Clone identification and genetic relationship among vine cacti from the genera Hylocereus and Selenicereus based on RAPD analysis

Scientia Horticulturae 100 (2004) 279–289

Clone identification and genetic relationship among vine cacti from the genera Hylocereus and Selenicereus based on RAPD analysis N. Tel-Zur a , S. Abbo b , D. Bar-Zvi a , Y. Mizrahi a,c,∗ a

c

Department of Life Sciences, Ben-Gurion University of the Negev, POB 653, Beer-Sheva 84105, Israel b Department of Field Crops, Vegetables and Genetics, The Hebrew University of Jerusalem, POB 12, Rehovot 76100, Israel The Institutes for Applied Research, Ben-Gurion University of the Negev, POB 653, Beer-Sheva 84105, Israel Accepted 3 September 2003

Abstract Clones of Hylocereus and of Selenicereus species were distinguished from each other by the banding pattern generated by one to nine 10-mer oligonucleotide primers in the random amplified polymorphic DNA (RAPD) reaction. RAPD analysis was also applied to estimate the genetic relationship among five Hylocereus and nine Selenicereus species. A dendrogram was constructed based on a data matrix of 173 polymorphic bands originated by nine primers. Two groups were identified, one consisting of Hylocereus species and the other consisting of Selenicereus species. These results are consistent with the accepted taxonomic classification of the genera studied. The principal coordinate analysis (PCO), i.e. the plot drawn on the basis of the RAPD data, clearly distinguished between three groups, namely, Hylocereus species, S. megalanthus and the rest of the Selenicereus species studied. PCO thus strongly support the notion that the tetraploid S. megalanthus is an exception among the Selenicereus group. The RAPD results support our hypothesis regarding the allopolyploid (rather than autopolyploid) origin of this species. © 2003 Elsevier B.V. All rights reserved. Keywords: DNA fingerprinting; Genetic relationship; Hylocereus; Pitahaya; Selenicereus; Vine cacti

1. Introduction During the past 15 years, a number of cactus species native to the warm and humid forests of northern South America, Central America and Mexico were introduced into Israel (Nerd and Mizrahi, 1997). Among these are the vine cacti of the genera Hylocereus (Berger) ∗

Corresponding author. Tel.: +972-8-6461966; fax: +972-8-6472984. E-mail address: [email protected] (Y. Mizrahi). 0304-4238/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2003.09.007

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Br. and R. and Selenicereus (Berger) Br. and R., which have a considerable economic potential as new exotic fruit crops due to their attractive edible fruits (Mizrahi and Nerd, 1999). At present, a number of these species are cultivated in the open in tropical countries (Barbeau, 1990; Cacioppo, 1990; Mizrahi et al., 1997). H. undatus is cultivated on commercial scale in Colombia, Nicaragua and Vietnam (Mizrahi et al., 1997), while S. megalanthus is cultivated in Colombia (Cacioppo, 1990; Mizrahi et al., 1997). In Israel, H. polyrhizus, H. undatus and S. megalanthus are cultivated in net-houses rather than in the open to prevent damage by the relatively high solar radiation prevailing in this area compared with that in their native habitats (Raveh et al., 1997). The genus Hylocereus consists of 16 species, and the genus Selenicereus includes 20 species (Barthlott and Hunt, 1993). Hylocereus species are characterized by triangular stems and medium to large (200–800 g) scale-bearing spineless fruits. Stems with four or more ribs and small to medium size (5–350 g) spiny fruits are typical of Selenicereus species (Britton and Rose, 1963). In both genera the flowers open in the evening and stay open one night only. The fruits contain numerous small soft seeds. In Latin America the fruits of all species of both genera share the same common name: “pitayas” or “pitahayas”. The fruits of S. megalanthus are sweet and tasty, where the fruit of all others species have an inferior taste. Cytological observations showed that H. costaricensis, H. ocamponis, H. polyrhizus, H. purpusii, H. undatus and S. grandiflorus are diploids, whereas S. megalanthus is tetraploid (Beard, 1937; Spencer, 1955; Lichtenzveig et al., 2000; Tel-Zur, 2001). Considering the morphological features of S. megalanthus, which are reminiscent of both Hylocereus and Selenicereus species, Britton and Rose (1963) classified it into a new genus named Mediocactus, thereby implying both an intermediate morphology and an intermediate taxonomic status. Based on the above data, we hypothesized that S. megalanthus may have originated by natural intergeneric hybridization (followed by chromosome doubling) between certain, unidentified yet, species of these two genera (Tel-Zur, 2001). The volume of fresh vine cacti fruits imported to Europe has increased more than 10-fold over the last 5 years. This produce is still based on wild genotypes, adopted from natural habitats and vegetatively propagated. Standardization of fruit supply could be achieved only through selection of elite material and vegetative propagation of selected clones based on horticultural characteristics, long shelf life and consumer taste preferences. In effort to improve fruit quality and horticultural characteristics of the vine cacti grown in Israel, a large interspecific and intergeneric crossing program was initiated several years ago (Lichtenzveig et al., 2000; Tel-Zur, 2001). In traditional breeding programs, germplasm identification is based on morphological characteristics. In our breeding project we encountered problems in identifying the different clones of the vine cacti due to the following reasons. First, morphological characteristics are influenced by environmental conditions. Second, different clones may be morphologically similar. Third, evaluation of fruit traits requires mature plants, thus implying waste of time and money before identification could be made due to a long juvenile phase. An alternative, relatively simple, identification technique is the use of molecular markers. In the present work we used a fast, cost-effective and simple system based on the random amplified polymorphic DNA (RAPD) technique (Williams et al., 1990; Tingey and del Tufo, 1993) for the evaluation of the genetic relationships among Hylocereus and Selenicereus species and the amplified DNA pattern polymorphism among clones in vine cacti.

