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RAPD ANALYSIS OF SHRUBSBELONGING TO THE GENUSCYTISUS AND ALLIES(GENISTEAE: FABACEAE),AS AN AID FOR TAXONOMICDISCRIMINATIONFernando González-Andrés a & Jesús-María Ortiz aa Departamento de Biología Vegetal, Escuela TécnicaSuperior de Ingenieros Agrónomos , UniversidadPolitécnica de Madrid , 28040 , Madrid , SpainPublished online: 19 Apr 2013.

To cite this article: Fernando González-Andrés & Jesús-María Ortiz (1995) RAPDANALYSIS OF SHRUBS BELONGING TO THE GENUS CYTISUS AND ALLIES (GENISTEAE:FABACEAE), AS AN AID FOR TAXONOMIC DISCRIMINATION, Israel Journal of PlantSciences, 43:4, 303-314, DOI: 10.1080/07929978.1995.10676617

To link to this article: http://dx.doi.org/10.1080/07929978.1995.10676617

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ISRAEL JOURNAL OF PLANT SCIENCES, Vol. 43, 1995, pp. 303-314

RAPD ANALYSIS OF SHRUBS BELONGING TO THE GENUS CYTISUS AND ALLIES (GENISTEAE: FABACEAE), AS AN AID FOR

TAXONOMIC DISCRIMINATION

FERNANDO GoNzALEz-ANDREs* AND JEsOs-MARiA ORTIZ Departamento de Biologfa Vegetal, Escuela Tecnica Superior de Ingenieros

Agr6nomos, Universidad Politecnica de Madrid, 28040 Madrid, Spain

ABSTRACT Twenty-five accessions belonging to the genus Cytisus and allied taxa (Gen­isteae: Fabaceae) were analyzed using RAPD (Random Amplified Polymorphic DNA) techniques in order to study the usefulness of this technique for characterization of species and genera and for estimation of its potential value for further populational studies and prebreeding programs. A high level of intrapopulational variability was detected. Nevertheless, when only common bands to all the studied individuals per accession were consid­ered for numerical analysis, the technique was useful for characterization at the specific and generic levels. The dendrogram obtained supports the generic arrangement proposed by Bisby (1981), helping to elucidate the difficult taxonomy ofthe studied taxa. The high intrapopulational variability indicates that RAPD could be useful for populational studies and also as molecular markers.

INTRODUCTION

Forest fires and "set-aside lands programs" are major problems in arid and semiarid Mediterranean lands (Le Houerou, 1993). The restoration ofthese ecosystems should be based on the establishment of a plant layer, preferably consisting of autochthonous species. Several authors, Le Houerou (1978), Correal (1988), and Sankary and Ranjhan (1989), have indicated that shrubs are essential in the restoration process. However, only a few shrubby species have been thoroughly studied. A study of restoration species should comprise: (i) a characterization survey (including taxonomic data and genetic markers) and (ii) evaluation of agronomic traits (Brown, 1989). The present work is part of a broader characterization study on shrubby legume species. The selected species belong to the tribe Genisteae, and are potentially useful for the previously mentioned aims.

Historically, the taxonomy of the tribe Genisteae (Adans.) Benth. has generated a great controversy. Bisby (1981) proposed the scheme presented in Table I, which is based on the morphological pattern agreed upon at the Genisteae Working-Party in Southampton, July 1977. However, other authors prefer a different generic arrangement.

*Author to whom correspondence should be addressed.

Received 30 July 1995 and in revised form 17 September 1995.

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304 F. GONZALEZ-ANDRES AND J.-M. ORTIZ Isr. J. Plant Sci.

Polhill (1976), on the basis of morphology, and Cristofolini and Chiapella (1977, 1984), on the basis of serology, consider that Chamaecytisus Link does not deserve the generic status. These authors consider it a section (Tubocytisus DC) of the genus Cytisus. The anatomical work of N orverto et al. ( 1994) supported the classification of Chamaecytisus as an independent genus. Teline Medic. has been another debatable taxon. Bentham (1865) classified it as a subgenus of Cytisus, based on the presence of a seed aril in both taxa. However, this character has been reported as artificial by Polhill (1976). Gibbs and Dingwall (1971) and Flora Europaea (Heywood and Ball, 1968) classified Teline as an independent genus, due to certain morphological differences. On the other hand, Polhill (1976), based on morphology as well, considered Teline as a section of Genista. This has been supported by the serosystematical studies of Cristofolini and Chiapella ( 1977,

