Genetic Relationships between Oleander
Accessions by Means of AFLP Profiling
E. Portis, C. Comino and S. Lanteri
Di.Va.P.R.A. – Plant Genetics and Breeding
University of Turin
Via L. da Vinci 44
10095 Grugliasco (TO)
Italy
(Nerium
oleander
L.)
A. Lenzi, P. Lombardi and R. Tesi
DI.S.A.T.
University of Florence
Piazzale delle Cascine 18
50144 Florence
Italy
Keywords: AFLP, Apocynaceae, diversity, DNA fingerprinting, Nerium oleander L.
Abstract
Oleander (Nerium oleander L.) is a Mediterranean evergreen shrub widely
grown as an ornamental for its abundant and long-lasting flowering as well as its
moderate hardiness. Genetic relatedness among 71 accessions, including commercial
varieties, different sources of the same varieties, and selections from the wild were
investigated using amplified fragment length polymorphism (AFLP). Genetic
similarities among accessions were calculated according to Jaccard’s Similarity
Index and used to construct a dendrogram based on the unweighted pair group
method, using arithmetic averages. Our results show that up to about 9 % molecular
genetic differentiation was detected among morphologically indistinguishable
provenances of the same variety. This can be partly attributed to scoring error but
mainly to somatic variation occurring during vegetative propagation. On the other
hand lower genetic distance values were sometimes found among varieties, which
differ in morphological characters and are thus commercialised with different
names. The possibility of considering the amount of genetic variation within a
variety as the threshold value for discrimination of initial varieties and essential
derivative varieties is discussed.
INTRODUCTION
Oleander (Nerium oleander L.) is a Mediterranean evergreen shrub characteristic
of watercourses, gravely places, and damp slopes. It is widely grown as an ornamental in
warm temperate and subtropical regions, due to its abundant and long-lasting flowering
and moderate hardiness (Kingsbury, 1964; Hardin and Arena, 1974). It is used for
screens, hedging along highways, planting along beaches and in urban areas as, by
removing suckers and leaving just a few stems, it can also be formed into very attractive
small trees. Oleander has flexible branches with green, smooth bark eventually turning to
dark grey. The leaves are 5 to 20 cm long, narrow, acuminated, or acute in the apex,
shortly petiolate, with a coriaceus dark-green blade. Some cultivars have white or yellow
variegated leaves. Flowers are produced in terminal heads and their colours vary from
deep to pale pink, lilac, carmine, purple, salmon, apricot, copper, orange, yellow and
white (Huxley, 1992). Oleander can be propagated by seed (Pagen, 1988) but, being
allogamous and highly heterozygous, it shows great variability in seedling populations.
Growers generally use cuttings.
Variety identification is mainly based on flower colour and shape, but other
discriminating characters are presence of foliage variegation and growth habit. Naming
and identifying oleander varieties is difficult, due mainly to sale of material under
unreliable names. Thus an accurate method for their identification and characterization is
necessary.
Recent developments in DNA marker technology provide means for cultivar
fingerprinting as well as for assessing genetic diversity and phylogenetic relationships.
The AFLP technique (Vos et al., 1995), which is based on selective amplification of
restriction fragments from a digest of total genomic DNA, has several advantages over
other marker systems currently in use. It does not require previous knowledge of the
Proc 21st IS on Breeding Ornamentals, Part II
Eds: G. Forkmann & S. Michaelis
Acta Hort 651, ISHS 2004
173
species genome, produces a large number of informative polymorphic markers per primer
pair, is highly sensitive, requires small amounts of DNA and has proved to be robust,
reliable and reproducible (Mueller and Wolfenbarger, 1999; Hodkinson et al., 2002).
To our knowledge, the method has not until now been used to analyse the oleander
genome. The objectives of the present study were to evaluate the usefulness of AFLP in
differentiating oleander varieties, and to determine genetic relationships in a sample of 71
accessions including commercial varieties, provenances within the same variety, and
selections from the wild.
