Relationships among Accessions of Cucurbita pepo
Based on ISSR Analysis

N. Katzir, E. Leshzeshen, G. Tzuri, N. Reis, Y. Danin-Poleg, and H.S. Paris

Department of Vegetable Crops, Agricultural Research Organization, Newe Ya'ar Research Center,
P.O. Box 1021, Ramat Yishay 30-095, Israel

Additional index words. marker, cluster analysis, polymerase chain reaction, PCR

Abstract. Eighteen accessions of C. pepo were compared using an inter simple sequence repeat (ISSR) multilocus marker system. A total of 155 bands were used for analysis. A sample originating from a wild gourd population in Tamaulipas, Mexico was the most distinct (average distance = 0.33). Two major clusters were observed, the first containing samples from various accessions of C. pepo ssp. ovifera (a wild Texan and a pear gourd, an acorn, two scallops, a crookneck, and a straightneck squash) and the second containing samples from various accessions of C. pepo ssp. pepo (an orange gourd, four pumpkins, a cocozelle, two vegetable marrows, and two zucchini squash). Of the accessions tested, the two most similar were the zucchini cultivars (average distance = 0.03).

We thank A. Genizi of the Department of Statistics, Agricultural Research Organization, Bet Dagan for his advice concerning SAS, and Y. Tadmor of Newe Ya'ar for his comments concerning this work. We also thank the following individuals for supplying to us gratis seed samples used for this study: Tom Andres of Bronx, New York; H.D. Wilson of Texas A&M University, College Station; G.P. Silvestri of Centro Cooperativo di Sperimentazione Agraria, Fidenza, Italy; G. Santini of Società Agricola Italiana Sementi, Cesena Italy; and a traveler to the former Yugoslavia whose identity is unknown to us. Contribution no. 135/98 from the Agricultural Research Organization, Bet Dagan, Israel.


Cucurbita pepo L. is highly polymorphic for fruit characteristics, even by cucurbit standards. Duchesne (1786) was the first to identify the boundaries separating this species from the other forms of Cucurbita then familiar to European naturalists and botanists. He correctly concluded that the four Cucurbita species that had been named and described by Linné (1753, 1767) were in fact merely four very different forms of one highly polymorphic species. Furthermore, based on the 90 cultigens he grew and observed, he classified C. pepo into five races or extended families: the orange gourd and its relatives; the smooth, thick-rinded gourds; the warted gourds; pumpkins and their relatives; and scallop squash and their relatives.

There have been many attempts at intraspecific classification of this species since then. Any such classification would on one hand have to be practical and workable, and on the other hand

have to be reflective of genetic relationships. Two recently proposed classifications have been based on fruit shape (Paris, 1986) and allozyme variation and seed morphology (Decker, 1988). The former classification considers C. pepo as containing eight edible-fruited cultivar groups: cocozelle, pumpkin, vegetable marrow, zucchini, acorn, crookneck, scallop, and straightneck (the gourds are not included). The latter considers C. pepo as consisting of two subspecies, C. pepo ssp. pepo and C. pepo ssp. ovifera (Decker, 1988). The result of combining the two classifications is a synthesis considering the first four groups named above as belonging to C. pepo ssp. pepo and the latter four belonging to C. pepo ssp. ovifera. The races of orange gourds, warted gourds, and pumpkins of Duchesne (1786) belong in C. pepo ssp. pepo while the races of thick-rinded smooth gourds and scallop squash belong in C. pepo ssp. ovifera (Decker, 1988).

Cucurbita pepo has not been the subject of extensive DNA analysis. Wilson et al. (1992), in a cladistic analysis of chloroplast DNA restriction-site mutations among nine samples of C. pepo, observed that they were divided into two groups, nearly in accordance with the subspecific classification of Decker (1988). Torres Ruiz and Hemleben (1991), using RFLP analysis of fragments of rDNA in eight C. pepo cultivars, observed that the band


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ing patterns of cultivars within the same group were similar but usually differed among cultivars from different groups, much in accordance with the cultivar group classification of Paris (1986).

Inter simple sequence repeat (ISSR) is a multilocus marker system useful for fingerprinting, diversity analysis, and genome mapping (Zietkiewicz et al. 1994; Godwin et al. 1997; Danin-Poleg et al. 1998). The ISSR method involves polymerase chain reaction (PCR) amplification of DNA using a single primer composed of a microsatellite sequence that is anchored at the 3¢ or 5¢ end by two to four arbitrary, often degenerated nucleotides. The primer targets SSRs that are abundant throughout the genome and does not require prior knowledge of a DNA sequence. Amplification products can be separated either on a polyacrylamide gel or on an agarose gel.

