Euphytica 136: 125–137, 2004.
© 2004 Kluwer Academic Publishers. Printed in the Netherlands.
125
Amplified fragment length polymorphism for variety identification and
genetic diversity assessment in oleander (Nerium oleander L.)
Ezio Portis1 , Cinzia Comino1, Anna Lenzi2 , Piero Lombardi2 , Romano Tesi2 &
Sergio Lanteri1,∗
1 Di.Va.P.R.A. – Plant Genetics and Breeding, University of Turin,
via L. da Vinci 44, 10095 Grugliasco (TO), Italy;
University of Florence, Piazzale delle Cascine 18, 50144 Florence, Italy; (∗ author for correspondence,
e-mail: sergio.lanteri@unito.it)
2 DISAT,
Received 25 August 2003; accepted 6 January 2004
Key words: AFLP, Apocynaceae, diversity, DNA fingerprinting, Nerium oleander L.
Summary
Oleander is a Mediterranean evergreen shrub found along watercourses, gravelly places and damp slopes. It is
grown widely 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). Nine
primer combinations yielded a total of 603 bands of which 241 were polymorphic. 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 the AFLP technique, which
can simultaneously and assay a large number of loci randomly distributed in the genome, is much more informative on the genetic relationship and origin of accessions than the limited number of morphological characters
conventionally used for variety discrimination. 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 essentially derived varieties is discussed.
Introduction
Oleander (Nerium oleander L., Family Apocynaceae)
is a Mediterranean evergreen shrub characteristic of
watercourses, gravelly places and damp slopes. It is
widely grown as an ornamental in warm temperate
and subtropical regions, due to its abundant and longlasting flowering and moderate hardiness (Kingsbury,
1964; Hardin & 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. In Northern regions it may be grown as an
indoor or patio plant.
Oleander has flexible branches with green, smooth
bark eventually turning to dark grey. Cut or broken
branches exude a thick, white sap (Font-Quer, 1979;
Schvartsman, 1979; Lampe & McCann, 1985; Pearn,
1987). 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). Each flower
is about 5 cm in diameter with five petals, although
some cultivars have double flowers. The fruit consists
126
Table 1. Some morphological characters of the 51 varieties and five selections from the wild in study. D: Diameter; W:
Width; SPAD (Soil Plant Analysis Development) chlorophyll levels detected in leaves
Variety
Album Plenum
Algiers1
Alsace
Altini
Angiolo Pucci1
Arad1
Arizona1
Aurora1
Biancaneve1
Bonfire
Capraia
Commandant Barthelemy
Dimona1
Elat1
Elfo1
Emilie
Fiesta Pienk1
Foliis Variegata
Hardy Red
Isle of Capri1
Italia
Jannoch
Luteum Plenum
Madame Leon Blum
Magaly
Margaritha
Maria Gambetta
Maurin des Maures1
Minouche (Ville d’Hyeres)2
Mishna1
Mont Blanc
Mrs. Roeding
Nana Rosso1
Nomade1
Palermo selection A
Palermo selection B
Palermo selection D2
Palermo selection E
Palermo selection F
Papà Gambetta1
Petite Pink1
Petite Red (Maravenne)2
Petite Salmon2
Petite White2
Pink Beauty
Professeur Granel
Corolla
Colour
Type
D (mm)
W (mm)
Leaf
SPAD
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
White
Red
Yellow
Pink
Pink-Red
Pale pink
Red
Pale salmon pink
White
Pale pink
Fuchsia pink
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
Double
Single
Single
Double
Single
Single
Single
Single
Single
Single
Double
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
56.7 ± 2.4
66.1 ± 2.21
45.1 ± 0.13
49.2 ± 0.41
62.9 ± 1.30
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
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
18.3 ± 0.33
30.1 ± 2.55
15.7 ± 0.67
16.0 ± 0.54
29.1 ± 0.56
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
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
58.6 ± 2.04
50.9 ± 2.56
58.0 ± 1.19
60.2 ± 0.31
63.9 ± 5.59
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
127
Table 1. Continued
Variety
Ré D ‘JR 95-3’1
Rosa Bartolini1
Roseum Plenum
Rosy Rey1
Sausalito1
Sister Agnes
Soleil Levant
Souvenir d’August Royer
Suor Luisa
Tito Poggi
Corolla
Colour
Type
D (mm)
W (mm)
Leaf
SPAD
Fuchsia pink
Pink with dark margins
Pink
Pale pink
Ivory yellow with pink margins
White
Dark salmon pink
Pale pink
Red
Pink
Single
Single
Double
Single
Single
Single
Single
Double
Single
Single
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
75.0 ± 4.07
57.1 ± 1.61
71.8 ± 3.95
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
34.0 ± 2.78
19.7 ± 0.33
24.1 ± 0.22
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
52.6 ± 2.68
68.2 ± 2.91
64.1 ± 2.09
1 compact habit. 2 dwarf.
of a narrow follicle 7.5 to 17.5 cm long which opens
to disperse fluffy seeds.