MATERIALS AND METHODS
Plant Material
The accessions under study are maintained at DISAT, University of Florence
(Lenzi et al., 1999; Lenzi and Tesi, 2000). Seventy-one accessions, including 51
commercial varieties (Table 1), different sources of the same varieties as well as 5
Sicilian selections obtained from the wild, were included in our analysis.
For each accession the growth habit (i.e. vigorous, compact or dwarf) and the
following morphological characters were recorded: corolla colour (measured using a
portable colorimeter NR-3000, Nippon Denshoku), type (double or single), diameter and
width; chlorophyll levels measured in three leaves of at least two plants using the portable
equipment SPAD-502 (Minolta). Examples of varieties characterized by single or doublecorolla flower are shown in Fig. 1.
DNA Extraction and AFLP Analysis
Samples were collected from young leaves and DNA was extracted from one leaf
per plant, and three plants per accession, according to Lanteri et al. (2001). The AFLP
protocol was essentially that of Vos et al. (1995) with minor modifications (Lanteri et al.,
2003). Briefly, 5 µl extracted DNA (400-500 ng) were digested with EcoRI and MseI and
ligated to adapters. Digested and ligated DNA fragments were first pre-amplified with
primer complementary to the adapters with an additional selective 3’ nucleotide
(EcoRI+A and MseI+C primers); subsequently selective amplification was carried out
using primer with two or three selective nucleotide. Nine AFLP primer combinations
(listed in Table 2) were chosen on the basis of a previous screening conducted in our
laboratory. Amplified fragments were separated by electrophoresis on a 5 % polyacrylamide sequencing gels and silver stained as described by Bassam et al. (1991).
Data Scoring and Analysis
Electrophoretic patterns were documented at the Gel Documentation System
(Quantity One Programme, BioRad). Each PCR product was assumed to represent a
single locus and data were scored as the presence (1) or absence (0) of a band for each
polymorphic band.
The polymorphism degree was calculated for each primer pairs by means of the
Polymorphic Information Content: PIC = 2f (1-f), were f is the percentage of plants where
the marker is present (Anderson et al., 1993):
Genetic similarity among accessions was calculated according to Jaccard’s
Similarity Index (JSI) (Jaccard, 1908) in all possible pair-wise comparisons:
JSIxy=a/(a+b+c), where a = number of bands shared from individuals x and y, b = number
of bands present in x and absent in y, c = number of bands present in y and absent in x;
thus, JSIxy=1 indicates identity between x and y, whereas JSIxy=0 indicates complete
diversity. The JSIs were used to construct a dendrogram using UPGMA (unweighted pairgroup method, arithmetic average). A co-phenetic matrix was produced using the
hierarchal cluster system and correlated with the original distance matrices for AFLP
data, in order to test for association between the cluster in the dendrogram and the JSI
matrix. All calculations and analyses were conducted using the appropriate routines of the
software NTSYS version 1.80 (Rohlf, 1993).
174
RESULTS
A total of 241 polymorphic bands (39.9 % of the total amplified bands), ranging
from 40 to 1500 bp, were scored. The average number of polymorphic bands per primer
combination was 26.8 ranging from 22 to 38 per priming pair (Table 2).
The JSI values ranged from 0.201 for ‘Rosy Rey’ and ‘Commandant Barthelemy’
to 1.00 for the provenances II and III of variety ‘Papà Gambetta’. The dendrogram based
on the similarity values generated using UPGMA (Fig. 2), shows that accessions ‘Rosy
Rey’ and ‘Palermo selection A’ were the most divergent, with respectively an average
genetic similarity of about 45 and 49 % to the others. The dendrogram separated the other
accessions into 4 main branches (A, B, C, D) with branch B being subdivided into three
major clusters: B1, B2 and B3. However, it was not possible to consistently correlate the
clustering based on AFLP data with morphological characters or growth habits usually
adopted for varietal identification. Although branch A includes only varieties with
compact habit and the dwarf ‘Petit Salmon’, other varieties with compact or dwarf habit
were distributed in the other clusters; Moreover although yellow cultivars were mainly
included in cluster B1, the yellow flowered ‘Sausalito’ and ‘Luteum Plenum’ were in
cluster A and B3 respectively. Interestingly most of the double-flowered varieties were in
cluster B2, and three of them in cluster B3.
