genetic variation analysis of the genus passiflora l. using rapd markers

Euphytica 101: 341–347, 1998. 341c 1998Kluwer Academic Publishers. Printed in the Netherlands.

Genetic variation analysis of the genusPassifloraL. using RAPD markers

Diego Fajardo1, Fernando Angel2;�, Mikkel Grum3, Joe Tohme2, Mario Lobo1,William M. Roca2 & Ines Sanchez11 Unidad de Recursos Geneticos, CORPOICA, Cali, Colombia;2 Biotechnology Research Unit, CentroInternacional de Agricultural Tropical, CIAT, A.A. 6713, Cali, Colombia;3 International Plant Genetic ResourcesInstitute, IPGRI, A.A. 6713, Cali, Colombia; (� author for correspondence)

Received 5 September 1997; accepted 27 November 1997

Key words:cluster analysis, genetic variation, molecular markers,Passiflora, RAPD markers

Summary

Genetic analysis based on Random Amplified Polymorphic DNA (RAPD) was carried out on 52 accessionsrepresenting 14 species of the genusPassifloraL. using 50 random 10-mer primers. A dendrogram constructedusing the Dice similarity coefficient and the UPGMA algorithm based on 626 reproducible polymorphic productsranging in size from 2.8 to 0.3 Kb revealed high levels of variation within and among species, and clustering ofaccessions according to species. Similarity coefficients ranged from 0.929 to 0.075 showing a diverse genepool inthe genus. Large intraspecific variation was found inP. ligularisandP. adenopodawhile P. edulisandP. maliformisexhibited a low level of intraspecific variation. The clusters based on RAPD markers correlate fairly well to thepresent classification scheme based on morphological description with two exceptions. The subgenusPassiflorawas split intoP. edulisand the rest of the members of the subgenusPassifloraseparated by the subgenusTacsonia;secondly the two species of the subgenusDecaloba, P. adenopodaandP. coriacea, were not clustered together onthe dendrogram of genetic relationships.

Introduction

The genusPassifloraL., is one of the 12 to 18 (Killip,1938; Holm-Nielsen et al., 1988) genera of the familyPassifloraceae, and is numerically and economicallythe most important genus of the family. It is distributedthroughout the tropics and subtropics, though the vastmajority is endemic to the New World. The principaleconomic interest is in the fruit production industry.About sixty species produce edible fruit andPassiflorais the genus with the largest number of edible species.However, a majority of the species are also of someinterest as ornamental plants given their spectacularexotic forms and colours. Some others are of phar-maceutical interest due to the sedative, antispasmod-ic, antibacterial (Perry et al., 1991) and insecticidal(Echeverry et al., 1991) secondary metabolites theycontain.Passiflorahave in recent years undergone astrong transformation in the sense of cultivation, distri-

bution and commercialization that may result in rapiderosion of their genetic variation (NRC, 1989).

At present, the genusPassiflora is an importantgenetic resource and the characterization and evalua-tion of wild and cultivated populations is seen as highpriority for Andean countries because of its high poten-tial for development and crop diversification. Strate-gies for conservation and improvement are needed tooptimize the use of this resource. Compared to sev-eral agricultural crops, relatively little is known aboutthe inheritance of important traits or about the geneticdiversity within and among different species ofPassi-flora. Recently developed DNA-based marker systemsoffer a reliable and neutral alternative to detect geneticpolymorphisms. Apart from marker systems such asRFLP, mini- and microsatellites, RAPDs have provedto be very useful for the analysis of a large number ofgenotypes (Halward et al., 1992; Parent et al., 1993;Yang & Quiros, 1993; Sharma et al., 1995; Machadoet al., 1996; Asemota et al., 1996).

