Genetic Resources and Crop Evolution 49: 247–252, 2002. 247 2002 Kluwer Academic Publishers. Printed in the Netherlands.

Genetic variation in wild and cultivated artichoke revealed by RAPDmarkers

1, 2 2 1*G. Sonnante , A. De Paolis , V. Lattanzio and P. Perrino1National Research Council (CNR), Istituto del Germoplasma, Via Amendola, 165/A, 70126 Bari, Italy;2 *CNR Istituto di Orticoltura e Colture Industriali, Via S. Loja, 85050 Tito, Pz, Italy; Author forcorrespondence

Received 16 August 2001; accepted in revised form 5 June 2001

Key words: Cynara cardunculus, Evolution, Genetic relationships, Genetic resources, RAPDs

Abstract

Determination of intraspecific genetic diversity is an important first step for the utilization of genetic resources andcan provide useful information on crop evolution. A living collection of Italian and foreign artichoke varieties andecotypes is maintained at the Germplasm Institute, Bari, Italy. A total of 32 accessions of cultivated (Cynaracardunculus L. var. scolymus (L.) Fiori), three of wild (Cynara cardunculus var. cardunculus L.) artichoke andtwo of cultivated cardoon (Cynara cardunculus var. altilis L.) were analysed in order to study genetic variationand relationships within the species, using RAPD markers. Cultivated accessions were selected according tomorphological variation and geographical distribution available in the collection. Fifty arbitrary decamer primerswere initially tested, 18 of which showed from 3 to 10 unambiguously interpretable fragments. An intra-accessionanalysis using 4 varieties and 8 polymorphic primers revealed that no RAPD variation was detected amongindividuals. Jaccard’s similarity index (JSI) was comprised among 1 and 0.693 when all accessions wereconsidered, on the other hand, within the cultivated artichoke, JSI ranged between 1 and 0.817. A UPGMAdendrogram based on the similarity matrix showed that wild artichokes were clearly separated from cultivatedaccessions. Moreover, within cultivated artichokes, several groups could be distinguished.

Introduction ribosomal genes of the same length (Tucci and Mag-gini 1986; Maggini et al. 1988).

Globe artichoke (Cynara cardunculus L. var. Artichoke is a diploid (2n 5 2x-34) outcrossingscolymus (L.) Fiori) is a relatively recent crop (about species, whose propagation is made mainly vegeta-1st century) traditionally cultivated in the Mediterra- tively. A high number of cultivars or ecotypes isnean Basin (Foury 1989). The same species includes present especially in Italy and other Mediterraneantwo more botanical varieties: C. cardunculus var. countries. However, sometimes the same variety takesaltilis, the cultivated leafy cardoon and C. cardun- a different name according to the place where it isculus var. sylvestris, the wild progenitor of artichoke cultivated, and this often engenders confusion. In(Rottenberg and Zohary 1996), which are completely order to allow a better varietal classification for theinterfertile with the cultivated globe artichoke (Bas- artichoke world germplasm collection held in Bari,nizki and Zohary 1994). Isozyme markers confirmed Italy, Dellacecca et al. (1976) prepared a list ofC. cardunculus var. sylvestris as the progenitor of descriptors based on 20 morpho-productive traits andcultivated artichoke since the similarity between these Porceddu et al. (1976) analysed 27 traits in 78 artich-two taxa is much higher than that between other wild oke varieties by means of multivariate analysis. AfterCynara species and artichoke (Rottenberg et al. a preliminary evaluation of 104 artichoke accessions1996). Artichoke and its wild progenitor also possess belonging to the Germplasm Institute collection,

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based on eight quantitative characters (Miccolis et al. reading at the spectrophotometer at 260 nm and then1989), Elia and Miccolis (1996) used cluster analysis confirmed on agarose gel.to measure relationships among these accessions.Hammouda et al. (1993) analysed only an Egyptian RAPD analysisvariety and the Romanian strain (cultivated in Egypt)for variation in flavonoid and polyphenol content. Amplification reactions were prepared in a total of 18

