1. Introduction

The family Cucurbitaceae belongs to the order Cucurbitales, class Magnoliopsida (subclass Rosidae). Although most of its species originated in the Old World[1], many originated in the New World, and at least seven genera in both hemispheres[2]. There is tremendous genetic diversity within the family in tropical, subtropical, arid deserts and temperate regions. Archaeological evidence indicated that cucurbits were present in ancient and prehistoric cultures.

Molecular markers can be an effective mean to determine genetic relatedness among cultivars and strains used in cucurbits breeding programmes. A number of studies have been designed to examine genetic diversity and phylogenetic relationships among cucurbit cultivars. Some of these studies used biochemical methods such as isoenzyme[3], while others used molecular approaches based on DNA[4] to evaluate genetic diversity in crop species. Most of the isoenzymes tested produced monomorphic patterns ([5,6].

Several methods are now available concerning molecular markers studies, and scientists can choose the method of particular interest, depending on available material andobjectives. Among the existing molecular approaches, random amplified polymorphic DNA (RAPD) is one of the most widely used molecular methods in genetic studies and has been applied for the less-known species. However, this method is less reproducible and shows a lower degree of variability as compared with to other methods. RAPD procedure provides a sufficient number of informative markers that could distinguish watermelon cultivars[7, 8]. In a study[9], genetic diversity and relatedness were examined among Citrullus lanatus var. lanatus, C. lanatus var. citroides, and C. colocynthis using RAPD analysis. RAPD markers were also used in the construction of an initial genetic linkage map for watermelon (Citrullus lanatus)[10], and to determine genetic relatedness among Asian watermelon cultivars and breeding lines[11]. Reference[12] used RAPD to study polymorphism and to construct a linkage map for watermelon (Citrullus lanatus (Thunb.) Matsum and Nakai by using 148 primers. Amnon et al.[13] used RAPD markers to study genetic diversity among accessions of some American cultivars of watermelon (Citrullus lanatus var. lanatus). The objectives of this study was studied the genetic diversity among some Cucurbits and the utility of the RAPD markers system for evaluating similarity.

2. Materials and Methods

Plant material: Accessions of ten cucurbit species were obtained from various sources (Table 1). For DNA extraction the plants were grown in pots.

RAPD analysis: For RAPD analysis DNA was extracted from cucurbits leaves according to[14]. Purity and concentration of DNA was tested spectrophotometrically at wave lengths of 260 and 280 nm (Eppendorf – Germany). RAPD reactions were performed using 13 random primers (Nine of these primers were 10-mers (Operon Technologies; OP, and University of British Columbia, UBC), while the other 4 were 12-mers (Bexnet, Co., Japan.) according to[15] using PCR thermocycler. Amplification was performed using touchdown annealing temperature along two or three steps, starting with the melting temperature of the primer and then reducing the temperature by 3 degrees for each step. After all cycles had been completed, 7 µl of the products were mixed with 1 µl of DNA loading buffer and loaded into 2% agarose gel. The agarose gel was then run in 1x TAE buffer at 100 V for one and a half hour. The gels were soaked in 3% ethidium bromide for 30- 60 minutes, and then placed on the UV- transilluminator for band visualization.

Cluster analysis: The RAPD fragments 200 to 1500 base pairs (bp) were visually scored as present (1) or absent (0). A dendrogram was constructed based on similarity matrix data by applying the UPGMA[16] and cluster analysis using the software package NTSYS – PC version 2[17].

3. Results and Discussion

The DNA concentration of the 10 accessions ranged between 80.8 and 352.1 µg/ml, and purity ranged from 1.7 to 2.3 (Table 2). The analysis of the 10 accessions with 13 RAPD primers produced a total of 227 reproducible bands (Table 3). Among these, 225 bands were polymorphic (99.1%) and ranged in size from 200 to 1500 bp. Heikal,et al.[18]reported that the total number of reproducible fragments among the fourteen genotypes of Cucurbita species by six primers reached 463 bands out of them 405 (87.5%) were polymorphic fragments. They found the levels of polymorphism among the fourteen genotypes of Cucurbita species are relatively high (69.8% - 97.0%), indicating the wide gene pool existence in the fourteen genotypes. The total number of bands per primer ranged between 9 and 24, with an average of 17.5 bands per primer (Table 3). The number of polymorphic bands per primer varied from 9 to 24, with an average of 17.3 bands (Table 3). The number of polymorphic bands produced per primer was relatively high among the species (Figs 1 and 2). This high level of RAPD markers polymorphism is in accordance with the results of[19] and[20] who reported that Cucurbita pepo and Cucurbita maxima are highly polymorphic species. However, DNA polymorphism has been reported as moderate to high in other allogamous genera such as Brassica, Allium, Asparagus and Cucumis.