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2. Materials and methods 2.1. Plant material and growth conditions The taxa used in this study and their origin are given in Table 1. Each clone was isolated from a single plant and vegetatively propagated. Plant husbandry details were the same as those described by Lichtenzveig et al. (2000). Cuttings from selected clones were allowed to root in continuously aerated tap water after they had been air-dried for 2–3 weeks in order to prevent rot of the cut end of the stem. 2.2. DNA isolation Fresh roots harvested from cuttings were used for DNA isolation. The reason for choosing roots rather than green tissue is their lower polysaccharides content, which generally interfere with the DNA isolation procedure. Total genomic DNA was isolated with the modified CTAB procedure (Tel-Zur et al., 1999) or with PUROGENE DNA Isolation Kit (Gentra, D-5000). The two methods gave similar DNA yield and purity as assessed by spectrophotometric analysis. The isolated DNA was stored at −20 ◦ C until use. 2.3. DNA amplification RAPD assays were carried out in 25 ␮l reaction mixtures containing 75 mM Tris–HCl, pH 9; 20 mM (NH4 )2 SO4 ; 0.01% Tween 20; 1.5 mM MgCl2 ; 200 ␮M each of dNTPs (dNTP set, R0181, MBI, Fermentas); 0.4 ␮M primer (Operon Technologies, CA, USA); 4–20 ng of genomic DNA and 1 U of Taq DNA polymerase (Super-Therm DNA polymerase, LPI-801, GMR, UK). The sequences of the primers applied are given in Table 2. Amplification was performed using an Omnigene Thermocycler (Hybaid, Teddington, UK). The reaction protocol included four cycles of 1 min of template DNA denaturation at 94 ◦ C, 3 min of primer annealing at 36 ◦ C and 3 min of primer extension at 72 ◦ C. In the next 30 cycles, the period of the denaturation was reduced to 30 s, the primer annealing to 45 s and the primer extension to 1 min. The amplified DNA products were resolved on 2% agarose gel, visualized by ethidium bromide staining, and photographed by a Polaroid DS-34 camera. The reproducibility of the amplification products was checked on a template DNA from at least two independent DNA isolations. Only bands that were consistently detected in all replications were considered for the analysis. 2.4. Data collection and analysis RAPD bands were scored from photographs into a binary data matrix as 1 (present) or 0 (absent). The genetic relationship among the species was estimated using the similarity index (S) proposed by Nei and Li (1979): S = 2NAB (NA + NB )−1 where NAB is the number of common bands shared by species A and B, NA the number of bands in species A and NB the number of bands in species B.