Table 1 Genera of the Genisteae according to Bisby (1981). Studied genera are represented in bold

characters

Subtribe

Lupininae

Genistinae

Group

"Cytisus group"

"Genista group"

"Outliers"

Genus

Lupin us

Argyrolobium Adenocarpus

Laburnum Hesperolaburnum Podocytisus Cytisophyllum Petteria Argyrocytisus Chamaecytisus Cytisus

Retama Genista

Echinospartium Stauracanthus Ulex

Calicotome Erinacea Spartium Gonocytisus

Section

Nubigena Corothamnus Spartopsis Chronanthus Lembotropis

Teline Pterospartum

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Vol. 43, 1995 RAPD ANALYSIS IN GENISTEAE 305

1984) and accepted by Bisby (1981). Cytisus sessilifolius L., the only species included in Cytisus L. sect. Cytisus by Polhill (1976), is an "unstable" taxon as well. Studies by Bisby and Nicholls (1977), Cristofolini and Chiapella (1977), and Greinwald et al. (1991) confirmed the earlier suggestion by Frodin (1965) that Cytisus sessilifolius L. should be transferred to a monotypic genus named Cytisophyllum Lang.

RAPD (Random Amplified Polymorphic DNA) has been used in DNA mapping programs as molecular markers linked to particular agronomical traits, and is also a useful tool for cultivar identification or characterization. This method has also been applied to populational studies: e.g., Francisco-Ortega et al. (1993) in the fodder tree Chamaecytisus proliferus (Fabaceae) and Heun et al. (1994) in wild accessions of Avena sterilis (Poaceae), etc. RAPD has been useful for characterization at the specific level in the genera Microseris (Van Heusden and Bachmann, 1992 a,b,c ), Clusia (Bohm et al., 1993), and Glycine (Williams et al., 1993). Demeke et al. (1992), working with the tribe Brassiceae, even pointed out that RAPD could also be adequate to distinguish between closely related genera.

The aim of this work was to study the usefulness of RAPD for the characterization, identification, and delimitation of wild Genisteae species at the specific and generic levels. A preliminary study of the infrapopulational variability detected was also carried out in order to study its potential value for further populational studies and pre breeding programs.

MATERIAL AND METHODS

PLANT MATERIAL

Plant material was obtained from seeds maintained at the germplasm bank of the Departamento de Biologfa Vegetal, Escuela Tecnica Superior de Ingenieros Agr6nomos, Universidad Politecnica de Madrid. All the selected taxa belong to the genus Cytisus and allies (Genisteae) and are currently being cultivated in an open air experimental plot. Table 21ists the taxa under investigation as well as their origin. Table 1 shows the generic grouping established by Bisby (1981), which has been adopted in this work. Species names are according to the Flora Europaea (Heywood and Ball, 1968). Seeds were germinated as described by Gonzalez-Andres et al. (1993), the seedlings grown in a greenhouse for three months and planted in the experimental plot with a randomized complete block design, to be used for several characterization and evaluation studies.

DNA EXTRACTION

Plant material for DNA extraction consisted of the central leaflet of a young healthy leaf, collected in the experimental field at the beginning of the second growing season. Three individuals per accession were sampled. The leaflet was thoroughly washed with running water and, finally, with distilled water. DNA extraction was performed as described by Edwards et al. (1991), modified as follows: Plant material was placed in an Eppendorf tube and macerated, using a manual grinder, with 400 111 of the extraction

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Table 2 Plant material

Ace. OTU# Species Origin No.

