Genetic diversity in passion fruit (Passiflora spp.) evaluated by RAPD markers

AGRICULTURE, AGRIBUSINESS AND BIOTECHNOLOGY

Genetic diversity in passion fruit (Passiflora spp.) evaluated by RAPD markers

Maria Lúcia Crochemore^*; Hugo Bruno Correa Molinari ; Luiz Gonzaga Esteves Vieira

Laboratório de Biotecnologia; Instituto Agronômico do Paraná (IAPAR); C. P. 481; 86001-970; Londrina - PR - Brazil

ABSTRACT

The objective of this study was to characterize the genetic diversity within a Passiflora collection by PCR-RAPD markers. Genetic analysis was performed in 70 accessions, representing 11 species of the genus Passiflora. The use of only five primers produced 136 reproducible polymorphic bands. The hierarchical classification showed high levels of dissimilarities between and within the species studied. A clear separation was obtained among species and accessions of P. edulis and P. edulis f. flavicarpa, which were forms of the high commercial value species. The fingerprints produced from the studied genotypes would allow the identification of improved varieties/populations in an easy, fast and inexpensive manner.

Key words:Passiflora spp., germoplasm, genetic diversity, RAPD-PCR

RESUMO

A diversidade genética de Passiflora spp. tem sido estudada basicamente através de características morfológicas e agronômicas, levando à uma classificação até espécie. A caracterização intraespecífica por meio destes descritores necessita de um ciclo integral de cultivo, e é pouco discriminante. O objetivo deste estudo foi caracterizar a variação genética de uma coleção de Passiflora através da técnica RAPD. Análises moleculares foram realizadas em 70 acessos, envolvendo 11 espécies do gênero Passiflora. A utlização de apenas cinco primers permitiu a obtenção de 136 fragmentos polimórficos. A classificação hierárquica obtida mostrou alto nível de dissimilaridade entre e dentro das espécies. Nítida separação foi obtida entre acessos de P. edulis e da forma flavicarpa, de alto interesse comercial, fracamente diferenciados pelas características morfológicas e agronômicas. A utilização desses marcadores na avaliação dos recursos genéticos de Passiflora, é atrativa para o melhorista e poderá muito auxiliar na identificação dos acessos, pela facilidade, rapidez e pelo baixo custo na condução das avaliações.

INTRODUCTION

Passiflora is the most important genus in the family Passifloriaceae and it is distributed within tropical and sub-tropical regions. Approximately, 460 species of the genus Passiflora have already been described. From these, about 90% have originated from the Americas and close to 150 species occur in Brazil (Sousa and Meletti, 1997). The economical importance of some species is associated to the quality of the fruits for consumption and medicinal properties.

The passion fruit is an allogamous plant and the edible fruit-producing species are diploids (2n=18), namely, the purple (P. edulis) and the yellow passion fruit (P. edulis f. flavicarpa) (Martin and Nakasone, 1970), which is cultivated in Brazil. The high interspecific compatibility in natural and artificial crossings among the diploid species leads to a wide genetic diversity distributed in different agro-climatic regions. Morphological characters used for taxonomical description of passion fruit have allowed a classification up to the species level, but not to an intraspecific discrimination. Besides fruit color, flavor and resistance to diseases, P. edulis and P. edulis f. flavicarpa do not show contrasting differences (Martin and Nakasone, 1970). Although still not much used in passion fruit genetic variability studies, RAPD markers have been shown to be polymorphic, allowing the characterization of the variability among and within species as well as for the identification of hybrids and parentals (Fajardo et al., 1998, Cassiano, 1998; Otoni et al., 1995). The characterization of the genetic diversity within a Brazilian Passiflora spp. collection using RAPD markers may permit the discrimination of plant introductions, the analysis of inter- and intraspecific variability and the detection of duplicated accessions resulting in a complementary tool for selection of progenitors for breeding programs. Also, the identification of cultivars, which are forms of the high commercial value species by molecular markers, may be a valuable support in view of Cultivar Protection Laws (Jondle, 1992). Although these molecular descriptors are not accepted for protecting intellectual property by the UPOV convention (International Union for the Protection of New Varieties of Plants), these techniques have an enormous potential for cultivars discrimination and deserve further investigation. The objective of this study was to characterize the genetic diversity within a Passiflora collection by RAPD markers.