The co-phenetic correlation coefficient (r-value) between the data matrix and the
co-phenetic matrix for AFLP data was 0.88, suggesting a very good fit between the
dendrogram clusters and the similarity matrices from which they were derived.
DISCUSSION
Many oleander varieties are now available and commercialised therefore their
accurate identification is becoming important. This appears to be the first report of the use
of a DNA-based polymorphism assay to identify genetic differences among oleander
varieties, which for commercial purposes are vegetatively propagated.
For clonally propagated ornamentals, varietal uniformity and stability are only
influenced by somaclonal variation, therefore testing authorities are studying the
possibility to apply molecular markers for assessing distinctness, uniformity and stability
(DUS) criteria for new varieties, and for the management of reference collections (De
Riek, 2001). Furthermore, molecular markers might find application in detecting
infringements of plant breeders’ rights and help in the discrimination of Essential Derived
Varieties (EDVs). As noted by De Riek (2001), a test based on the molecular genetic
relatedness between an initial variety (IV) and an EDV is very informative, as even if
both varieties have completely different flower shape or colour, they may share most of
their genome.
In this study we applied the AFLP technique, since it has proved to be powerful in
detecting similarities in the genome of related cultivars and has been applied to
assessment of genetic conformity and for testing essential derivation in numerous
ornamental plants (Van Huylenbroeck et al., 2001; Tomkins et al., 2001; Carr et al.,
2003).
To obtain unambiguous attribution of accessions to a variety, we characterized
each accession by the growth habit and morphological characters usually adopted for
varietal identification, and we confirmed that different provenances within the same
variety were always indistinguishable. Two accessions, ‘Rosy Rey’, with compact habit
and single pink flowers, and ‘Palermo selection A’, with the same flower characters but a
more vigorous habit, were highly genetically differentiated from all the others. Indeed, in
both of them, we detected four exclusive bands, which might be converted into STS
(sequence tagged site) markers of great values for varietal fingerprinting. Interestingly,
among Sicilian selections, ‘Palermo selection A’ was the only one, which did not cluster
with other commercial varieties, and this might confirm its derivation from autochthonous
instead of naturalised germplasm.
The other 69 accessions could be grouped in four main branches of which branch
B was further subdivided into three major clusters. On the whole, it was not possible to
175
correlate morphological characters usually adopted for variety identification with the
clustering obtained with molecular data. Varieties with different corolla colour or size as
well as growth habit were quite uniformly distributed among the clusters. Interestingly
varieties with double corolla were always included in clusters B2 and B3 and this
supports the hypothesis of their different origin and introduction at the end of the 17th
century from India (Pagen, 1988), although both single and double corolla types, together
with their hybrids, are now present in nature.
The weak correlation between morphological and molecular data is not surprising,
considering that the limited number of characters used for variety discrimination is
encoded by a limited number of genes, which can originate new phenotypes as a
consequence of simple mutation events or non-heritable changes: i.e. transposons or
epigenetic effects. Vice versa, by means of AFLP markers, we were able to
simultaneously and randomly assay a large number of loci in the genome.