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 (Ude et al., 2002). The AFLP technique, 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 (Vos et al., 1995; Reeves et al., 1998; Ridout
& Donini, 1999). It does not require previous knowledge of the 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 & Wolfenbarger, 1999; Hodkinson et
al., 2000, 2002).
To our knowledge, DNA markers have 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 representative of the most common
Table 2. List of the eight varieties of oleander including
different provenances
Variety
Accession
Provenances
Papà Gambetta
I
II
III
IV
V
VI
I
II
III
IV
I
II
III
I
II
I
II
I
II
I
II
I
II
France
Latium (Italy)
Marches (Italy)
Liguria∗ (Italy)
Liguria∗ (Italy)
Sicily (Italy)
Latium (Italy)
France
Marches (Italy)
Liguria (Italy)
Latium (Italy)
Marches (Italy)
Liguria (Italy)
Marches (Italy)
Tuscany (Italy)
France
Marches (Italy)
Latium (Italy)
Tuscany (Italy)
Marches (Italy)
France
Latium (Italy)
Tuscany (Italy)
Maria Gambetta
Luteum Plenum
Emilie
Magaly
Pink Beauty
Tito Poggi
Madame Leon Blum
∗ Accessions IV and V of Papà Gambetta came from the
same region, but from different nurseries.
commercial varieties, provenances within the same
variety, and selections from the wild.
128
Materials and methods
Plant material
The accessions under study are maintained at DISAT,
University of Florence (Lenzi et al., 1999; Lenzi
& Tesi, 2000). The collection contains 51 varieties
commercialised by Italian and French nurseries as
well as 5 Sicilian selections obtained from the wild
(Table 1). Eight varieties included different provenances as reported in Table 2. Seventy one accessions
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 were measured in three leaves of at
least two plants using the portable equipment SPAD502 (Minolta). Examples of flower form and colour in
oleander varieties are shown in Figure 1.
DNA extraction and AFLP analysis
One young leaf was collected from three plants per
accessions and the three pooled leaves used for DNA
extraction according to Lanteri et al. (2001). The
AFLP protocol was essentially that of Vos et al. (1995)
with minor modifications (Lanteri et al., 2003).
Restriction and ligation were done concurrently by
adding 5 µl extracted DNA (400–500 ng DNA) to 45
µl buffer (10 mM Tris-HCl pH 7.5; 10 mM MgAc,
50 mM KAc) containing 5 units EcoRI, 5 units MseI, 2
units T4 DNA ligase (New England BioLabs, Beverly,
MA), 5 pmol EcoRI adapter, 50 pmol MseI adapter
and 0.2 mM ATP. The mixture was then incubated at
37◦ for 4h and diluted 10 times in 0.1× TE (1 mM
Tris-HCl, 0.1 mM EDTA pH 8).
We used two consecutive PCRs to selectively
amplify the EcoRI-MseI DNA fragments. The preselective amplification (first PCR) was performed using 5 µl of the above mentioned diluted mixture added
to a 15 µl mixture giving a final concentration of
10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2 ,
0.2 mM of each dNTPs (Sigma-Aldrich), 40 ng of
EcoRI and MseI adapter-directed primers, each possessing a single selective base (EcoRI+1 and MseI+1
primers), and 1 unit of Taq polymerase (Promega).
PCR reactions were performed with the following profile: 94 ◦ C for 60 s, 25 cycles of 30 s denaturing at
94 ◦ C, 30 s annealing at 55 ◦ C and 60 s extension
at 72 ◦ C, ending with 10 min at 72 ◦ C to complete
extension. After checking for the presence of a smear
of fragments (100–1000 bp in length) by agarose electrophoresis, the amplification product was diluted 40
times in 0.1× TE.