MATERIALS AND METHODS

Plant Material

Seventy introductions of the IAPAR's Passiflora spp. Germplasm Collection, comprising 11 species and two not-classified introductions were used. Their origin and identification are shown in Table 1. Seedlings propagated by budding or grafting were grown under greenhouse facilities.

DNA extraction

Young leaf tissues (30-40 mg) from each introduction were harvested at the same plant growth stage. The leaves were oven-dried (37ºC) for 12 h and then macerated until a fine dust. DNA extraction was performed as described by Molinari and Crochemore (2001). DNA concentration was estimated in a fluorimeter as described by the manufacturer (Hoefer Scientific Instruments).

RAPD analysis

RAPD reactions were performed according to Williams et al. (1990) in a final volume of 25 ml containing 25 ng genomic DNA, 2.5 ml buffer 10X [200 mM Tris (pH 8.4), 500 mM KCl), gelatin 0.01%, 1.5 mM MgCl₂, 0.25 mM of each dNTP, 0.2 mM primer and 1 U Taq polymerase. The amplifications were performed in a thermocycler (MJ Research, Inc.) using the following program: 1 cycle of 4 min at 94ºC followed by 37 cycles of 1 min at 93ºC, 1 min at 45ºC and 1 min at 72ºC, with a final extension of 6 min at 72ºC.

Amplification products were analyzed by electrophoresis at 5V/cm in agarose gel (1.4%). The gel was stained in 0.5 mg/ml ethidium bromide solution and photographed with the KODAK EDAS 120 system.

From a screening using 42 primers (Operon Technologies, Inc. Kits A, B, AE1 and C16 oligonucleotides), five primers were shown to be highly polymorphic and were used in this study (Table 2). Only amplified consistent DNA bands, in the approximate range of 0,1–3 kilobases (kb) were scored.

Statistical analysis

Jaccard's similarity indexes (Jaccard, 1901) were calculated with data provided by the observation of the presence (1) or absence (0) of bands between pairs of accessions using the formula D_(ij) = a/(a+b+c), where: a is the number of fragments shared by accessions i and j; b is the number of fragments present in i and absent in j; c is the number of fragments present in j and absent in i. A hierarchical classification (dendrogram) was obtained from the similarity matrix using the UPGMA (Unweighted Pair Group Mathematical Average) method. The mean similarities and variances detected in the hierarchical classification were calculated. All calculations were performed using the SAS Statistical Program (SAS Institute, 1998).

RESULTS AND DISCUSSION

Table 2 summarizes the number, average and size range of amplified fragments obtained with the five primers used to compare the Passiflora collection. A total of 136 amplified fragments were selected. Depending on the introduction-primer combination, between 1 and 11 distinct RAPD products were amplified, ranging in size from approximately 120 to 3000 bp. Each RAPD amplification was prepared at least three times to determine reproducibility of the patterns.

Genetic diversity

High dissimilarity was observed among the introductions. The hierarchical classification obtained by the clustered method showed three large groups (Fig. 2). The values of mean dissimilarity, range and variances of all possible pairwise comparisons within these groups are shown on Table 3. As expected, the highest dissimilarity was observed among the species of Group I. This group is composed by 14 accessions, comprising the species: P. cocinea, P. alata, P. macrocarpa, P. edulis, P. alata x P. macrocarpa P. caerulea, P. serrato-digitata, P. giberti and P. suberosa, two accessions of P. edulis (Kenya and Madeira Island) and the accession Passiflora sp. (2). The lowest dissimilarity in Group I was found between accession BG25 (P. alata) and BG05 (P. alata x P. macrocarpa). On the other hand, the highest dissimilarity (0.97) was found between accession BG11 (P. suberosa) and BG23 (P. edulis) from Group II.