Our objective was to obtain a better understanding of the genetic composition and relationships among accessions of C. pepo. We describe herein some of the problems inherent and difficulties we encountered in DNA analysis of this highly polymorphic species. Herewith we also present the results we obtained based on analysis of the DNA of 18 accessions of this species, using the ISSR method.

Materials and methods

The 18 accessions sampled, their affiliation as

to subspecies and cultivar group, and the sources of their seeds are presented in Table 1.

DNA was isolated from young leaf tissue of single plants using modifications of the minipreparation procedure described by Fulton et al. (1995). PCR amplification was performed using DNA of single plants or bulks of DNA from several plants for each accession.

ISSR analysis was performed using 20 primers of oligonucleotide set no. 9 obtained from the University of British Columbia, Nucleic AcidProtein Service Unit, Vancouver, B.C., Canada (primer nos. 808, 809, 810, 817, 827, 828, 840, 841, 842, 845, 846, 847, 848, 850, 851, 853, 855, 856, 857, and 858). PCR reaction mixtures for separation on agarose gel (nonradiolabelled) contained 30 ng of plant genomic DNA, 2 mm of Mg2+, 7.5 pmol of primer, 166 µm of each of the dNPTs, 1x Taq Buffer (Advanced Biotechnologies, U.K.), and 0.5 unit of Taq DNA polymerase (Advanced Biotechnologies), in a total volume of 25 µL. The amplification program was as follows: 7 min at 94 °C, 30 s at 94 °C, 45 s at 45 °C, and 2 min at 72 °C for 35 cycles on a thermocycler (PTC-100; MJ Research Inc.). PCR products produced were separated by electrophoresis in 1.2% agarose gel (Techcomp Ltd., Hong Kong) for 4 to 5 h and stained with ethidium bromide. The size marker used was PBR 322 Alw441/MVA1 (MBI Fermentas). PCR reaction mixtures for separation on sequencing gel (radio

Table 1. Sources of seeds of the Cucurbita pepo accessions studied.

Number Accession Subspecies Group Seed source

1 Ranger ovifera Crookneck Dessert, El Centro, Calif.

2 Golden Girl ovifera Straightneck Harris, Rochester, N.Y.

3 Golden Bush Scallop ovifera Scallop Dessert, El Centro, Calif.

4 Benning's Green Tint ovifera Scallop Northrup King, Minneapolis, Minn.

5 Table Queen ovifera Acorn Northrup King, Minneapolis, Minn.

6 Striped Pear ovifera (gourd) Stokes, St. Catherines, Ontario, Canada

7 Wild Texas ovifera (gourd) H.D. Wilson, Texas A&M, College Sta., Texas

8 Wild Mexico pepo (gourd) T.C. Andres, Bronx, N.Y.

9 Orange Ball pepo (gourd) Stokes, St. Catherines, Ontario, Canada

10 Yugoslavia 7 pepo Pumpkin (Unidentified traveller)

11 Small Sugar pepo Pumpkin Ledden, Sewell, N.J.

12 Connecticut Field pepo Pumpkin Ledden, Sewell, N.J.

13 Tondo di Nizza pepo Pumpkin Ingegnoli, Milan, Italy

14 Beirut pepo Veg. marrow Hazera', Haifa, Israel

15 Vegetable Spaghetti pepo Veg. marrow Sakata, Yokohama, Japan

16 Striato d'Italia pepo Cocozelle C.C.S.A., Fidenza & S.A.I.S., Cesena, Italy

17 Fordhook Zucchini pepo Zucchini Burpee, Warminster, Pa.

18 Black Zucchini pepo Zucchini Gurney, Yankton, S.D.

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Table 2. Number of bands obtained from each of the five ISSR primers used in data analysis of 18 accessions of Cucurbita pepo.

Primer No. bands Polymorphic Monomorphic

809 18 14 4

810 39 31 8

841 32 14 8

842 49 48 1

855 27 21 6

Total 155 128 27

single plants. In all but one case, the polymorphism detected within an accession was lower than the polymorphism detected between accessions. The exception was the cocozelle accession, 'Striato d'Italia', one of whose genotypes differed markedly from the others and may have been a contaminant in the seed stock.