Provenances within the same variety always clustered together, according to our
data, although limited genetic differentiation among them was detected. The range of
genetic differentiation was about 3 % among the three accessions of ‘Luteum Plenum’
and two accessions of ‘Magaly’ and ‘Tito Poggi’, and even lower (about 2 %) for five of
the six ‘Papà Gambetta’ and three of the four ‘Maria Gambetta’. However, ‘Maria
Gambetta’ accession III was genetically differentiated at about 5 % from other
provenances of the same variety and an analogous value was detected between the two
accessions of ‘Emilie’. Interestingly, ‘Papà Gambetta’ accession V was more genetically
similar to ‘Rosa Bartolini’ than to the other accessions within the same variety, from
which a genetic distance of 9 % was detected; an analogous value was found between the
two accessions of ‘Madame Leon Blum’ and of ‘Pink Beauty’. By comparing AFLP
profiles of identical clones and replicate samples we estimated that the scoring error in our
analyses was about 2 %, which is consistent with that estimated in other studies (Mueller
and Wolfenbarger, 1999; Hodkinson et al., 2002); higher values can thus be attributed to
somatic variation occurring during vegetative propagation.
The highest genetic distance among morphologically indistinguishable
provenances of the same varieties, i.e. 9 %, may be considered the threshold value due to
somaclonal variation occurring over time. Thus the distance of about 9 % between ‘Tito
Poggi’, ‘Madame Leon Blum’ and ‘Aurora’, which are phenotypically very similar,
suggests that they share the same genetic background and presumably the same origin.
Indeed, Pagen (1988) reports that ‘Tito Poggi’ is a selection with darker flowers of
‘Madame Leon Blum’, while for Filippi (1997) states that the two varieties might be
retraced to the same variety and are both very similar to ‘Soleil Levant’, which from our
data was indeed 9 % distant from the others.
A distance of about 4 % was detected among ‘Roseum Plenum’, ‘Palermo
selection F’ and ‘Foliis Variegata’, all of them with double pink flowers but the last
differing in the presence of leaf variegation, which can thus be attributed to mutation of a
common ancestor; furthermore, distances lower than 9 % were found between ‘Magaly’
and ‘Pink Beauty’, both with simple pale pink flowers, as well as between ‘Jannoch’ and
‘Suor Luisa’, both with single red flowers.
Notwithstanding its wide popularity and commercialisation there is no Official
Variety Register for oleander. Our data demonstrate that when two varieties, although
morphologically distinguishable and commercialised with different names, show a
molecular genetic differentiation lower or analogous to that detectable among
provenances of the same variety, they might be considered as EDVs; at least, molecular
markers should function as a strong indication to competing breeders to prove the origin
of their new selections.
Literature Cited
Anderson, J.A., Churchill, G.A., Autrique, J.E., Sorells, M.E. and Tanksley, S.D. 1993.
Optimizing Parental Selection For Genetic-Linkage Maps. Genome 36: 181-186.
Bassam, B.J., Caetano-Anolles, G. and Gresshoff, P.M. 1991. Fast and sensitive silver
176
staining of DNA in polyacrylamide gels. Analytic Biochemistry 19: 680-683.
Carr, J., Xu, M., Dudley, J.W. and Korban, S.S. 2003. AFLP analysis of genetic
variability in New Guinea impatiens. Theoretical and Applied Genetics 106: 15091516.
De Riek, J. 2001. Are molecular markers strengthening plant variety registration and
protection? Acta Hort 552: 215-223.
Filippi, O. 1997. Guide de reconnaissance des variétés de lauriers-roses. Pépinière Filippi,
Meze (France).
Hardin, J.W. and Arena, J.M., 1974. Human poisoning from native and cultivated plants,
2nd ed. Kingsport, Tennessee, Duke: University Press.
Hodkinson, T.R., Chase M.W. and Renvoize, S.A. 2002. Characterization of a genetic
resource collection for Miscanthus (Saccharinae, Andropogoneae, Poaceae) using
AFLP and ISSR PCR. Annals of Botany 89: 627-636.
Huxley, A., ed-in-chief. 1992. The New Horticultural Society Dictionary of Gardening,
vol. 3. MacMillan, London.
Jaccard, P. 1908: Nouvelles recherches sur la distribution florale. Bull. Soc. Vaud. Sci.
Nat. 44: 223-270.
Kingsbury, J.M. 1964. Poisonous plants of the United States and Canada. Englewood
Cliffs, NJ Prentice Hall.