Reference[21] found high level of the polymorphism among the different Cucurbita species (97.44%). However, in case of Citrullus lanatus,[22] reported an average of polymorphic bands produced per primer was 21.2.

To estimate genetic diversity among the accessions, genetic similarity coefficients were calculated, and a dendrogram was constructed (Fig 3). The range of DNA similarity in the dendrogram was between 0.15 and 0.62. Reference[23] reported a range 0.13 - 0.41 of dissimilarity in 31 landraces of Cucurbita moschata using 31 random primers. The wide range of dissimilarity values suggests that germplasm collection represent a genetically diverse population.The dendrogram consisted of three major clusters. The first cluster consisted of one group (Group І) which comprises the species: Cucurbita moschata and C. pepo which both have a DNA of a genetic similarity of 38.6 %The second cluster consisted of four groups as follows: Group І comprises the species Luffa aegyptiaca which has a genetic similarity of 20.6 %. Group II comprises the closely related species Cucumis melo var. reticullatus and C. melo var. flexuous with a genetic similarity of 62 %. Group III consists of Cucumis sativus. This group is similar to group II in their genetic similarity (37.8 %). Group IV consists of Ctenolepis cerasiformis with a genetic similarity of 26.6 %.

The third cluster consisted of two groups. The first group comprises the species Citrullus lanatus and Colocynthis vulgaris which had a genetic similarity of 36.2 %. The second group consists of Coccinia grandis which has a genetic similarity of 19.4 %. Reference[24] analysed accessions of Cucurbita moschata with 11 SRAP primers and reported a total of 148 reproducible bands. Among these, 98(66.2%) were polymorphic and ranged in size from 140 to 950 bp. The total number of bands per primer ranged between 9 and 21, with an average of 13.5 bands per primer. They also analysed accessions of the same plant with 6 AFLP primers and identified a total of 156 reproducible bands which ranged in size from 60 to 380 bp. Of these, 134 (85.9 %) were polymorphic. They found a range of 14 to 41 amplified bands per primer, with an average of 26 bands.

Reference[25] analysed accessions of three African edible-seeded cucurbits (Citrullus lanatus L. Matsumra & Nakai, Cucumeropsis mannii L. Naudin and Cucumis melo var. agrestis L. Naudin.), with 11 ISSR primers on DNA extracted from an accession of the three species and observed 66 bands with 4 to 11 bands per primer.

Reference[13] analysed accessions of Citrullus lanatus var. lanatus using 138 RAPD primers that were initially screened among some cultivars of watermelon. Of the 138 RAPD primers, only 35 primers produced polymorphic RAPD patterns. Of the later, 25 primers produced 288 reproducible RAPD bands that ranged in size from 100 to 3000 bp.

Reference[13] also analysed accessions of Citrullus lanatus and C. colocynthis with 30 RAPD primers that were used for genetic diversity among the two species. The primers produced 662 bands that could be rated with high confidence and ranged in size from 100 to 2650 bp. The number of bands produced by each primer in the above study was relatively high (an average of 22 bands per primer).

Reference[26] analysed molecular variation in accessions of Cucumis melo L. and C. metuliferus by 18 RAPD primers and found a total of 176 bands. The number of polymorphic bands was 75 in Cucumis metuliferus and 96 in Cucumis melo. In general, the cluster analysis grouped the accessions according to many morphological traits and showed a clear separation between the species. The RAPD marker revealed considerable genetic diversity among the species, a finding which strongly agrees with the great morphological variability observed among the cucurbits in this study. Similar results were observed for the oriental Cucurbita moschata using RAPD marker[27]. RAPD marker systems proved to be useful for analyzing the genetic diversity of some genera of the family Cucurbitaceae. The knowledge of the diversity of this germplasm will facilitate its use in breeding programmes and the improvement of management of large collections of the species of this family.