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Table 1 Source and code number of the plant material studied Species

Code

Source

H. costaricensis (Weber) Br. & R.

89-023a

Huntington Botanical Gardens, USA

H. ocamponis (Salm-Dyck) Br. & R.

94-031a

Palermo, Botanical garden, Italy

H. polyrhizus (Weber) Br. & R.

89-028a 97-401 97-402 97-403 97-404

Huntington Botanical Gardens, USA UCLA, CA, USA UCLA, CA, USA UCLA, CA, USA UCLA, CA, USA

H. purpusii (Weingart) Br. & R.

89-025a

Huntington Botanical Gardens, USA

H. undatus (Harwort) Br. & R.

87-601 88-027a 89-024 89-026 92-051 92-052 92-053 94-001 95-004

Private garden, Israel Wild, Colombia Huntington Botanical Garden, USA Huntington Botanical Garden, USA Private garden, Israel Huntington Botanical Garden, USA Private garden, Israel Seedling, Vietnam Tabasco Plantation, Mexico

S. grandiflorus (L.) Br. & R.

92-080 94-032a 96-683

Private garden, Israel Palermo, Botanical garden, Italy Hebrew University, Botanical garden, Jerusalem, Israel

S. megalanthus (K. Schum. ex Vaupel) Moran

88-023a 88-025 88-026 90-001a 90-002 90-003 93-003-B

Wild, Ecuador Wild, Ecuador Wild, Ecuador Plantation, Colombia Plantation, Colombia Plantation, Colombia Wild, Colombia

S. atropilosus Kimnach

98-320a

Rainbow Gardens Nursery, Vista, CA, USA

S. coniflorus (Weingart) Br. & R.

98-321a

Rainbow Gardens Nursery, Vista, CA, USA

S. macdonaldiae (Hooker) Br. & R.

98-325a

Rainbow Gardens Nursery, Vista, CA, USA

S. rubineus

98-328a

Rainbow Gardens Nursery, Vista, CA, USA

S. wercklei (Weber) Br. & R.

98-329a

Rainbow Gardens Nursery, Vista, CA, USA

S. innesii Kimnach

98-324a

Rainbow Gardens Nursery, Vista, CA, USA

S. murrillii Br. & R.

98-330a

Rainbow Gardens Nursery, Vista, CA, USA

a Clone

selected to test genetic relationship among Hylocereus and Selenicereus species.

The similarity matrix was processed by the NEIGHBOR program on PHYLIP 3.5 C (Felsenstein, 1993). Genetic distance estimates based on pair-wise comparisons served as elements in the proximity matrix in a cluster analysis by the unweighted pair group method with arithmetic averages (UPGMA) and plotted with the use the DRAWGRAM program

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Table 2 Sequences of the primers applied Primer

Sequence 5 –3

OPA-8 OPA-9 OPA-10 OPA-11 OPA-12 OPA-15 OPA-16 OPA-17 OPA-20

GTGACGTAGG GGGTAACGCC GTGATCGCAG CAATCGCCGT TCGGCGATAG TTCCGAACCC AGCCAGCGAA GACCGCTTGT GTTGCGATCC

on PHYLIP 3.5 C. The resulting clusters were represented as a dendrogram. To evaluate the strength of the resulting branches, bootstrap probabilities were calculated using 1000 bootstrap resampling data with the program Free-Tree (http://www.natur.cuni.cz/flegr/programs/ +++freetree). The RAPD data were also evaluated by principal coordinate analysis (PCO) as an average distance, using the MVSP package (Kovach, 1999). 3. Results 3.1. Clone identification in Hylocereus and Selenicereus species Clones of H. polyrhizus, H. undatus, S. grandiflorus and S. megalanthus were analyzed using one to nine primers (Table 3). Five H. polyrhizus clones were analyzed. The products of six primers, OPA-4, -9, -12, -16, -17 and -20, were monomorphic; therefore, these primers could not be used for Table 3 Polymorphism among clones of two Hylocereus and two Selenicereus species revealed by RAPD primers Differentiated clones H. polyrhizus 89-028, 97-401, 97-404 97-402, 97-403 H. undatus 87-601, 88-027, 89-024, 89-026, 92-051, 92-052, 92-053, 94-001, 95-004 S. grandiflorus 92-080, 94-032, 96-683 S. megalanthus 88-023, 88-025, 88-026, 90-001, 90-002, 90-003 88-023, 93-003-B

Differentiating primer

Fragment sizes (bp)

No. of amplified fragments

No. of polymorphic fragments

OPA-11 OPA-15

400–3600 400–3600

10 11

3 3

OPA-8

500–6500

11

6

OPA-9

550–1500

10

4

OPA-16

300–3600

10

6

OPA-17

500–2500

12

2

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Fig. 1. (A) RAPD profiles of H. undatus clones obtained with the primer OPA-8 (5 -GTGACGTAGG-3 ). Lanes: (1) DNA size marker lambda DNA/Eco471 (AvaII); (2) Clone 87-601; (3) Clone 94-001; (4) Clone 92-052; (5) Clone 92-051; (6) 89-026. (B) RAPD profiles of S. grandiflorus clones obtained with the primer OPA-9 (5 -GGGTAACGCC-3 ). Lanes: (1) DNA size marker lambda DNA/Eco471 (AvaII); (2) Clone 94-032; (3) Clone 92-080; (4) Clone 96-683. (C) RAPD profiles of S. megalanthus clones obtained with the primer OPA-16 (5 -AGCCAGCGAA-3 ). Lanes: (1) DNA size marker lambda DNA/Eco471 (AvaII); (2) Clone 90-001; (3) Clone 88-025; (4) Clone 88-026; (5) Clone 88-023; (6) Clone 96-665. Primes indicate independent replicates of DNA isolation.