Lanteri, S., Di Leo, I., Ledda, L., Mameli, M.G. and Portis, E. 2001. RAPD variation
within and among populations of globe artichoke cultivar “Spinoso sardo”. Plant
Breeding 120: 243-246.
Lanteri, S., Acquadro, A., Quagliotti, L. and Portis, E. 2003. RAPD and AFLP
assessment of genetic variation in a landrace of pepper (Capsicum annuum L.), grown
in North-West Italy. Genetic Res. and Crop Evol. 50: 723-735.
Lenzi, A., A. Palandri, A. Bovelli and R. Tesi 1999. L’oleandro (Nerium oleander L.) per
la coltura in contenitore. Colture Protette 9: 101-112.
Lenzi, A. and Tesi, R. 2000. La collezione di oleandri del D.I.S.A.T. Proceedings of the
Congress “Il germoplasma toscano: tutela e valorizzazione”, 129-136.
Mueller, U.G. and Wolfenbarger, L. 1999. AFLP genotyping and fingerprinting. Trends
in Ecology and Evolution 14: 389–394.
Pagen, F.J.J. 1988. Oleanders. Nerium L. and the oleander cultivars. Agricultural
University Wageningen, The Netherlandes. pp. 113.
Rohlf, F.J. 1993. NTSYS-PC numerical taxonomy and multivariate analysis system.
Version 1.8. University of New York, Stony Brook, NY.
Tomkins, J.P., Wood, T.C., Barnes, L.S., Westman, A. and Wing, R.A. 2001. Evaluation
of genetic variation in the daylily (Hemerocallis spp.) using AFLP markers.
Theoretical and Applied Genetics 102: 489-496.
Van Huylenbroeck, J., Coart, E., Janneteau, F. and De Riek, J. 2001. Identification of
woody ornamentals by AFLP. Eucarpia Meeting. Ornamental Symposium, "Strategies
for New Ornamentals”, Melle, Belgium.
Vos, P., Hogers, R., Bleeker, M., Reijand, M., Van de Lee, T., Hornes, M., Fritjers A.,
Pot, J., Paleman, J., Kuiper M., and Zabeau, M. 1995. AFLP: A new technique for
DNA fingerprinting. Nucleic Acids Research 23: 4407-4414.
177
Tables
Table 1. Some morphological characters of the 51 commercial varieties in study. D:
Diameter; W: Width; SPAD (Soil Plant Analysis Development) chlorophyll levels
detected in leaves.
Variety
Album Plenum
Algiers 1
Alsace
Altini
Angiolo Pucci 1
Arad1
Arizona1
Aurora1
Biancaneve1
Bonfire
Capraia
Commandant Barthelemy
Dimona1
Elat1
Elfo 1
Emilie
Fiesta Pienk1
Foliis Variegata
Hardy Red
Isle of Capri1
Italia
Jannoch
Luteum Plenum
Madame Leon Blum
Magaly
Margaritha
Maria Gambetta
Maurin des Maures 1
Minouche (Ville d’Hyeres) 2
Mishna 1
Mont Blanc
Mrs. Roeding
Nana Rosso 1
Nomade 1
Papà Gambetta 1
Petite Pink 1
Petite Red (Maravenne) 2
Petite Salmon 2
Petite White 2