RAPD markers have demonstrated to be effective mL volume containing 50 mM KCl, 10 mM Tris-HClin studies on genetic variation, for identifying geno- pH 9.0, 1.5 mM MgCl , 0.1 mM each of the nucleo-2

types, for population analysis and phylogenetic tides (dATP, dCTP, dGTP, dTTP), 0.2 mM primer, 0.8studies in several plant species (Francisco-Ortega et unit of Taq polymerase and 20 ng DNA. Amplifica-al. 1993; Sonnante et al. 1997; Yang and Quiros tion was performed using the following programme:1993). Tivang et al. (1996) have evaluated the level of one cycle at 94 8C for 29 followed by 35 cycles atvariation in two breeding populations and three cul- 94 8C for 450, 37 8C for 450, 72 8C for 900 and a cycletivars of artichoke, one of which vegetatively prop- at 728 for 59.agated, using RAPD markers. The products obtained were analysed on a 1.5%

The objectives of the present paper were to ascer- agarose gel with 1 3 TAE buffer. The gel was stainedtain if RAPD markers could be used to determine with ethidium bromide and photographed under UVgenetic diversity in an artichoke collection and to light.assess the genetic relationships among wild, culti-vated artichoke and cardoon. Data analysis

The electrophoretic patterns were visually analysedMaterial and methods and DNA bands were scored as present (1) or absent

(0). The matrix obtained was entered into thePlant material NTSYS-pc programme (Rohlf 1993) and Jaccard’s

similarity index (JSI) was calculated for each pair ofThirty-two cultivated artichoke accessions and one samples. A UPGMA dendrogram was constructedcardoon (accession No. 37) were obtained from the based on JSI.living collection of the Germplasm Institute (CNR),Bari, Italy. Wild artichokes (33) and (34) and onecultivated cardoon (36) were kindly provided by Prof. Results and discussionMauromicale, Catania, Italy. The other wild accession(35) was collected by Dr P. Medagli, Lecce, Italy. All A total of 50 decamer primers were initially tested,the material analysed is listed in Table 1. some were eliminated because they did not show any

Portions of young leaves were collected, frozen in amplification, others because the banding pattern wasliquid N and stored at 280 8C. not interpretable. Eventually, 18 primers were chosen2

(OPO04, OPO07, OPO17, OPP02, OPP03, OPP04,DNA extraction OPP11, OPP12, OPQ6, OPQ12, OPQ18, OPV10,

OPV16 and OPV17 from Operon Technologies, Inc.;DNA was extracted according to Paz and Veilleux UBP6, UBP7, UBP10, UBP24 from Figliuolo and(1997) with some modification. An amount of about Spagnoletti Zeuli (1994)) for the screening of all0.3 g was ground in liquid N and 1 mL extraction samples. In Figure 1 an example of some electro-2

buffer (0.1 M Tris-HCl pH 8, 1.4 M NaCl, 0.02 M phoretic profiles is shown, with primer OPO17. TheEDTA pH 8, 2% CTAB) was added prior to incuba- analysis of amplification patterns has revealed from 3tion at 60 8C for 1 h. to 10 DNA fragments for each primer. Out of a total

After a chloroform-isoamyl step, DNA was precipi- of 113 bands unambiguously scored, 69 (61%) weretated with cold isopropanol and resuspended in TE. polymorphic.Samples were digested with RNAse and extracted Four varieties: ‘Violetto di Toscana’ (8), ‘Spinosoonce with phenol-chloroform and once with chloro- Sardo’ (11), ‘Romanesco’ (19) and ‘Green Globe’form/ isoamyl. DNA was precipitated with EtOH and (32) were evaluated for intravarietal variation byresuspended in TE. Concentration was calculated by analysing five plants for each clone with eight poly-

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Table 1. List of the material analysed.*VARIETY ORIGIN GROUP

1 Catanese I-Catania CAT-A2 Locale di Mola I-Bari CAT-A3 Di Ogni Mese I-Pisa CAT-E4 Masedu I-Sassari CAT-B5 Precoce Violetto di Chioggia I-Padova VIO-D6 Locale di Cuneo I-Cuneo OUT-C7 Moretto I-La Spezia VIO-D8 Violetto di Toscana I-Pistoia VIO-D9 Testa di Ferro I-Pistoia OUT10 Verde di Pesaro I-Pesaro —11 Spinoso Sardo I-Sassari SPI-C12 Spinoso di Sciacca I-Agrigento SPI-C13 Spinoso di Palermo I-Messina SPI-C14 Spinoso 2 I-Cagliari —15 Sakiz Turkey —E16 Bayrampasa Turkey OUT-E17 Mazzaferrata I-Pescara ROM-E18 Castellammare I-Roma ROM-E19 Romanesco I-Latina ROM-E20 Campagnano I-Napoli ROM-E21 Nostrano Tipo Nero I-Arezzo OUT-E22 Precoce di Jesi I-Ancona OUT-E23 Violetto di Putignano I-Bari —D24 Verde di Putignano I-Bari —D25 Bianco Tarantino I-Taranto OUT-C26 Macau I-Sassari ROM-E27 Tudela Spain OUT-E28 Violetto di Provenza I-Sassari CAT-A29 Banafsigi Egypt CAT-B30 Baladi Egypt CAT-B

