke genome sequence (
Here we propose the karyo-morphological and molecular karyotype and the first ideogram of both cultivated cardoon and globe artichoke. Our results enable the identification of chromosomes pairs 3, 5 and 8 and the discrimination of acrocentric chromosomes in the complement of the two crop. Their karyotype revealed close affinity, but also chromosome structural variation among the two C. cardunculus varieties. Differences have been detected in the number of acrocentric chromosome, with cardoon showing an additional chromosome pairs, and also in the DNA content of the two varieties. The proposed karyotypes could help future anchoring of pseudomolecules from globe artichoke genome sequencing to chromosomes and contribute in locating important genes involved in the divergent evolution and domestication of C. cardunculus, for example, those associated with the development of different leaf structure and flower architecture.
Andrea Gennaro is employed with the European Food Safety Authority (EFSA), the present paper is published under the sole responsibility of the authors. The positions and opinions presented in this paper are those of the author alone and are not intended to represent the views or scientific works of EFSA.
The Authors thank Dr. Tim Langdon (IBERS Aberystwyth University, Aberystwyth, UK) for his suggestions and Dr. Cushla Metcalfe (CSIRO Agriculture, Queensland Bioscience Precinct St Lucia, Brisbane, Australia, respectively) for proof reading the manuscript.
The authors acknowledge the ‘CAR-VARVI’ project, financially supported by the Italian Ministry of Agriculture, Food and Forestry Policies.