Pink Beauty
Professeur Granel
Ré D ‘JR 95-3’ 1
Rosa Bartolini 1
Roseum Plenum
Rosy Rey 1
Sausalito 1
Sister Agnes
Soleil Levant
178
Corolla
Colour
White
Red
White with a pink hue
Red
Ivory yellow
Pink
Ivory yellow with a pink hue
Pink
White
Fuchsia Pink
Pink
Fuchsia pink-Red
Pink
Pink
White with a pink hue
Pink
Pink
Pink
Fuchsia pink-Red
Pale yellow
Fuchsia pink-Red
Red
Pale yellow
Pink
Pale pink
Pink-fuchsia
Yellow
Fuchsia pink
Fuchsia pink
Pale pink
White
Pale salmon pink
Pink with dark margins
Pink
Pink–Red
Pale pink
Red
Pale salmon pink
White
Pale pink
Fuchsia pink
Fuchsia pink
Pink with dark margins
Pink
Pale pink
Ivory yellow with pink margins
White
Dark salmon pink
Type
Double
Single
Single
Single
Single
Single
Single
Single
Single
Single
Single
Double
Single
Single
Single
Single
Single
Double
Single
Single
Single
Single
Double
Single
Single
Single
Single
Single
Single
Single
Double
Double
Single
Single
Single
Single
Single
Single
Single
Single
Double
Single
Single
Double
Single
Single
Single
Single
D (mm)
55.8 ± 1.27
51.0 ± 5.90
60.3 ± 1.17
58.9 ± 1.06
62.3 ± 1.67
38.4 ± 2.22
52.4 ± 1.71
57.4 ± 1.90
50.7 ± 4.33
78.1 ± 2.41
51.7 ± 7.64
67.2 ± 2.02
53.8 ± 2.14
48.8 ± 0.84
51.1 ± 1.07
60.3 ± 1.84
60.3 ± 3.2
59.8 ± 1.62
55.4 ± 1.28
45.0 ± 0.4
56.2 ± 1.06
54.6 ± 1.95
55.6 ± 1.68
70.6 ± 1.64
64.6 ± 1.64
56.0 ± 0.69
69.6 ± 0.59
55.2 ± 0.62
46.6 ± 1.29
36.8 ± 0.96
59.4 ± 1.90
61.4 ± 0.95
43.6 ± 0.69
46.2 ± 2.36
63.6 ± 1.31
44.3 ± 1.26
48.7 ± 0.84
39.0 ± 0.50
41.7 ± 1.5
63.6 ± 1.46
49.7 ± 1.58
67.6 ± 0.30
56.7 ± 1.44
62.5 ± 2.43
44.2 ± 2.45
47.7 ± 1.41
61.3 ± 1.53
67.4 ± 1.31
W (mm)
24.8 ± 0.78
13.2 ± 1.17
20.4 ± 0.59
18.9 ± 0.80
20.1 ± 0.48
10.0 ± 0.05
14.9 ± 0.51
20.2 ± 1.35
17.8 ± 2.58
23.8 ± 0.11
18.5 ± 3.54
30.3 ± 2.22
13.1 ± 0.96
13.7 ± 2.33
19.2 ± 0.84
23.0 ± 0.51
23.0 ± 0.9.
25.8 ± 2.13
21.4 ± 0.44
15.0 ± 0.1
20.4 ± 0.44
19.9 ± 1.31
27.1 ± 2.56
23.9 ± 0.68
22.3 ± 0.69
22.6 ± 0.11
21.0 ± 0.33
18.3 ± 0.19
15.4 ± 0.30
10.7 ± 0.58
28.8 ± 2.47
27.3 ± 1.53
15.6 ± 1.71
16.2 ±1.41
22.0 ± 0.33
16.4 ± 0.73
16.9 ± 1.28
10.3 ± 1.20
12.3 ± 0.6
23.0 ± 0.67
17.8 ± 1.75
21.2 ± 0.29
15.9 ± 0.87
29.5 ± 1.39
11.9 ± 0.48
12.6 ± 0.71
22.6 ± 1.24
20.6 ± 0.73
Leaf
SPAD
73.1 ± 3.38
52.8 ± 1.99
73.0 ± 1.70
58.2 ± 2.25
68.5 ± 1.53
72.2 ± 3.68
68.0 ± 1.85
67.2 ± 3.81
67.3 ± 9.45
72.4 ± 0.63
65.7 ± 2.59
57.7 ± 3.13
62.4 ± 0.29
61.6 ± 6.09
57.8 ± 2.69
64.7 ± 2.08
55.5 ± 4.72
79.3 ± 1.68