˜31 Nato Argentina OUT-E32 Gren Globe Thornless California (USA) —E33 C. cardunculus var. sylvestris I-Enna —34 C. cardunculus var. sylvestris I-Messina —35 C. cardunculus var. sylvestris I-Porto Cesareo —36 C. cardunculus var. altilis Bianco avorio I-Catania —37 C. cardunculus var. altilis I-Bari —* GROUP: for each accession, the first abbreviation refers to the belonging to groups as in Porceddu etal. (1976), and precisely, CAT 5 Catanesi, VIO 5Violetti, SPI 5 Spinosi, ROM 5 Romaneschi, OUT5 not belonging to any group. The second abbreviation refers to the groupings as in Elia and Miccolis(1996); I 5 Italy.

morphic primers. No variation was observed within propagated for about 25 years, and this may be theeach clone (data not shown). Tivang et al. (1996) reason why no variation was detected.found RAPD heterogeneity in two breeding popula- Dellacecca et al. (1976), based on morpho-pro-tions and three varieties, two of which were seed ductive traits, found four main artichoke groups. Thepropagated and the other, ‘Green Globe’, was vegeta- ‘Spinosi’ group includes varieties with long thornstively propagated. The variability observed in ‘Green on bracts and leaves; the ‘Violetti’ group comprisesGlobe’ was attributed to heterogeneity among clones, types with violet-coloured heads harvested in thewhich is consistent with its multiclonal composition early springtime; the ‘Romaneschi’ group, including(De Vos 1986). In our case, the ‘Green Globe’ ana- the big, spherical headed varieties harvested late inlysed was obtained from the living collection of the the spring; the ‘Catanesi’ group, characterised byGermplasm Institute, where the same clone has been varieties with elongated heads harvested for a longer

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Figure 1. RAPD amplification products obtained with primer OPO17. From left to right: ‘Di Ogni Mese’ (3), ‘Spinoso di Sciacca’ (12),‘Spinoso di Palermo’ (13), ‘Spinoso 2’ (14), ‘Romanesco’ (19), ‘Campagnano’ (20), ‘Verde di Putignano’ (24), ‘Banafsigi’ (29), ‘Baladi’(30), Wild artichoke (33), Wild artichoke (34), Wild artichoke (35), Cardoon (37), ‘Violetto di Toscana’ (8), ‘Spinoso Sardo’ (11), ‘GreenGlobe’ (32)

period, from the late autumn to the spring. A certain (19), with which JSI is 0.863 and 0.872; however,number of accessions was not included in any of the ‘Romanesco’ often shows similarity values similarfour groups, since they showed intermediate or and sometimes higher with other artichoke acces-different characters. Elia and Miccolis (1996) ana- sions, especially with the variety ‘Di Ogni Mese’lysed 104 artichoke accessions belonging to the (3), JSI 5 0.906.Germplasm Institute collection on the basis of eight The three accessions belonging to the ‘Violetti’quantitative characters. Cluster analysis identified types, ‘Precoce Violetto di Chioggia’ (5), ‘Moretto’five large groups of accessions: ‘Early’ (group A), (7), ‘Violetto di Toscana’ (8) form a separate group‘Medium-early’ (B), ‘Late with small head’ (C), including, more distantly apart, ‘Spinoso 2’ (14),‘Late-violet’ (D) and ‘Late with big head’ (E). Most which is a type with small and spiny heads and veryof the plant material analysed in this study was thorny leaves.included in the two papers mentioned above, as Another group includes the ‘Spinosi’ types (11,indicated in Table 1. 12, 13) together with a Spanish, Tudela (27) and two

The data obtained from RAPD analysis provide Turkish accessions analysed: ‘Sakiz’ (15) andJaccard’s similarity index (JSI) comprised between 1 ‘Bayrampasa’ (16). Going up in the dendrogram, a(for the two thorny types, 12 and 13) and 0.693 big cluster is subdivided into two groups. The