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Table 1. Origin and accession number of the different accessions of the genusPassiflora, used for the RAPD analysis

Subgenus Section Species Accession Origin

Passiflora Lobatae caeruleaL. 394007 Rionegro (Antioquia)

394018 EMBRAPA (Brazil)

Astrophea Botryastrophea spinosa(P. & Endl.) Mast. 394073 Puerto Berrıo (Antioquia)

Distephana vitifoliaH.B.K. 394086 Cocorna (Antioquia)

Passiflora Incarnatae edulisfv. flavicarpaDegener. 394009 EMBRAPA (Brazil)

394075 Belem de Para (Brazil)

edulisSims. 394020 Sta Rosa de Osos (Antioquia)

394037 Guarne (Antioquia)

Passiflora Tiliaefoliae maliformisL. 394001 Rionegro (Antioquia)

394035 Rionegro (Antioquia)

394048 Armero (Tolima)

Decaloba Pseudodysosmia adenopodaD.C. 394005 Rionegro (Antioquia)


394027 Caqueza (Cundinamarca)

394077 San Vicente (Antioquia)

394078 La Union (Antioquia)


Decaloba Cieca coriaceaJuss. 394080 Tamesis (Antioquia)

Tacsonia Colombiana antioquiensisKarst. 394069 La Estrella (Antioquia)

Tacsonia Bracteogama cumbalensis(Karst.) Harms 394028 Tunja (Boyaca)

Passiflora Tiliaefoliae ligularisJuss. 394003 Rionegro (Antioquia)

394006 Urrao (Antioquia)


394023 Cajibıo (Cauuca)

394025 El Tambo (Narino)








394063 Cajibıo (Cauca)

394067 Armero (Tolima)




Tacsonia Bracteogama mollissima(HBK) Bailey 394021 Tenerife (Valle del Cauca)


394029 Sta Rosa de Osos (Antioquia)

394030 Villamarıa (Caldas)

394032 El Cerrito (Valle del Cauca)

394054 El Cerrito (Valle del Cauca)

394055 San Bernardo (Cundinamarca)

394057 San Bernardo (Cundinamarca)

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Table 1. Continued

Subgenus Section Species Accession Origin

Tacsonia nd* sp india** 394015 Rionegro (Antioquia)

394016 La Ceja (Antioquia)


394083 Sonson (Antioquia)

394084 Retiro (Antioquia)

Tacsonia Pogendorffia pinnatistipulaCav. 394033 Sta Rosa de Osos (Antioquia)

Tacsonia Pogendorffia xrosea 394047 Usme (Cundinamarca)

� not determined.�� Based on morphological description, these five accessions are hybrids betweenP. mollissimaandone or two species of the sectionColombianathat have not yet been determined (P. Moller Jorgensen,personal communication). In subgenusTacsonianatural hybridization is common. In Colombia thesespecimens are named ‘curuba india’. In this paper it is designated assp. india.

The majority of taxonomic studies onPassifloratodate have been based on morphological characteriza-tion (Vanderplank, 1991). Some biochemical studieshave been reported. Escobar (1988) used analyses offlavonoids in the classification of the species of the sub-generaTacsonia. Nonetheless, inconsistencies remainamong the proposed classifications. The use of molec-ular markers, in this case RAPD markers, for the firsttime as a tool in genetic characterization and study ofthe genetic variation within and among differentPassi-flora spp. will help elucidate the variation found with-in this genus. In the present study molecular markerswere used to study the genetic variation and relatednessamong different species and to determine the geneticsimilarities among accessions within species.

Materials and methods

Plant material

A total of 52 accessions from collections establishedat the Experimental Station La Selva, Rionegro, ofthe Colombian Corporation for Agricultural Research(CORPOICA) were employed in this study. All acces-sions were collected from the regions of Antioquia,Boyaca, Cauca, Cundinamarca, Narino, Tolima andValle del Cauca of the Colombian Andes, except oneaccession ofP. caerulaeand two accessions ofP. edulisfv. flavicarpa, provided by EMBRAPA, Brazil (Table1). Young leaves were harvested from one plant ofeach accession and frozen in liquid nitrogen; DNAextraction was as described by Dellaporta et al. (1983)yielding 150�g of total DNA per gram of fresh leaftissue.

RAPD analysis

Polymerase chain reaction (PCR) was accomplishedin a PTC-100 programmable thermal controller (MJResearch, Inc.) as follows: initial strand separationat 94�C (5 min), 35 cycles of 36�C (1 min)/72�C(2 min)/94�C (1 min), final extension at 72�C (7 min),as described by Williams et al. (1990). PCR reactionvolume was 25�l. The fifty arbitrary decamer primers(Operon) randomly selected are listed in Table 2. Theamplification products were electrophoresed in 1.4%agarose gels and visualized by ethidium bromide stain-ing and photographed under UV light.