Group II was exclusively formed by eight introductions of P. edulis, a cross between P. edulis and the introduction Passiflora sp. (1). This group showed a mean dissimilarity of 0.67 (Table 3). This high variation observed within P. edulis was corroborated by Cassiano (1998) but was not detected in the studies done by Fajardo et al. (1998) probably due to the small number of accessions studied by these authors. Two introductions, originally classified as P. edulis - one from Kenya (BG04) and the other from Madeira Island (BG19) - were assigned to Group I. However, due to their close similarity to alata-macrocarpa and P. caerulea respectively, we inferred that these introductions were misclassified in the original germoplasm collection. The introduction named Passiflora sp. (1), was grouped very close to a wild Brazilian introduction (d=0.56) of P. edulis, and probably it was closed related to this species.

Group III was formed by 46 introductions of P. edulis f. flavicarpa, native of several states of Brazil (Table 1). The LM introductions had been selected according to their desirable agronomical characteristics. On the other hand, IA introductions were introduced genotypes, without any specific selected characteristic. Despite being formed only by P. edulis f. flavicarpa, this group presented a mean dissimilarity of 0.40 (Table 3).

The grouping method (UPGMA) allowed a perfect distinction between the introductions LM and IA showing that the RAPD markers were useful even to discriminate closely related genotypes.

P. edulis f. flavicarpa was likely to be a population selected from P. edulis based on agronomical characteristics, like fruit color. The classification obtained evidences the genetic ascendancy of P. edulis over P. edulis f flavicarpa, supporting the origin of P. edulis f. flavicarpa as a mutation of P. edulis (Pope and Degener, cited by Martin and Nakasone, 1970) and not from a cross of P. edulis with any of the species herein studied. Our results have shown that could be possible to distinguish several Passiflora species as well as separate accessions of P. edulis and P. edulis f. flavicarpa with only five primers. Molecular markers are very useful in an early breeding program for allowing germoplasm screening at any developmental stage of the plants or when a large number of individuals need to be evaluated.

Fingerprinting

The analysis of the RAPD products profile showed highly discriminative amplified fragments among the 70 introductions studied. An example of banding patterns given by DNA amplification products after agarose gel electrophoresis is presented in Fig. 1.

The primer OPA-04 showed the highest discriminative power and could be used to clearly discriminate the group flavicarpa from all other accessions. This primer revealed a specific band (700 bp) for all accessions of P. alata and P. macrocarpa and it also identified P. foetida by a 640 bp fragment. The primer OPB-08 separated P. edulis from P. edulis f. flavicarpa and also revealed one specific amplified fragment for Passiflora sp. (2) and one for P. coccinea. The lack of a OPB-18-440bp fragment, present in all introductions of the collection was characteristic for the P. suberosa. Moreover, three specific amplified fragments revealed with only primer OPB-18 were found in Passiflora sp. (2). The primer OPB-19 presented a specific amplified fragment (550bp) for P. giberti and the primer OPB-20 revealed four fragments that specifically discriminated Passiflora sp. (2) and P. giberti. Specific RAPD products were not found in P. caerulea, P. serrato-digitata and, therefore, it was not possible to discriminate these introductions from all others of the germoplasm collection.

ACKNOWLEDGEMENTS

We thank Neusa C. Stenzel from IAPAR for providing the germoplasm collection.

Received: December 26, 2001

Revised: August 01, 2002

Accepted: July 10, 2003

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*

Author for correspondence

Publication Dates

• Publication in this collection
  10 Feb 2004
• Date of issue
  Dec 2003

History

• Accepted
  10 July 2003
• Reviewed
  01 Aug 2002
• Received
  26 Dec 2001