Subsequently selective amplification (second PCR)
was carried out using primers with three selective
nucleotides. Initially, 16 primer pairs, originated by
the combination of 4 EcoRI primers and 4 MseI
primers, were tested in four oleander accessions. From
this pilot study, the following nine primer combinations were selected, on the basis of clearness and
reproducibility of electrophoretic patterns, and applied to all samples: E32/M49 (AAC/CAG), E32/M50
(AAC/CAT), E32/M62 (AAC/CTT), E33/M49 (AAG/
CAG), E33/M50 (AAG/CAT), E33/M60 (AAG/CTC),
E35/M50 (ACA/CAT), E35/M60 (ACA/CTC), E35/
M62 (ACA/CTT). The EcoRI and MseI adapters and
primers were synthesized by Invitrogen Life Technologies. Selective PCR reactions were performed with
the following profile: 94 ◦ C for 60 s, 36 cycles of 30 s
denaturing at 94 ◦ C, 30 s annealing and 60 s extension
at 72 ◦ C, ending with 10 min at 72 ◦ C to complete
extension. Annealing was initiated at 65 ◦ C which was
then reduced by 0.7 ◦ C for the next 12 cycles and
maintained at 56 ◦ C for the subsequent 23 cycles.
Reproducibility for each primer pair was checked
by running the AFLP protocol at different DNA concentrations; a threshold of 20 ng DNA/µl before digestion was the lowest concentration which avoided appearance of artifacts or disappearance of some bands.
Electrophoresis of the PCR product
Amplification products were mixed with 15 µl of
formamide-dye (98% formamide, 10 mM EDTA,
0.01% w/v bromophenol blue and 0.01% w/v xylene
cyanol), denatured at 95 ◦ C for 4 min and separated
by electrophoresis on 5% denaturing polyacrylamide
sequencing gels (5% acrylamide-7 M Urea 19:1) in
1×TBE buffer. The gels were pre-run for about 30 min
before 4.5 µl of the mix was loaded. Gels were run at
110 W for about 2.5 h.
Staining
Gels were silver stained (Bassam et al., 1991). The
gel was fixed in 10% acetic acid for 30 min, washed
twice with a large quantity of ultrapure water for 5
min, transferred to a silver impregnation solution (1g
L−1 AgNO3 , 0.056% formaldehyde) for 30 min and
then rinsed with ultrapure water for 5 s. All steps were
129
Figure 1. Example of flower types and colours among oleander varieties (A). Flower and leaf of ‘Minouche’ (B) and ‘Commandant Barthelemy’
(C).
130
performed with slow agitation on a shaker. Image development was carried out with manual agitation for
1 to 3 min in developer (30 g L−1 Na2 CO3 , 0.056%
formaldehyde, 400 µg L−1 sodium thiosulphate). To
stop development and fix the gel, 10% acetic acid was
added directly to the developing solution, and shaking
continued for 2–3 min. The gel was then rinsed briefly
in ultrapure water and dried at room temperature.
Data scoring and analysis
AFLP amplifications were repeated at least twice in
order to test their consistency. Electrophoretic patterns
were documented using the Gel Documentation System (Quantity One Programme, BioRad). Each PCR
product was assumed to represent a single locus and
only reproducible polymorphic bands were scored as
present (1) or absent (0).
AFLP data were evaluated by means of Shannon’s
Index, Marker Index and Polymorphic Information
Content.
Shannon’s Index (H’j ) (Shannon and Weaver,
1949) for each locus was calculated as follows: H’j =
–pi log2pi , where pi is the frequency of the presence
or absence of fragment. In order to compare the level
of diversity detected by different primer combinations,
we partitioned diversity for each primer combinations
by subtotalling H’j .
Marker Index (MI) was calculated according to
Powell et al. (1996) as the product of two functions:
Expected Heterozygosity (Hn ) and Effective Multiplex
Ratio (EMR). Hn of a locus is defined as: 1 – pi 2 ,
where pi is the frequency of the presence or absence of
the fragment (band). EMR of a primer was defined as:
βn, were β is the percentage of polymorphic loci and
n is the number of loci detected per primer (Milbourne
et al., 1997).
The polymorphic information content (PIC) was
calculated by applying the simplified formula for the
Expected Heterozygosity (Anderson et al., 1993):
PIC = 2f (1-f), were f is the percentage of plants where
the marker is present.
A binary matrix for cluster analysis was prepared
using the NTSYS-pc (numerical taxonomy and multivariate analysis system) version 1.80 package (Rohlf,
1993). Genetic similarity among accessions was calculated according to Jaccard’s Similarity Index (JSI)
(Jaccard, 1908) in all possible pair-wise comparisons,
using the SIMQUAL (similarity of qualitative data)
routine. The JSI was defined as: 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 divergence.
The JSIs were used to construct a dendrogram using
UPGMA (unweighted pair-group method, arithmetic
average) through the SAHN (Sequential, agglomerative, hierarchical, and nested cluster analysis) routine.
A co-phenetic matrix was produced using the hierarchical cluster system, by means of the COPH routine,
and correlated with the original distance matrices for
AFLP data, in order to test for agreement between the
cluster in the dendrogram and the JSI matrix.