˜(between 34 and 20 and between 33 and 17) when smaller one includes ‘Nato’ (31), ‘Violetto di Putig-all the samples are considered. When only the nano’ (23), ‘Verde di Putignano’ (24) and ‘Precocecultivated types are examined, as expected, the di Jesi’ (22), which belong to the group C or D inminimum value of similarity is higher (JSI 5 0.817 Elia and Miccolis (1996). The bigger group com-between 19 and 25). prises a set of accessions possessing a ‘Romanesco’-

The UPGMA dendrogram (Figure 2) shows two type head (17, 18, 20, 32), plus ‘Locale di Cuneo’main clusters, one including all the cultivated arti- (6), and another cluster including ‘Catanese’-typechokeandcardoonaccessions, theotheronecomprising artichokes (2, 4, 9, 28, 1, 29, 30), plus ‘Di Ognithe wild artichokes (33, 34, 35). The two clusters Mese’ (3) and ‘Macau’ (26).split at a JSI of 0.740. The tree node at JSI of 0.865 Summing up, from the bottom to the top, theseparates, from the other accessions, the two culti- dendrogram contains, with some exceptions, the wildvated cardoons which are grouped with ‘Romanesco’ artichokes, the ‘Violetti’ types, the cultivated car-

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genetically closer to each other than to the wild type.Even though thornless varieties are considered moreevolved than spiny ones, these have been preferredin some areas because they are considered moretasty.

For horticultural crops, domestication took placelater than for seed crops and artificial selection byman has represented the main force, which hasalmost completely suppressed natural environmentalselection and natural selection by competition(Hanelt 1986).

Conclusion

RAPDs revealed to be useful markers for the studyof genetic diversity in artichoke. Even though wildand cultivated artichoke and cultivated cardoon arecompletely interfertile and belong to the samespecies, the wild material was clearly separated fromthe cultivated one, in fact some bands were typicalonly of the wild accessions. On the other hand,cardoon was genetically more similar to the culti-Figure 2. UPGMA dendrogram based on Jaccard’s similarity

index. vated artichoke. Except for two spiny types (12 and13) which showed identical banding profiles, all the

doon, the ‘Spinosi’, the ‘Romaneschi’ and the other cultivated artichoke accessions showed a JSI‘Catanesi’ types. The probable artichoke phylogeny lower than one and therefore could be distinguishedincludes the wild progenitor C. cardunculus var. from one another. With some exceptions, it wassylvestris as a starting point, followed by the culti- possible to separate some groups in the dendrogram,vated spiny types (Barbieri 1959), then the violet corresponding to the main artichoke groups previous-types which possess a less spiny head, up to the most ly detected on the basis of morpho-productiveevolved thornless types: ‘Romaneschi’ and ‘Catan- characters. The clusters, therefore, reflected more theesi’, which include the varieties cultivated on a large morphological characteristics than the geographicalscale (Bianco 1990). origin of the accessions.

Cultivated cardoon is grown on a minor scale forits succulent young leaves (Dellacecca 1990). It iscompletely interfertile and there is a morphological Acknowledgementscontinuous variation between the cultivated artichokeand cardoon. In fact, sometimes also artichoke We wish to thank Prof. G. Mauromicale and Dr P.leaves, like cardoon ones, are used for human Medagli for providing us the wild material, Dr D.consumption and in Southern Italy even wild artich- Pignone for useful discussion, M. S. Pace for techni-oke young leaves and stalks, as well as capitula are cal assistance for the maintenance of the artichokecooked and eaten (Caneva et al. 1997). The culti- living collection, and F. Losavio for graphic assis-vated forms represent, therefore, the result of two tance. This work was partially supported by fundsdifferent selection pressures operated by man on the from kP.O. CNR/MURST ‘Ricerca, Sviluppo Tec-same crop species (Basnizki and Zohary 1994). nologico ed Alta Formazione’ 1994–1999—ProgettoDuring the selection process, man preferred non ‘Interventi per la salvaguardia e la valorizzazione

`spiny heads and leaves, but probably, after some della biodiversita di specie vegetali sottoutilizzate etime, the two kinds of selections, for capitula from di razze autoctone di animali domestici a limitataone side and fleshy leaves from the other, diverged, diffusione’l. Responsible for the project: Ersilioleading to the two cultivated forms which are Desiderio.

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