8242 OJ Cytisophyllum sessilifolium (L.) Lang Madrid B.G. (Spain) 4226 02 Cytisus baeticus (Webb) Steudel Monchique (Portugal) 8251 03 Cytisus grandiflorus DC. Trevelez (Granada; Spain) 8232 04 Cytisus heterochrous Webb ex Colmeiro Vistabella (Castellon; Spain) 4236 05 Cytisus reverchonii (Degen & Hervier) Bean s• Cazorla (Jaen; Spain) 8219 06 Cytisus reverchonii (Degen & Hervier) Bean Sagra Pass (Granada; Spain) 8220 07 Cytisus reverchonii (Degen & Hervier) Bean Santiago Espada (Jaen; Spain) 8222 08 Cytisus scoparius (L.) Link La Barranca (Madrid; Spain) 8230 09 Cytisus scoparius (L.) Link Pto. Morcuera (Madrid; Spain) 2555 10 Cytisus striatus (Hill) Rothm. Villagarcfa (Pontevedra; Spain) 8221 11 Cytisus villosus Pourret Corse (France) 8224 12 Chamaecytisus glaber (L.fil.) Rothm. Vacnitot B.G. (Hungary) 8237 13 Chamaecytisus glaber (L.fil.) Rothm. Madrid B.G. (Spain) 8234 14 Chamaecytisus hirsutus (L.) Link Madrid B.G. (Spain) 8223 15 Chamaecytisus podolicus (Blocki) A. Klaskova Vacratot B.G. (Hungary) 8226 16 Chamaecytisus ruthenicus (F.ex Wol.) Klaskova Vacratot B.G. (Hungary) 8236 17 Chamaecytisus ruthenicus (F.ex Wol.) Klaskova Madrid B.G. (Spain) 8225 18 Chamaecytisus supinus (L.) Link Vacratot B.G. (Hungary) 8240 19 Genista canariensis L. Madrid B.G. (Spain) 8228 20 Genista linifolia L. La Almoraima (Cadiz; Spain) 8109 21 Genista monspessulana (L.) L.A.S. Johnson Malaga (Spain) 8239 22 Genista monspessulana (L.) L.A.S. Johnson Madrid B.G. (Spain) 8215 23 Genista monspessulana (L.) L.A.S. Johnson La Almoraima (Cadiz; Spain) 8216 24 Genista monspessulana (L.) L.A.S. Johnson Jimena-Ubrique(Cadiz;Spain) 5476 25 Genista tenera (Jacq. ex Murr.) 0. Kuntze Madeira (Portugal)

Ace. No.: Accession number at the Germplasm Bank in the Departemento de Biologia Vegetal. Universidad Politecnica de Madrid. B.G.: Botanical Garden.

buffer (200 mM Tris HCI pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5% sodium dodecyl sulfate, 1% 28-mercaptoethanol), then vortexed for 5 s. The .::xtracts were centrifuged at 13,000 rpm for 5 min at room temperature, and 300 J.Ll of the supernatant transferred onto a fresh sterile Eppendorf tube. Isopropanol, 300 J.Ll, was added to the supernatant and gently mixed to help pellet formation. The supernatant was discarded and the pellet washed with a 76% v/v ethanol/ 10 mM ammonium acetate solution, up to four times. The washing duration was: 5 min-60 min-60 min-5 min. Finally the pellet was dissolved in Tris-EDTA (1:10). The desired final concentration was 7-10 ng DNNJ.Ll and 100-300 J.Ll of Tris-EDT A was needed. DNA concentration was measured with a TKO 100 (Hoefer) fluorimeter.

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Vol. 43, 1995 RAPD ANALYSIS IN GENISTEAE 307

PCR REACTION AND ELECTROPHORESIS

A 25 jll standard PCR reaction was carried out in a Perkin Elmer Cetus 480 Thermal Cycler. The PCR mix was 10 mM Tris-HCl pH 8.2, 1.9 mM MgC1

2, 50 mM KCl,

0.0008% (w/v) gelatin (included with the lOx buffer of Perkin Elmer), 0.5 mM each of dATP, dCTP, dGTP, and dTTP (from Perkin Elmer), 0.2 mM primer (from Operon Technologies, Alameda, CA), approximately 30-35 ng DNA template, and 1 unit Taq DNA polymerase (from Perkin Elmer). The thermal cycle was (94 "C for 30 s) x 1, (92 ·c for 30 s, 35 ·c for 1 min, 72 ·c for 2 min) x 50, and then the mix was stored at 4 ·c. Amplified mix, 20 jll, was loaded onto a 1.5% agarose gel and run at a constant intensity of 70 rnA for about 5 h. Boehringer Mannheim Molecular Weight Marker Type VI was used as marker. Sharp bands became visible after staining the gel with ethidium bromide during 45 min. An MP-4 Land camera (Polaroid) was used to score bands. Out of the 40 primers tested, 'A3' (5'AGTCAGCCAC3'), 'A18' (5'AGGTGACCGT3'), '06' (5'CCACGGGAAG3'), '07' (5'CAGCACTGAC3'), and '013' (5'GTCAGAGTCC3') were used for the final study. Reproducibility of results was evaluated by replicating every amplification twice .