Bulk DNA had been prepared in two ways: joint extraction from leaves collected from several

labelled) containing: 30 ng of plant genomic DNA, 1 mm of Mg2+, 7.5 pmol of primer, 277 µm of dATP, dTTP, dGTP, 3.3 µm of dCTP, 0.1 µL of 3000 Ci/mmol [a-33P] dCTP, 1¥ Taq Buffer (Advanced Biotechnologies), 1 unit of Taq DNA polymerase (Advanced Biotechnologies), in a total volume of 15 µL. The PCR conditions were the same as above but for 27 cycles. PCR products (3 µL/lane) were separated on a DNA sequencing gel containing 6% polyacrylamide, 8 m urea and 1¥ TBE, at 60 W constant power for 3.5 to 5.5 h. After drying, a Kodak MR film was exposed to the gels. Only those bands that were reproducibly present or absent were considered.

Cluster analysis and similarity procedures were obtained from Statistical Analysis Systems (SAS). The ISSR amplification for the 18 C. pepo accessions using five primers yielded 155 bands, 128 polymorphic and 27 monomorphic (Table 2). The amplification products were separated on a sequencing gel. The ISSR bands were treated as binary, absent or present (scored as 0 or 1, respectively). For the similarity analysis, variation among samples was evaluated through pairwise comparison by calculating two times the number of shared fragments divided by the total number of polymorphic bands in the profile. An index equivalent to that of Nei and Li (1979) was obtained.

Results and discussion

The 20 ISSR primers were applied to amplify DNA from the 18 C. pepo accessions. DNA representing each accession was pooled from several plants. To ensure that this bulk DNA represents well the accession, DNA of single plants from several accessions was amplified using five primers. Amplification patterns of the bulk DNA were compared with the amplification patterns of the

Figure 1. Inter-simple sequence repeat (ISSR) polymerase chain reaction (PCR) polymorphism using primer no. 842 obtained (A) on an agarose gel and (B) on a sequencing gel. M = size marker PBR322 Alw441/MVA1.

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plants and separated extractions from single plants that were pooled, using equal quantities of DNA. We observed that a better representation of the different plants was achieved using the latter method.

Amplification products were first separated on an agarose gel (Figure 1a). Of the 20 primers used, 13 (65%) yielded polymorphic patterns among the 18 accessions when separated on agarose gels. Four (20%) were not polymorphic while the remaining three failed to amplify a clear product. An average of 10 amplification products per primer for each accession were separated on an agarose gel. Polymorphic patterns included one to five clearly polymorphic products.

Amplification products of five primers that had yielded polymorphic patterns on agarose gel were also tested on sequencing gels (Figure 1b).

The resolution of the sequencing gel was three times greater than that of the agarose gel. An average of 30 clear reproducible amplification products per primer for each genotype were detected on a sequencing gel with a minimum of 10 polymorphic products (Table 2). Clear amplification products that appeared in at least two PCR experiments were scored. A total of 155 bands were observed, of which 128 were polymorphic and 27 monomorphic. Cluster analysis was conducted using SAS, producing the dendogram depicted in Figure 2.

In the dendogram (Figure 2), 'Wild Mexico', collected in the wild in Tamaulipas, northeastern Mexico by Andres (1987), is the most distinct

Figure 2. Dendogram obtained for 18 accessions of Cucurbita pepo. Numbers 1­18 are in accordance with the accession numbers listed in Table 1.

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(average distance = 0.33). Also observable is a dichotomy, with the 'Wild Texas' and 'Pear' gourds, acorn, scallop, crookneck and straightneck squash in one cluster and the 'Orange Ball' gourd, pumpkins, cocozelle, vegetable marrow, and zucchini squash in the other cluster. This result is very much in accordance with the subspecific classification of Decker (1988), one cluster representing C. pepo ssp. ovifera and the other C. pepo ssp. pepo. The two most similar accessions were the two zucchini cultivars, average distance = 0.03.

Our results are based on only eighteen accessions and five ISSR primers of this highly polymorphic species. Considerably more accessions and more primers will have to be employed in order to allow for a clearer understanding of the relationships within the respective subspecies. Comparison of results obtained from different clustering methods is warranted.

Our findings indicate that DNA analysis using ISSR markers can serve as an efficient, reliable tool in this endeavor. The study presented here suggests as well that where multilocus markers are advantageous, ISSR is a potentially useful technique for postcontrol and legal protection (Danin-Poleg et al., 1998).

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Danin-Poleg, Y., G. Tzuri, N. Reis, and N. Katzir. 1998. Application of inter-SSR markers in melon (Cucumis melo L.). Cucurbit Genet. Coop. Rpt. 21:25­28.

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Duchesne, A.N. 1786. Courge, Cucurbita. In: J.B.P.A. de M. de Lamarck (ed.). Encyclopédie Méthodique Botanique 2:148­159.

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Nei, M. and W.-H. Li. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA 76:5269­5273.

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