Data analysis

RAPD assays were performed in duplicate and onlythose patterns obtained clearly twice were scored.Presence or absence of fragments were recorded as1 (present) or 0 (absent) and treated as binary char-acters. Resulting matrices of molecular data for allprimers were submitted for analysis. Pairwise similari-ties were computed and analyzed with NTSYS (Rohlf,1994) version 1.80, using the Dice similarity coeffi-cient (Dice, 1945). The dendrogram was constructedusing the UPGMA algorithm (Sneath & Sokal, 1973).

Results and discussion

RAPD-PCR reactions were performed with 52 plantgenotypes representing 14 species. The amplificationproducts obtained were highly polymorphic in thespecies and genotypes analyzed. A total of 626 poly-morphic fragments from all genotypes and primers

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Table 2. 10-mer oligonucleotides used as primers in the RAPD assay

No. Sequence (50-30) No. Sequence (50-30) No. Sequence (50-30)

AA4 AGGACTGCTC AD7 CCCTACTGGT AM4 GAGGGACCTC

AA6 GTGGGTGCCA AD10 AAGAGGCCAG AM7 AACCGCGGCA

AA9 AGATGGGCAG AD13 GGTTCCTCTG AM10 CAGACCGACC

AA10 TGGTCGGGTG AD14 GAACGAGGGT AM13 CACGGCACAA

AA14 AACGGGCCAA AG6 GGTGGCCAAG AN5 GGGTGCAGTT

AA15 ACGGAAGCCC AG8 AAGAGCCCTC AN6 GGGAACCCGT

AB3 TGGCHCACAC AH3 GGTTACTGCC AN7 TCGCTGCGGA

AB11 GTGCGCAATG AH5 TTGCAGGCAG AN8 AAGGCTGCTG

AB14 AAGTGCGACC AH6 GTAAGCCCCT AN17 TCAGCACAGG

AC3 CACTGGCCCA AH8 TTCCCGTGCC G13 CTCTCCGCCA

AC4 ACGGGACCTG AH9 AGAACCGAGG M13 GGTGGTCAAG

AC7 GTGGCCGATG AH10 GGGATGACCA P6 GTGGGCTGAC

AC9 AGAGCGTACC AI5 GTCGTAGCGG V7 GAAGCCAGCC

AC10 AGCAGCGAGG AI6 TGCCGCACTT X4 CCGCTACCGA

AD1 CAAAGGGCGG AI11 ACGGCGATGA Y1 GTGGCATCTC

AD4 GTAGGCCTCA AI13 ACGCTGCGAC Y2 CATCGCCGCA

Y4 GGCTGCAATG

Y7 AGAGCCGTCA

Figure 1. RAPD fragments of nineteen different genotypes generated by the primer AB11. The amplification products were separated on 1.4%agarose gels in 1� TBE buffer. Inter- and intraspecific polymorphisms are observed. A, I, L, N-Q:P. ligularis; B, E-G, T:P. mollissima; C: P.adenopoda; D: P. cumbalensis; H: P. pinnatistipula; J: lambda-Pst I; K, S:P. maliformis; M: P. edulisfv.flavicarpa; R: P. xrosea.

could be unambigously identified and were used forthe computation of genetic similarities (Figure 1).Means and ranges of genetic distances withinPassiflo-ra species, as revealed by RAPD analysis, are shownin Table 3.P. ligularisandP. adenopoda, showed wideintraspecific variation. In contrast,P. edulisandP. mal-iformisexhibited a low level of intraspecific variation.However, because the sample size of our study wassmall, a more detailed analysis using a larger numberof accessions is necessary to confirm these patterns ofintraspecific variation, particularly inP. maliformisandP. edulisin which three and four accessions respective-ly were studied.

Similarity coefficient was computed from 626 poly-morphic bands, Similarities between accessions wereestimated by the Dice algorithm using the NTSYS.PCcomputer program (Rohlf, 1988). The Dice algorithmis identical to that of Nei & Li (1979), i.e. 2Nxy/Nx +Ny, where Nxy is the number of bands shared by acces-sions X and Y, respectively. The similarity coefficientsobtained were used to construct a dendrogram with theUPGMA method (Figure 2). Pairwise Dice coefficientof similarity values between accessions ranged from0.929 (betweenP. mollissimaaccessions 394029 and394030) to 0.012 (P. coriaceaaccession 394080withP.caeruleaaccession 394018). Cluster analysis showedsimilarity coefficients from 0.929 to 0.075 demonstrat-