A RAPD band presence/absence profile was recorded. Only reproducible bands were considered. In addition, a presence/absence data matrix was prepared and used for the numerical analysis. Only bands common to the three individuals were considered for the data matrix.

The numerical analysis was carried out by cluster analysis. The similarity matrix was calculated using the Jaccard coefficient. UPGMA was used for clustering (Sneath and Sokal, 1973). The software package NTSYS-pc (Rohlf, 1990) was used for all the analyses.

RESULTS

A total of 40 primers were tested (Operon Technologies series A and 0). From them, only A3, A18, 06, 07, and 013 yielded a number of clear and reproducible bands through all the studied accessions.

Figure 1 shows an example ofthe.results obtained. Reproducibility from one amplifi­cation to another was fairly good. From the qualitative viewpoint, most of the bands appeared in the two amplifications performed. Unstable bands (less than 4% of the total) were mainly those with extreme molecular weight. On the other hand, from the quantita­tive viewpoint, band intensity was not always consistent. For the band presence/absence profile, only reproducible bands were recorded.

Table 3 shows-the total number of bands of the three individuals studied per population vs. the number of bands common to those three individuals, and the percentage of bands in common. This is a preliminary approach to the intrapopulational variability. The average percentage for all the accessions ranges from 75% in primer A3 to approximately 50% in the rest of the primers. For every accession it is possible to find one or more primers useful in detecting variability within the accession. In general, the Genista accessions produced a higher percentage of common bands than the rest of the genera.

Table 4 shows the presence/absence data matrix. Only bands common to the three

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308 F. GONZALEZ-ANDRES AND J.-M. ORTIZ Isr. J. Plant Sci.

Fig. 1. Patterns of RAPD bands obtained. Results of tbe two performed amplifications for Ace. # 1 (Cytisophyllum sessilifolium) and #4 (Cytisus heterochrous) (Primer Al8) and Ace. #13 (Chamaecytisus glaber) and #24 (Genista monspessulana) (Primer 06) are shown.

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Vol. 43, 1995 RAPD ANALYSIS IN GENISTEAE 309

Table3 Number of common bands for the three individuals studied per accession vs. the total number of

bands and percentage of common bands

PRIMER '/13' PRIMER 'A18 PRIMER '06' PRIMER 'OT pRIMER '013'

on 1 1 2 1 3 1 l2 I 3 1 I 2 I 3 1 I 2 I 3 1121 3 1 2 2 100 1 3 33 1 4 25 3 6 50 1 2 50 2 2 4 50 4 5 80 1 7 14 4 6 ffl 1 1 100 3 1 4 25 3 8 38 2 4 50 1 3 33 1 2 50 4 5 5 100 4 6 ffl 4 5 80 3 4 75 1 5 20 5 3 3 100 6 6 100 2 6 33 1 4 25 . 6 4 4 100 4 6 ffl 1 4 25 1 7 14 2 5 40 7 3 5 60 4 7 57 3 6 50 1 5 20 4 5 80 8 3 4 75 1 6 17 1 4 25 1 3 33 0 5 0 9 4 4 100 4 8 50 3 5 60 4 4 100 2 2 100

10 2 2 100 2 6 33 1 6 17 2 6 33 1 2 50 11 3 6 50 2 6 33 4 7 57 1 4 25 2 4 50 12 4 8 50 1 6 17 3 6 50 2 3 67 1 3 33 13 3 7 43 2 5 40 4 7 57 2 3 67 1 2 50 14 3 7 43 3 6 50 3 8 38 2 3 67 1 2 50 15 1 1 100 1 2 50 2 4 50 1 2 50 1 1 100 16 1 5 20 3 5 60 3 3 100 1 2 50 2 2 100 17 1 1 100 3 5 60 2 5 40 1 3 33 2 2 100 18 2 2 100 1 2 50 3. 6 50 2 4 50 1 3 33 19 3 3 100 2 2 100 4 4 100 4 4 100 2 2 100 20 5 5 100 2 3 67 2 4 50 5 6 83 3 3 100 21 3 3 100 2 2 100 2 5 40 3 3 100 3 3 100 22 3 4 75 1 2 50 3 5 60 3 4 75 3 3 100 23 3 3 100 1 2 50 1 5 20 3 5 60 3 3 100 24 3 3 100 2 2 100 3 6 50 2 3 ffl 3 3 100 25 3 4 75 2 3 67 3 5 60 2 3 67 3 3 100