60.8 ± 2.37
55.0 ± 1.18
60.5 ± 5.27
61.5 ± 4.95
69.8 ± 6.20
81.5 ± 0.92
61.1 ± 4.58
51.1 ± 9.43
56.0 ± 10.21
60.9 ± 0.44
57.7 ± 1.55
60.4 ± 1.13
68.3 ± 3.76
61.7 ± 1.92
61.6 ± 6.09
54.6 ± 2.75
67.9 ± 1.78
55.1 ± 0.60
49.8 ± 2.43
49.8 ± 3.25
55.5 ± 0.81
69.8 ± 3.62
67.0 ± 0.85
51.3 ± 0.70
65.8 ± 1.77
54.1 ± 2.15
52.6 ± 4.47
70.8 ± 2.86
56.2 ± 1.21
76.2 ± 1.97
Souvenir d’August Royer
Suor Luisa
Tito Poggi
1
Double 75.0 ± 4.07
Single 57.1 ± 1.61
Single 71.8 ± 3.95
Pale pink
Red
Pink
34.0 ± 2.78
19.7 ± 0.33
24.1 ± 0.22
52.6 ± 2.68
68.2 ± 2.91
64.1 ± 2.09
compact habit; 2 dwarf
Table 2. Summary of AFLP primer combination characteristics. Total number of bands
(TNB), number of polymorphic bands (NPB), percentage of polymorphic bands (P%),
Polymorphic Information Content (PIC).
Primer combination
Eco+AAC / Mse+CAG
Eco+AAC / Mse+CAT
Eco+AAC / Mse+CTT
Eco+AAG / Mse+CAG
Eco+AAG / Mse+CAT
Eco+AAG / Mse+CTC
Eco+AAG / Mse+CAT
Eco+ACA / Mse+CTC
Eco+ACA / Mse+CTT
Total
average
TNB
58
67
75
64
80
66
63
63
67
603
67.0
NPB
24
25
27
27
38
24
22
24
30
241
26.8
P%
41.4
37.3
36.0
42.2
47.5
36.4
34.9
38.1
44.8
PIC
0.306
0.284
0.289
0.220
0.284
0.266
0.314
0.368
0.297
39.8
0.292
Figures
A
B
Fig. 1. Examples of varieties characterized by single or double-corolla flower.
179
Fiesta Pienk
Biancaneve
Elat
Arad
Dimona
Elfo
Nana Rosso
Petite Pink
Sausalito
Petite Salmon
Nomade
Mishna
Maria Gambetta II
Maria Gambetta I
Maria Gambetta IV
Maria Gambetta III
Palermo sel. E
Angiolo Pucci
Isle Of Capri
Arizona
Aurora
Tito Poggi II
Tito Poggi I
Madame Leon Blum II
Madame Leon Blum I
Soleil Levant
Papà Gambetta VI
Papà Gambetta III
Papà Gambetta II
Papà Gambetta I
Papà Gambetta IV
Papà Gambetta V
Rosa Bartolini
Maurin des Maures
Foliis Variegata
Palermo sel. F
Roseum Plenum
Margaritha
Altini
Palermo sel. B
Mont Blanc
Alsace
Magaly II
Magaly I
Pink Beauty I
Sister Agnes
Pink Beauty II
Souvenir d’August Royer
Professeur Granel
Bonfire
Commandant Barthelemy
Re’D’JR 95-3’
Album Plenum
Mrs Roeding
Algiers
Petite White
Luteum Plenum II
Luteum Plenum III
Luteum Plenum I
Jannoch
Suor Luisa
Petite Red (Maravenne)
Palermo sel. D
Hardy Red
Minouche
Italia
Capraia
Emilie II
Emilie I
Palermo sel. A
Rosy Rey
0.45
0.50
0.55
0.60
0.6 5
0.70
0.75
0.80
0.85
0.90
0.95
A
B1
B
B2
B3
C
D
1.00
Jaccard’s Similarity Index
Fig. 2. Dendrogram obtained from UPGMA cluster analysis of AFLP data generated by
the 9 primer combinations tested.
180