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Figure 2. Dendrogram of genetic similarities betweenPassifloragenotypes obtained with the Dice index and the UPGMA clustering methodfor 52 accessions ofPassifloraevaluated with 50 arbitrary primers.

ing a diverse genepool (Figure 2). Large variation wasfound within P. ligularis, even within theP. ligularisfrom one region, Urrao, Colombia (Table 1). This isall the more surprising because Urrao is the regionin Colombia where production ofP. ligularis is mostcommercialized and market forces would be expect-ed to lead to widespread cultivation of a few geno-types. Hence, there is a still great potential to improvethis species through selection and recombination.P.

ligularis also showed significant variation for cpDNAusing RFLPs (I. Sanchez et al., in preparation). It isclear that in the case ofP. ligularis a diverse gene poolis present. The genetic similarity among five accessionsof P. sp. india, all from the same region, ranged from0.905 to 0.637. Similar data were found inP. mollissi-ma(0.902–0.688) despite the samples being from fourgeographically distinct regions of the country (Table1).

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Table 3. Genetic distance withinPassifloraspecies basedon RAPD markers

Species Number of Mean Range

accessions

P. ligularis 17 0.545 0.112–0.731

P. adenopoda 6 0.573 0.247–0.789

P. sp. india 5 0.261 0.095–0.363

P. mollissima 8 0.231 0.098–0.312

P. edulis 4 0.615 0.522–0.683

P. maliformis 3 0.556 0.547–0.570

The analysis successfully separated species accord-ing to taxonomic classification based on morphologicaldescriptors (Figure 2). All species of the subgenusTac-soniawere grouped together and the dendrogram iden-tified P. mollissimaandP. sp. indiaas a single clusterwithin Tacsonia. The single accession ofP. xroseaclus-tered withP. mollissima. Sodiro (cited in Killip, 1938)proposed thatP. xroseais in fact a hybrid betweenP.pinnatistipulaandP. mollissima. As the dendrogramplacesP. xroseabetween the two supposed parents,our results do not refute this theory. On the other hand,the dendrogram splits the subgenusPassiflorainto twogroups withTacsoniain the middle. Ignoring theTac-sonia, the divisions within the subgenusPassiflorafitreasonably well with the traditional taxonomy.P. ligu-laris andP. maliformis, which both belong to the sec-tion Tiliaefoliaeare placed together, whileP. caeruleaandP. eduliswhich each belong to different sections ofthe subgenus are clearly separated from the previoustwo species. The four other species, all wild, are placedmuch further apart.P. coriaceaandP. adenopoda, thatboth belong to the subgenusDecaloba, though to sep-arate sections, are placed very far apart. Killip (1938),placed the subgenusDistephanabetween the subgen-eraTacsoniaandPassiflora, but the present analysisplaces it outside the cluster of the two groups (Figure2).

In summary, the clusters based on RAPD markerscorrelate fairly well to the present classification schemebased on morphological description with the exceptionof the placement ofTacsoniawithin the subgenusPas-sifloraand that the two species of the subgenusDecalo-ba, P. adenopodaand P. coriacea, were not placedtogether. The robustness of the dendrogram in relationto the computational methods applied is demonstrat-ed by the fact that different estimates of the relativegenetic similarity and different clustering methods didnot change the general structure of the phenogram. To

investigate the sensitivity of the dendrogram againstchanges in the computational methods, additional den-drograms were computed with a different formula forthe relative genetic similarities (simple match) and twoother clustering methods (single linkage and completelinkage). The combinations of similarity indices andclustering procedures resulted in eight very similardendrograms (results not shown). Neither the generaltopology of the clusters nor the differences of relativedistances within the clusters were altered. Althoughthe amount of data in the present study is not sufficientto discuss the significance of the most distant clus-ters, includingvitifolia, spinosaandcoriacea, severalfeatures of the dendrogram correspond to the presenttaxonomy.

Knowledge of genetic distances could be useful inbreeding programs, particularly in the selection of par-ents for hybridization. RAPDs offer information onintraspecific variation and interspecific genetic rela-tionships of use in the breeding of individual species.An understanding of genetic similarity among speciescan facilitate introgression of useful genes (e.g. resis-tance genes) from one species to another. This paperrepresents an important preliminary study in the analy-sis of genetic diversity within the Andean species ofthe genusPassiflora. Future studies using additionalaccessions representing more species and a broadergeographic distribution will provide a greater under-standing of the native genetic resources of this promis-ing genus.