1: Number of common bands for the three individuals studied per accession. 2: Total number of bands for the three individuals studied. 3: Percentage of common bands.

individuals studied per accession were considered for this matrix. A total of74 amplifi­cation products appeared for the five primers studied. Fifty-six of them were shared by at least two accessions. Eight bands were shared by the three Cytisus reverchonii accessions, 8 by the two Cytisus scoparius accessions, 5 by all the Chamaecytisus ruthenicus populations, and 8 bands were present in the two Chamaecytisus glaber accessions. Finally, 9 bands were shared by the four Genista monspessu/ana accessions. One band was present in all the Cytisus species (band 9, primer A3) and another one in all the Chamaecytisus species (band 11, primer 07). This last band appeared only in the genus mentioned.

Figure 2 shows the dendrogram obtained after cluster analysis of the total data matrix. At a similarity level of 0.15, five branches are clearly distinguished, which correspond to Cytisophyllum sessilifolium, the Cytisus species, Genista linifolia, the rest of the Genista accessions, and Chamaecytisus spp. However, Cytisus heterochrous is included within the Genista branch, although its similarity level with the rest of the Genista species is quite low (0.16). On the other hand, the similarity between Genista

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310 F. GONzALEZ-ANDRES AND J.-M. ORTIZ Isr. J. Plant Sci.

Table 4 Data matrix for the primers studied. Only common bands to all the individuals per population

were considered

GENUS IC' Cytlsus Chamaecyt/sus Genlsta IOTU • 1 2 3 4 5 8 7 8 9 10 11 12 13 14 15 18 17 18 19 20 21 22 23 24 25

1-,P,..,R--,. "'"BAN'""'"""D+-:-,M..,.,.W~.

4 20850000000000100100000000000 ~ ~m 8 8 8 8 8 8 8 8 8 8 8 ~ V 8 A 8 8 ~ 8 ~ 8 8 8 8 ~ I ~~~ ~ ~ ~ 1 ~ ~ ~ ~ ~ ~ ~ ~ 1 ~ & 8 8 8 ~ & X ~ ~ ~ ~ ~~ ~~ 8 8 8 8 8 8 8 8 ~ 8 8 8 8 8 8 A ~ 8 ~- 8 1 ~ ~ ~ ~ 12 11145 0 8 8 8 8 01 0 0o 0 0 0 01 00 00 0 8 0 0 0 1 0 0 8 00 8 n ~~ss~88ss88ss8&s8sss~sss88 ~9 m 8 A 8 A ~ ~ 1 l 1 ~ ~ 8 8 8 8 8 8 ~ ~ ~ A A ~ A 8 18 683 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

i 4 5

~ 9

2176 0 0 0 0 1 1 ~ 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ~B~~ 8 ~ ~ 8 8 g 0 8 g I g 8 g 8 g g g g g g g 8 8 g 8 1m 8 ~ 8 ~ 1 ~ A 8 l A 8 8 8 8 8 8 8 ~ 8 8 8 8 ~ 8 1~~~ 8 8 ? A 8 8 8 8 8 A & 8 8 8 8 8 8 8 8 ~ ~ 8 8 8

A18 11 m~~1~8AA~~~8888888A88888888 ~ 8 g0 8 ~ ~ ~ ~ 8 8 8 8 8 8 8 R 8 8 8 8 ~ 8 8 8 8 ~ Rll8o88~888888~8~B~88~8A8~~A ~~ 8 8 8 ~ 8 8 8 8 8 8 8 O O O 8 ? ? 8 8 ~ 8 8 8 8 8 ~~} 8 8 8 8 8 g 8 g 8 g 8 8 ~ ~ ~ ~ ~ ~ 8 8 g 8 8 g 8

