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. 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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