Acknowledgements

The authors acknowledge to the Inter-AmericanDevel-opment Bank (IDB) for financial support, Clara Medi-na from CORPOICA, La Selva, for supplying us withPassifloraaccessions; Dr. M. Fregene, J. Rauscher andC. Iglesias for their valuable comments and J. Gutierrezfor technical assistance.

References

Asemota, H.N., J. Ramser, C. Lopez-Peralta, K. Weising & G. Kahl,1996. Genetic variation and cultivar identification of Jamaicanyam germplasm by random amplified polymorphic DNA analy-sis. Euphytica 92: 341–351.

Dellaporta, S.L., J. Wood & J.B. Hicks, 1983. A plant miniprepara-tion: version II. Plant Mol Biol Rep 1: 19–21.

Dice, L.R., 1945. Measures of the amount of ecologic associationbetween species. Ecology 26: 297–302.

euph4619.tex; 13/05/1998; 9:10; v.7; p.6

347

Echeverry, F., G. Cardona, F. Torres, C. Pelaez, W. Quinonez &E. Renteria, 1991. Ermain: an insect deterrent flavonoid fromPassiflora foetidaresin. Phytochemistry 30 (1): 153–156.

Escobar, L.K., 1988. Flora de Colombia. 10. Passifloraceae. Institutode Ciencias Naturales. Museo de Historia Natural. Facultad deCiencias, Universidad Nacional, Bogota, Colombia.

Halward, T., T. Stalker, E. LaRue & G. Kochert, 1992. Use of single-primer DNA amplifications in genetic studies of peanut (ArachishypogeaL.). Plant Molec Biol 18: 315–325.

Holm-Nielsen, L., P.M. Jorgensen & J.E. Lawesson, 1988. Passiflo-raceae. In: G. Harling & L. Anderson (Eds), Flora del Ecuador.University of Goteborg, Copenhagen.

Killip, E.P., 1938. The American species of Passifloraceae. FieldMuseum of Natural History, Chicago.

Machado, M.A., H.D. Coletta Fillo, M.L.P.N. Targon & J. Pom-peu Jr., 1996. Genetic relationship of Mediterranean mandarins(Citrus deliciosaTenore) using RAPD markers. Euphytica 92:321–326.

N.R.C. (National Research Council), 1989. Lost Crops of the Incas:little-known plants of the Andes with promise for worldwidecultivation. National Academy Press, Washington, D.C.

Parent, J.G., M.G. Fortin & D. Page, 1993. Identification de culti-vars de framboisier par l’analyse d’ADN polymorphe amplifie auhasard (RAPD). Can J Plant Sci 73: 1115–1122.

Perry, N.B., G.D. Albertson, J.W. Blunt, A.L. Cole, M.H. Munro& J.R. Walker, 1991. 4-Hydroxy-2-cyclopentenone: an anti-pseudomonas and cytotoxic component fromPassiflora tetranda.Planta Medica 57 (2): 129–131.

Rohlf, F.J., 1994. NTSYS-pc Numerical taxonomy and multivariateanalysis system (version 1.80). State University of New York.

Sharma, S.K., I.K. Dawson & R. Waugh, 1995. Relationships amongcultivated and wild lentils revealed by RAPD analysis. TheorAppl Genet 91: 647–654.

Sneath, P.H.A. & R.R. Sokal, 1973. The principles and practiceof numerical classification. Numerical Taxonomy. Freeman, SanFrancisco, Ca.

Vanderplank, J., 1991. Passion flowers. Cambridge, Massachusetts:The MIT Press, Massachusetts Institute of Technology.

Williams, J.G.K., A.R. Rubelik, K.J. Livak, J.A. Rafalski & S.V.Tingey, 1990. DNA polymorphisms amplified by artitrary primersare useful as genetic markers. Nucleic Acids Res 18: 6531–6535.

Yang, X. & C. Quiros, 1993. Identification and classification ofcelery cultivars with RAPD markers. Theor Appl Genet 86: 205–212.

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genetic variation analysis of the genus passiflora l. using rapd markers

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