06

07

a 14 15 19 18 19 1 2 3 4 5 6 8 9 10 12 13 14 15 16 17 18 19 1 2 5 6 7 9 10 11 13 15 ~9 ~3 1

~ 6 7 8

013 9 10 11 a 14

2176 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 l~~ 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 A ~ 1538 0 0 8 8 0 8 0 0 0 0 0 8 8 00 0 0 0 1 0 0 0 8 8 0 0 1436 0 0 0 0 0 0 R 0 08 08 R 0 0 0 R 0 1 mB888~A8l8~~88~6 ~8A8~~888 1~~ 8 8 ~ ' 8 8 8 8 8 8 8 ~ ~ ' 8 ~ ~ ~ 8 ~ 8 8 8 8 8 867 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 8 1 1 1 1 0 8340000101111111011011 00001 H~ ~ ~ 8 8 8 8 8 8 ? 8 8 8 ~ ~ ~ ? ~ 8 ~ 8 ~ 8 8 A 8 ~~ 8 8 ~ ' 8 8 8 8 8 8 6 8 8 8 8 8 8 8 8 8 8 8 8 8 8 m8888888888?8&88888?886888 ~~g~ ~ l 6 8 A ~ ~ 6 8 ~ ~ 8 8 8 8 8 8 8 8 8 8 8 8 8 ~ l~~~ 8 8 8 6 8 8 8 8 ~ 8 8 8 8 8 8 8 8 8 ~ ~ ~ ~ 1 ~ O

1m ~ 8 & 8 8 8 8 8 ~ 8 8 8 8 8 8 & 8 ~ 8 ~ & 8 8 8 8 ~~~ 8 ~ 8 8 8 8 8 g ~ 8 8 ~ ~ ~ ? O ~ O 8 8 8 8 8 8 8 \~~~888&88~&8888888881&8888 m888?8888888?~~8888A8~A~~8 m888?88888888888888?68888A 26450000 01000000000000000000 m~8&88 8~88&8888888°8 ~~,~~~8 m~8888 8?8~88888888o8~68888 l~~ 8 8 A 8 A ~ 8 8 ~ 8 8 8 8 8 8 8 8 8 ~ 6 ~ 6 ~ ~ \06333 8888 88888~888~??8686~~~8 m868~ ~88~8~~?~8??~8888886 7391000 00000000000000000001

C*: Cytisophyllum. tMissing numbers correspond to noncommon bands. OTU# according to Table 2.

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Vol. 43, 1995 RAPD ANALYSIS IN GENISTEAE 311

linifolia and the rest of the accessions studied is only 0.07. The Chamaecytisus species showed the lowest similarity level with respect to the rest of the accessions (0.05). The similarity between Cytisophyllum sessilifolium and Cytisus-Genista was 0.11. Finally, the similarity between these two last groups was 0.15. When several populations per species were studied, the grouping obtained agrees with the previously recognized taxa. Two branches are clearly distinguished at a similarity level of 0.24 for the Chamaecytisus genus. These two branches correspond to the species with racemose and capitate inflorescence, respectively (Heywood and Ball, 1968). On the other hand, Cytisus reverchonii, C. scoparius, and C. striatus accessions form a fairly lax group, although the similarity among accessions of the same species is always higher than the similarity among accessions belonging to different species. Within the Cytisus group, C. villosus shows the lowest similarity level with respect to the rest of the accessions (0.17).

DISCUSSION

The reproducibility level from the qualitative viewpoint is similar to that obtained by other authors (Heun et al., 1994). Likewise the lack of reproducibility of quantitative differences was also observed by other researchers (Heun and Helentjaris, 1993).

Similarity level

0 DO 0 25 0. 50 0 75 1 . 0 0 ~------~--------~------~------~

r--------------------0-1 CytisOphyllum r---------------------------0 ----c::===05 r 07 r- L_ __________________ 06

~ rf I r----J L,___---08 Cytisus ~ LL-------------------------09 L----------------------------10

03 y 1

y'------_ ___,1 I L-------22

r----~~~ ::~-- L_ ___________ 2

L_ _____ :~~~~~~~~~~~:::::::::::::::::;=!ii = r---------------------------15 L_ _________________________ 1

Fig. 2. Dendogram obtained after cluster analysis of total data matrix (Table 4) (UPGMA method). Numbers correspond to OTU# (Table 2). Cp: genus Cytisophyllum, Cy: genus Cytisus, Ch: genus Chamaecytisus, Ge: genus Genista.