Clustering ability test
The clustering abilities of nine selected AFLP primer
combinations were tested to determine the optimal
number of primer pairs needed to discriminate the
maximum number of oleander accessions. The primer
combination with the highest ability to cluster the 71
accessions was first analysed alone (PC1.J = primer
combination 1, based on Jaccard’s Similarity Index)
and then in combinations with the other PCs with
progressively lower discrimination power. The last
combination (PC9.J) comprised all the nine PCs used
in the study.
Results
Morphological characterization
Data on morphological characterization of the 51 varieties and five selection from the wild in study are
reported in Table 1.
Primer selection and AFLP analysis
Preliminary tests were conducted using the preamplification products in order to define the conditions
that would yield distinct amplified fragments on the
sequencing gel. We tested different combinations of
selective primers, with three selective nucleotides at
the EcoRI end (EcoRI+3) and from one to three selective nucleotides at the MseI end (from MseI+1
to MseI+3). On the whole from 4 to 6 selective
nucleotides were tested. Only the primer combinations characterised by 6 selective nucleotides produced
scoreable bands and were used in this study; the others
resulted in a smear or yielded too many fragments for
accurate scoring.
131
Nine of the 16 primer combinations gave clear
and reproducible amplification patterns. Among the
7 discarded primer combinations, three amplified too
many bands for accurate scoring; although it could
be argued that these primer combinations are highly
informative, the difference in electrophoretic mobility
between bands was very small and increased the risk
of misalignment. The other four primer combinations
discarded (all of them characterized by the presence
of the Eco+ACG primer) were not very informative
as they yielded small numbers of bands often not
uniformly distributed in the gel.
A total of 241 polymorphic bands (39.9% of
the total amplified bands), ranging from 40 to 750
bp, were scored (Table 3). The average number of
polymorphic bands per primer combination was 26.8
ranging from 22 to 38 per priming pair (Table 3).
The Shannon Index, Polymorphic Information
Content and Marker Index for each primer combination are also reported in Table 3. Primer combination E35/M60 showed the highest values for H’j and
PIC, while primer combination E33/M50 showed the
highest value for MI and allowed to distinguish 52 of
the 56 varieties and selections from the wild analysed,
and 59 of the 71 accessions studied. Primer combination E33/M49, gave the lowest values for H’j , PIC and
MI.
Five of the nine primer combinations used made
possible the detection of 15 unique/distinctive bands
(i.e. fragments present in only one accession, Table 3).
Eight of the 56 varieties and selections were characterized by distinctive bands (Table 4). An example of an
AFLP profile is shown in Figure 2.
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 in the
B3 out-groups respectively. Interestingly most of the
double-flowered varieties were in cluster B2, and three
of them in B3 out-groups.
The co-phenetic correlation coefficient (r-value)
between the data matrix and the co-phenetic matrix
for AFLP data was 0.88, suggesting a good fit between
the dendrogram and the similarity matrix from which
it was derived.
Genetic relatedness
Many oleander varieties are now available and commercialised, therefore their accurate identification is
becoming important; however, at present, classification is based on a limited number of characters, mainly
shape, size or colour of the corolla, presence of foliage variegation and growth habit. 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,
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’ (see
Table 2). The values among all accessions are available on request from the authors.
The dendrogram based on the similarity values
generated using UPGMA (Figure 3), 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 two major clusters: B1, B2 and a few out-groups
(B3). However, it was not possible to consistently
correlate the clustering based on AFLP data with
Test for clustering ability of primer combinations
The number of variety and accession subsets resulting from the successive clustering analyses with the
nine primer combinations are shown in Table 5. The
number of oleander varieties clustered by using different primer pair was found to increase from 52 in
PC1.J, to 56 in PC4.J. The clustering power reached
a maximum point of 70 subsets (accessions) after the
AFLP data of the sixth primer combination were added. The addition of the other primer combinations
(PC7.J, PC8.J, PC9.J) resulted in minor modification
of the dendrogram (data not shown).
