Abstract: Cytological and molecular cytogenetic studies were conducted on the two accessions of Cucumeropsis mannii Naudin to establish the true somatic chromosome count, investigate the occurrence and number of ribosomal RNA genes in its genome as well as investigate any cytological and cytogenetic variation in the two cultivars of the monospecific taxon. Chromosomal counts from squashed root tips stained with FLP orcein and DAPI stained mitotic chromosomes showed a somatic count 2n = 24 for this species. The chromosomes were small in size measuring 3-4.5 μm long and mostly varied from submetacentric to subacrocentric in shape. Both 18S-5.8S-25S and 5S rRNA genes were observed in the genome. Four sites, comprising two major and two minor, of 18S-5.8S-25S rRNA genes were present on four of the largest chromosomes (4.5 μm long) while two sites of the 5S rRNA genes were observed on two other individuals of the largest chromosomes. This report establishes the correct somatic chromosome count as well as the ribosomal rRNA features of this taxon. It also shows no cytological variation between the two cultivars of this species.
INTRODUCTION
Cucumeropsis mannii Naudin (Annales des Sciences Naturelles; Botanique, Sér. 5,5: 1866; Synonym: Cucumeropsis edulis (Hooker fil.) Cogn., Basionym: Cladosicyos edulis Hook. f.) is a monoecious and monospecific taxon in the Cucurbitaceae (Order: Violales and Subclass: Dillenidae). It originates and is endemic to West and Central Africa (Okoli, 1984). Being partially drought-resistant, cultivation of C. mannii is mostly around the tropical rain forest regions of West and East Africa. The plant is flagelliflorous and is often cultivated alongside Dioscorea spp. (yam), Abelmoschus esculentus (L.) Moench. (okro) and Telfairia occidentalis Hooker fil. (fluted pumpkin). The seeds are usually planted close to small trees, shrubs, fences or other similar support, which act as stakes for it.
In regions where it occurs, C. mannii is of great economic importance (Zoro Bi et al., 2003; Andres, 2004). It serves mainly as food; the most regularly used part of it being the seed. The seeds (milky in colour) can be eaten roasted. Otherwise, the testa can be removed and the oily white proteinous cotyledons are gathered, pulverized and used as a native soup thickener. This species is nutritious (Okigbo, 1975) and also medicinally important (Gill, 1992; Sofowora, 1986). The cultivar, which has oblong-rounded fruit with more robust seeds, appears more favoured as a delicacy than the one with more cylindrical fruit with slimmer seeds. Rodents more readily savour the fruit and seeds of the oblong-rounded fruit type than the cylindrical type. The fruits and seeds are boiled and eaten along with yam (Dioscorea spp.) while decoction of the leaves are used as mild laxative and contains steroids, tryptophans, vitamin C and carbohydrates (Gbile, 1986).
Though very important, this domesticated species has attracted only little attention in research and crop improvement. Hence little is known about its scientific prospects. For example, not much is known about the cytology and cytogenetics of this taxon. Though cytological work in the Cucurbitaceae is reportedly difficult (Whitaker, 1974; Jeffrey, 1980), a count, 2n = 2x = 22, was reported for C. mannii. King (1974) opined that the cell chemistry of Cucurbitaceae is different from other plant taxa. Poor stainability and small chromosome size combined with the absence of a standard nomenclature of genes did not encourage cytogenetic investigations in the Cucurbitaceae (Roy et al., 1991). However, occurrence of fairly large chromosomes has been reported for some species, in particular Cucumis spp., which is amenable to detailed karyotypic investigations (Ramchandran et al., 1983). But the chromosomes in most genera are relatively small (Okoli, 1984).
Beyond routine chromosomal characterization, molecular characterization of plant and animal species has contributed much desirable information required for their characterization, exploitation, conservation and improvement. Hence, occurrence and distribution of ribosomal RNA genes (i.e., rDNA) has been investigated and reported in many species. The ribosomal RNA gene has been widely investigated in animals, including the Diptera, Glossina (i.e., tsetse fly, Willhoeft, 1997) and a wide variety of wild and cultivated plants. The number of sites, pattern of distribution, strength of signal and other attributes of rDNA has been used to characterize, classify and discriminate between species as well as show taxonomic relationships and to explain phylogeny of hybrids, cultivars, species and genera (Osuji et al., 1998). To date, rDNA studies have not been conducted in the Cucurbitaceae.