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312 F. GONZALEZ-ANDRES AND J.-M. ORTIZ Isr. J. Plant Sci.

In our RAPD study, a high level of intrapopulational variability was found. RAPD can detect even the change in one base pair or a limited number of bases. Therefore, RAPD has been reported to be very adequate for infraspecific or populational studies, or as molecular markers. However, its value for taxonomic studies at the specific or higher levels is rather unpredictable in plants from various genera or families (Bachmann, 1992; Thormann and Osborn, 1992). From the high intrapopulational variability ob­tained in our results, we can conclude that the RAPD technique is adequate for popula­tional studies. Moreover, RAPD could be potentially useful as markers for agronomic traits. Further studies are currently being carried out regarding these aspects. On the other hand, only the bands common to the three individuals studied per accession have been recorded for the numerical analysis at the specific and generic levels. The reason is that noncommon bands are typical of the individual rather than of the accession, and our objective was to study the taxonomic relationships between accessions representing different taxa.

Numerical analysis revealed that RAPD are useful for the characterization at the specific level in the studied taxa because the similarity between the different accessions of one given species was always higher than the similarity existing between different species accessions. The main branches obtained in the cluster analysis correspond to the genera recognized by Bisby (1981). Therefore, RAPD analyses support the taxonomic generic arrangement proposed by Bisby. This agrees with other characterization studies carried out with the same taxa, regarding stem anatomy (Norverto et al., 1994), seeds morphometry (Gonzalez-Andres and Ortiz, 1995), adult plant morphometry, and isoen­zymes (Gonzalez-Andres, 1995). On the other hand, Genista linifolia is not placed in the main Genista group but in an independent branch, and Cytisus heterochrous is included in the Genista group. With respect to G. linifolia, adult plant morphological studies as well as seed morphometry also indicated the peculiar characteristics of this species in comparison to the other species studied from the same genus. Intermediate floral characters between Cytisus and Genista were shown by Cytisus heterochrous. Conse­quently, some authors have considered it as a Genista species (Genista patens D.C.). RAPD results confirm the intermediate position of this species. On the other hand, Cytisus villosus has been reported as involved in the link between Cytisus and Chamaecytisus genera (Bisby, 1981). Although in our study it is included in the Cytisus branch, its similarity with the rest of the Cytisus species is the lowest.

The low similarity level between Chamaecytisus and Cytisus supports the conclusion that both taxa should be considered different genera, as proposed by Bisby (1981), although this viewpoint was not accepted by Cristofolini and Chiapella (1977, 1984). These authors, based on serology, prefer to consider Chamaecytisus as section, namely Tubocytisus, of the genus Cytisus. However, most of the works published during the last few years give Chamaecytisus generic status (Francisco-Ortega et al., 1993; Shevera, 1993). Frodin (1965) was the first author to recognize that Cytisophyllum sessilifolium should be considered as a monotypic genus different from Cytisus. Further morphologi­cal (Bisby and Nicholls, 1977) and biochemical (Cristofolini and Chiapella, 1977; Greinwald et al., 1991) studies confirmed this situation. The clear discontinuity between

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Vol. 43, 1995 RAPD ANALYSIS IN GENISTEAE 313

Cytisophyllum sessilifolium and the rest of the Cytisus species has also been supported by our molecular approach. Teline is considered an independent genus by some authors (Heywood and Ball, 1968; Gibbs and Dingwall, 1971), or a section within Genista by others (Polhill, 1976; Bisby, 1981). Anyway, nowadays it is clear that it should be considered definitely independent from Cytisus. Nevertheless in our study, although Genista sect. Teline and Cytisus are located in independent branches, their similarity is greater than that existing between any other two genera. This situation was not sup­ported by other morphological or isoenzymatic studies (Gonzalez-Andres, 1995; Gonzalez-Andres and Ortiz, 1995). Probably, as recognized by Thormann and Osborn (1992), this could be due to nonhomology of fragments of identical size, amplified from accessions of poorly related taxa.

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