Discussion
132
Table 3. Summary of AFLP primer combination characteristics. Total number of bands (TNB),
number of polymorphic bands (NPB), percentage of polymorphic bands (P%), Shannon index
(H’j ), Polymorphic Information Content (PIC), Marker Index (MI), number of different varieties
and selections from the wild identified (NV), number of different accessions identified (NA), and
number of exclusive bands (NEB) obtained per primer combination
Primer
combination
TNB
NPB
P%
H’j
PIC
MI
NV
NA
NEB
58
67
75
64
80
66
63
63
67
24
25
27
27
38
24
22
24
30
41.4
37.3
36.0
42.2
47.5
36.4
34.9
38.1
44.8
0.678
0.636
0.651
0.525
0.632
0.603
0.685
0.785
0.663
0.306
0.284
0.289
0.220
0.284
0.266
0.314
0.368
0.297
3.038
2.653
2.805
2.505
5.124
2.326
2.412
3.369
3.990
49
46
49
47
52
50
40
51
51
54
50
52
52
59
55
46
58
55
0
3
0
4
5
1
2
0
0
603
67.0
241
26.8
39.8
0.651
0.292
3.136
48.3
53.4
E32/M49
E32/M50
E32/M62
E33/M49
E33/M50
E33/M60
E35/M50
E35/M60
E35/M62
Total
average
15
1.66
Table 4. List of varieties and selections in which exclusive bands were detected;
number of exclusive bands (NEB); primer combinations (PCs)
Variety
NEB
PCs
Palermo selection A
Rosy Rey
Palermo selection B
Dimona
Minouche (Ville d’Hyeres)
Papa Gambetta
Roseum Plenum
Souvenir d’August Royer
Total
4
4
2
1
1
1
1
1
15
E33/M49 (2) – E33/M50 – E35/M50
E32/M50 – E33/M50 (2) – E35/M50
E32/M50 – E33/M60
E32/M50
E33/M50
E33/M50
E33/M49
E33/M49
Table 5. Primer combinations, number of polymorphic bands (NPB), number of different varieties and selections from the wild (NV) and
number of different accessions (NA) they are able to distinguish
Dendrogram
Primer combinations
NPB
NV
NA
PC1.J
PC2.J
PC3.J
PC4.J
PC5.J
PC6.J
PC7.J
PC8.J
PC9.J
E33/M50
E33/M50. E35/M60
E33/M50. E35/M60. E35/M62
E33/M50. E35/M60. E35/M62. E33/M60
E33/M50. E35/M60. E35/M62. E33/M60. E32/M49.
E33/M50. E35/M60. E35/M62. E33/M60. E32/M49. E32/M62.
E33/M50. E35/M60. E35/M62. E33/M60. E32/M49. E32/M62. E33/M49
E33/M50. E35/M60. E35/M62. E33/M60. E32/M49. E32/M62. E33/M49. E32/M50
E33/M50. E35/M60. E35/M62. E33/M60. E32/M49. E32/M62. E33/M49. E32/M50. E35/M50
38
62
92
116
140
167
194
219
241
52
53
55
56
56
56
56
56
56
59
61
64
67
69
70
70
70
70
133
Figure 2. AFLP pattern obtained by primer combination E33/M50 in 35 of the 51 oleander varieties analyzed. Arrows show presence of
exclusive bands in varieties ‘Papà Gambetta’ and ‘Rosy Rey’.
134
Figure 3. Dendrogram obtained from UPGMA cluster analysis of AFLP data generated by the 9 primer combinations tested.
135
molecular markers might find application in detecting infringements of plant breeders’ rights and help
in the discrimination of Essentially 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 will 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 the assessment of genetic conformity and for testing essential derivation in numerous ornamental plants
(Barcaccia et al., 1999; Loh et al., 1999, Leus et al.,
2000; Van Huylenbroeck et al., 2001; Tomkins et al.,
2001; Carr et al., 2003).
In order to apply the technique, we tested a wide
range of primer combinations. The need for this has
been previously noted (Qi and Lindhout, 1997; Castiglioni et al., 1999; Lima et al., 2002; Lanteri et al.,
2004). Among the primer pairs tested, seven were discarded, due to the not clearly interpretable and not
reproducible electrophoretic patterns obtained, while
9 were chosen and applied for molecular characterization.
Pejic et al. (1998) reported that 150 polymorphic
bands make possible a reliable estimate of genetic
similarities among genotypes within the same species;
indeed we found that the maximum resolution of our
70 subsets (accessions) clustered by UPGMA analysis was obtained using 6 primer combinations, which
made possible the amplification of 167 polymorphic
bands. Nevertheless our study was based on the detection of 241 polymorphic bands, which permitted more
accurate estimates of the genetic relationships being
studied.
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 com-
mercial 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 two major clusters. On the whole, it
was not possible to 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 cluster B2 and in B3 out-groups; 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 and 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 an average 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 & Wolfenbarger, 1999; Hodkinson et al., 2002); higher values
can thus be attributed to somatic variation occurring
during vegetative propagation.
136
The lowest genetic similarity among morphologically indistinguishable provenances of the same varieties, i.e. JSI = 0.879 between ‘Papà Gambetta’ accessions V and I, 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 commercialization, 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.
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