distinguishing between any of the five clones. The primers OPA-15 and -11 revealed differences among the studied H. polyrhizus clones (Fig. 1A). All nine H. undatus clones analyzed could be distinguished by the first primer tested, OPA-8. Therefore, no further primers were tested with this taxon. In S. grandiflorus, three clones were distinguished by the primer OPA-9 (Fig. 1B). Among seven S. megalanthus clones analyzed, differences were found between six clones, 90-001, 90-002, 90-003, 88-023, 88-025 and 88-026, using primer OPA-16 (Fig. 1C). Primer OPA-17 revealed differences between clone 88-023 and clone 93-003-B. The products of primers, OPA-1, -8, -9, -10, -12, and -18 were monomorphic.

Hylocereus species

Hylocereus species costaricensis ocamponis polyrhizus purpusii undatus Selenicereus species atripilosus coiflorus grandiflorus innesii macdonaldiae megalanthus Colombia megalanthus Ecuador murrillii rubineus wercklei

Selenicereus species

costaricensis

ocamponis

polyrhizus

purpusii

undatus

atripilosus

coiflorus

grandiflorus

innesii

macdonaldiae

– 0.590 0.583 0.587 0.658

– 0.507 0.599 0.564

– 0.640 0.790

– 0.603

–

0.411 0.351 0.482 0.284 0.413 0.514

0.429 0.392 0.563 0.409 0.445 0.535

0.450 0.414 0.405 0.486 0.395 0.530

0.460 0.372 0.408 0.325 0.428 0.590

0.463

0.568

0.518

0.433 0.475 0.535

0.409 0.455 0.515

0.386 0.461 0.523

megalanthus Colombia

0.457 0.398 0.486 0.451 0.379 0.530

– 0.690 0.525 0.634 0.649 0.479

– 0.519 0.625 0.726 0.559

– 0.617 0.549 0.496

– 0.548 0.514

– 0.521

–

0.277

0.533

0.483

0.560

0.547

0.504

0.552

0.952

–

0.310 0.454 0.521

0.424 0.491 0.552

0.648 0.572 0.559

0.639 0.564 0.652

0.593 0.517 0.500

0.694 0.654 0.681

0.603 0.595 0.672

0.500 0.551 0.623

0.532 0.518 0.657

megalanthus Ecuador

murrillii

rubineus

wercklei

– 0.564 0.580

– 0.693

–

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Table 4 Pair-wise similarity matrix (Nei and Li, 1979) of the Hylocereus and Selenicereus species studied using nine DNA primersa

a The primers used were: OPA-8, -9, -10, -11, -12, -15, -16, -17 and -20.

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3.2. Genetic relationship among Hylocereus and Selenicereus taxa Five Hylocereus species and nine Selenicereus species (Table 1) were chosen for the study, one representative clone from each species. In S. megalanthus two clones collected from different geographic area, clone 88-023 from Ecuador and clone 90-001 from Colombia, were tested. Nine primers were used, OPA-8, -9, -10, -11, -12, -15, -16, -17 and -20, generating a total of 173 polymorphic fragments. Primers OPA-9, -15 and -16 generate unique monomorphic bands (one in each primer). One to thirteen polymorphic bands per genotype were generated by each primer, with an average of 8.1 per primer. The least informative primer was OPA-9, with an average of 5.3 bands per genotype, and the most informative primer was OPA-12, with an average of 10 bands per genotype. In order to quantify the level of polymorphism detected by RAPD, a distance matrix comprising pair-wise similarity indices was constructed according to Nei and Li, 1979 (Table 4). The average genetic similarity within Hylocereus and Selenicereus species was 0.612 for Hylocereus species and 0.585 for Selenicereus species. A high genetic similarity index (0.952) was found between the two S. megalanthus, clone 88-023 from Ecuador and clone 90-001 from Colombia, studied. The dendrogram constructed according to UPGMA cluster analysis shows two majors clusters, one containing the Hylocereus species and the second containing the Selenicereus species (Fig. 2). The Hylocereus cluster was classified into four subgroups: one comprising H. ocamponis, the second comprising H. costaricensis, the third comprising H. purpusii, and the last comprising H. polyrhizus and H. undatus. In the Selenicereus cluster there are three subgroups: S. megalanthus clones, S. grandiflorus, and the remaining seven species. The results of the bootstrap analysis on the RAPD data indicate the branches with bootstrap values up to 56%. In line with the dendrogram, three clear groups were also distinguishable by PCO analysis (Fig. 3), with the five Hylocereus species in one cluster, the two S. megalanthus clones, and the rest of the Selenicereus species in the third cluster.