Table 1. Plant accessions of cucurbits used in the study No. Species Distribution and Description 1 Coccinia grandis Wild. Nile banks, Khartoum State 2 Colocynthis vulgaris Wild. Nile banks, wadis and valleys in Khartoum State. Fruit edible by camels; the pulp is extracted and used in medicine. 3 Ctenolepis cerasiformis Wild. Nile banks, Khartoum State 4 Cucumis melo var.flexuous Widespread. Cultivated, Faculty of Agric, Dept of Horticulture. Fruits edible as salad or pickled 5 Cucumis sativus Widespread. Cultivated, Faculty of Agric, Dept of Horticulture. Fruits edible as salad or pickled 6 Cucumis melo var. reticullatus Widespread. Cultivated, Faculty of Agric, Dept of Horticulture. The fresh flesh of the fruit is eaten as dessert. 7 Luffa aegyptiaca Wild. Widespread in Khartoum state.Used as bath sponges and for cleaning purposes. 8 Cucurbita pepo Widespread. Cultivated, Faculty of Agric, Dept of Horticulture. The immature fruits are eaten as a fresh vegetable; mature fruits used for baking, making jam and pies or forage for livestock. 9 Citrullus lanatus Cultivated. Widespread in Khartoum State. Edible. Dried parched seeds are chewed. 10 Cucurbita moschata Widespread. Cultivated, Faculty of Agric, Dept. of Horticulture. The immature fruits are eaten as a fresh vegetable; mature fruits used for baking, making jam and pies or forage for livestock.

Table 2. DNA concentration and purity of cucurbits accessions No. Species Concentration (µg/ml) Purity 1 Coccinia grandis 80.8 1.9 2 Colocynthis vulgaris 154.6 2.3 3 Ctenolepis cerasiformis 193.0 1.7 4 Cucumis melo var. flexuous 186.0 1.8 5 Cucumis sativus 352.1 1.8 6 Cucumis melo var. reticullatus 228.0 1.8 7 Luffa aegyptiaca 201.2 1.8 8 Cucurbita pepo 294.3 2.1 9 Citrullus lanatus 274.8 1.8 10 Cucurbita moschata 152.1 1.7

Table 3. Number of total and polymorphic fragments using RAPD marker. Primer names are according to manufacturer’s identification system (Operon technology;OP, and University of British Columbia, UBC) No. Primer Sgquence(5 – 3) Temperature(℃) Total number of bands Number of polymorphic bands Percentage of polymorphism 1 A0 1 AGC AGC GCC TCA 42.4 16 15 93.8 2 A02 GCC AGC TGT ACG 42.4 18 18 100 3 A0 3 TGC CTC GCA CCA 42.4 17 17 100 4 A04 GCC CCG TTA GCA 42.4 23 23 100 5 OPB 05 TGCGCCCTTC 33.0 22 22 100 6 UBC106 CGTCTGCCCG 37.1 17 17 100 7 UBC115 TTCCGCGGGC 37.1 14 14 100 8 UBC152 CGCACCGCAC 37.1 24 24 100 9 UBc155 CTGGCGGCTG 37.1 24 24 100 10 UBC157 CGTGGGCAGG 37.1 17 16 94.1 11 UBC199 GCTCCCCCAC 37.1 16 16 100 12 UBC222 AAGCCTCCCC 33.0 10 10 100 13 UBC228 GCTGGGCCGA 37.1 9 9 100 Total 227 225 99.1 Average 17.5 17.3 98.9

Figure 1. Agarsoe gel showing polymerase chain reaction (PCR) products of 10 accessions of Cucurbits (a)using UBC157 primer (left) and UBC155 primer (right) and (b) using UBC 222 primer (left) and UBC OPB 05 primer (right)

Figure 2. Agarsoe gel showing polymerase chain reaction (PCR) products of 10 accessions of Cucurbits (a) using A0 2 primer (left) and A01 primer (right) and (b) using A0 4 primer (left) and A0 3 primer (right)

Figure 3. Dendrogram analysis of 10 species of the family Cucurbitaceae