4. Discussion In this work we have used a reliable RAPD system for vine cacti. The RAPD patterns observed in the vine cacti may suggest extensive heterozygosity in this group. This suggestion is especially supported by the fact that for H. undatus a single primer generated enough polymorphic bands, thereby nine clones studied were distinguishable. RAPD analysis of highly heterozygous species is expected to underestimate genetic distance, since RAPD markers are dominant. Further analysis using co-dominant markers, such as restriction fragment length polymorphism (RFLP) or simple sequence repeats (SSRs), may be required to support this observation. Clones of S. megalanthus are indistinguishable visually because of the very small intraspecific morphological variation. The RAPD profiling technique proved useful in distinguishing between the studied S. megalanthus materials. The dendrogram as well the PCO results are in line with the current taxonomical classification of this species, found to be remotely related to the other species of the Selenicereus cluster. The RAPD results agree with our hypothesis regarding the Hylocereus × Selenicereus hybrid nature of this tetraploid species, also supported by earlier cytological observations (Lichtenzveig et al.,

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Fig. 2. Dendrogram constructed according to UPGMA cluster analysis, based on the similarity index of Nei and Li (1979), showing the genetic relationships among Hylocereus and Selenicereus species. Scale at the bottom is UPGMA coefficient of similarity. Values above branches indicate bootstrap confidence level of at least 59%, based on 1000 bootstrap resampling.

2000; Tel-Zur, 2001). Interestingly, our RADP results like the above cytological observations, are in agreement with the suggested intermediate taxonomic status (Britton and Rose, 1963). In our previous work we showed that controlled hybridization between species and genera of vine cacti is possible, resulting in fertile interspecific and intergenetic hybrids (Lichtenzveig et al., 2000; Tel-Zur, 2001; Tel-Zur et al., 2003). In the wild, the formation of spontaneous interspecific and intergeneric hybrids requires geographic overlapping of the different taxons. In view of the geographic proximity between the native habitats of vine cacti, interspecific and intergeneric hybridization may be expected. Indeed, a clone was recently collected from the wild in Mexico (Ortiz, 2000), whose morphology suggests an

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Fig. 3. A PCO-plot showing genetic relationships among Hylocereus and Selenicereus species based on RAPD data. Axis 1 and axis 2 described 25.5 and 16.7% of the total variance, respectively.

intergeneric Hylocereus × Selenicereus hybrid origin. It is worth mentioning that this clone looks very much like the synthetic triploid we obtained by controlled crossing of these two genera (Tel-Zur, 2001). There is a great deal of confusion regarding the taxonomy of vine cacti in the literature. For example, accessions of the yellow pitaya S. megalanthus were introduced to Israel as Hylocereus triangularis and Hylocereus undatus (Weiss et al., 1995; Mizrahi and Nerd, 1999). Cacioppo (1990) used the name Cereus triangularis Haw. for S. megalanthus, and Infante (1992) referred to this species as Mediocactus coccineus. We hope that the present study will contribute to the clarification of the taxonomy of this group in addition to providing a new molecular tool for future breeding. The virtually unlimited number of primers and the technical simplicity of the analysis make RAPD an attractive tool for germplasm fingerprinting, for evaluating the genetic diversity of wild populations, and for segregating populations derived from crosses between parents in different fruit and horticultural trials. With the appropriate genetic designs, this approach may facilitate the establishment of linkages between economically important traits and DNA markers. Such markers, in turn, may facilitate selection of outstanding vine cacti genotypes.

Acknowledgements The authors thank Mr. E. Naus and Mr. J. Mouyal from Ben-Gurion University of the Negev for their skilful technical assistance, Mr. P. Becht from University of Kaiserslautern, Germany, who worked with S. megalanthus clones and Ms. Dorot Imber for editing the

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manuscript. The study was partially supported by “UCLA-BGU Program of Academic Cooperation”.

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