This species has been quite neglected and currently faces the threat of extinction. Considering its ethnobotanic value and genetic endowments, it is important to conduct proper genomic characterization of the C. mannii germplasm. Data so-generated would help to improve its utility and enhance exploitation of its genepool as a ready source of novel genes for both intra-and interspecific improvement of related economically viable species. Hence, the aim of this study was to establish the correct chromosome number and to investigate the occurrence and number of sites of the two major ribosomal RNA genes in this species.
MATERIALS AND METHODS
The plant materials used for this research were gathered from farms in different parts of south eastern Nigeria. The accessions were established in the Botanic garden at the Regional Center for Bioresources and Biotechnological Research, University of Port Harcourt, where they were maintained for over ten years. Healthy roots, about 10 mm long were generated by plating seeds, previously sterilized with 30% chloral solution, on wet filter paper in a dark cabinet and by germinating seeds in vivo.
| Fig. 1: | Mature fruits of C. mannii showing structural variation
between the fruits and seeds of the two cultivars. One cultivar has oblong-round
fruits with slightly larger seeds (a) while the other has cylindrical fruit
with longish and narrow seeds (b) |
Cytological studies: Mature fruits were obtained from plants representing the two varieties of C. mannii. Fresh viable seeds were extracted from mature fruits (Fig. 1), air-dried for two weeks and germinated to generate fresh healthy roots. Root tips were pretreated in 2 mM 8-hydroxyquinoline for 3 h and fixed in 3:1 v/v ethanol acetic acid for 24 h. Root tips were then stored in 70% ethanol prior to acid hydrolysis with 5% (2 min) and 9% (3 min) HCl followed by squashing in FLP orcein (Osuji, 2003). Mitotic slides were observed under Leitz Diaplan microscope.
Sites of ribosomal RNA genes: The method used for chromosome spreading followed Osuji et al. (1997, 1998). Fresh primary roots were harvested from chloral-sterilized seeds germinated by plating on moist filter papers. The roots were pretreated in 2 mM 8-hydroxyquinoline for 3 h and fixed in 3:1 ethanol acetic acid for 12 h. Fixed root tips were stored in 70% ethanol prior to hydrolysis. Roots were washed thrice in 1xenzyme buffer (0.01 M citric acid-sodium citrate, pH 4.6) for 9 min with shaking to remove fixative and preservative. Tips measuring about 4-5 mm were excised and hydrolyzed in 1.5 mL enzyme mix (2% cellulose, Onuzuka R10, Yakult Honsha Co., Tokyo and 20% liquid pectinase, from Aspergillus niger, Sigma) in enzyme buffer (Osuji et al., 1997; Schwarzacher et al., 1989) for 1 h at 37°C.
Digested root tips were washed thrice with 1 x enzyme buffer and squashed in a drop of 60% glacial acetic acid under No. 1 (18x18 mm) coverslip. About 1 mm of the actual tip of the root was squashed in a drop of 60% glacial acetic acid. The slides were flattened out each with a firm thumb pressure. The coverslip was removed after freezing on dry ice and the slides were air-dried.
On each slide, the area containing the chromosome spread was treated with 100 μL of 100 μg mL-1 DNase-free RNase in 2xSSC (20xSSC, 3 M sodium chloride plus 0.3 M trisodium citrate) solution. The slide was covered with a plastic coverslip and incubated at 37°C for 1 h. Slides were washed thrice, each for 5 min, in 2xSSC (on a shaker) and treated with 10 μg mL-1 pepsin, covered with a plastic coverslip, incubated at 37°C for 30 min and washed for 5 min in 2xSSC (shaking). Washed slides were pre-fixed with 4% paraformaldehyde twice each for 5 min, washed twice in 2xSSC (for 5 min each), dehydrated in 70% alcohol for 5 min and air-dried.
One of the two probes used was pTa71, isolated from Triticum aestivum (Gerlach and Bedbrook, 1979). It has a 9 kb EcoRI fragment containing 18S-5.8S-25S rRNA genes and intergenic spacer segments. The other probe was pTa 794 with a complete 410 bp 5S rRNA gene unit and spacer region also isolated from T. aestivum (Gerlach and Dyer, 1980). The probe pTa71 was labeled with biotin-11-dUTP (Boehringer Mannheim) while pTa794 was labeled with digoxigenin-11-dUTP (Boehringer Mannheim). The hybridization mixture comprised 20-30 ng of probe DNA (0.5 μL pTa71 and 1 μL pTa794), 15 μL 100% formamide, 6 μL 50% dextran sulphate, 10% sodium dodecyl sulphate, in water), 2 μg salmon sperm DNA, 3 μL 20xSSC in a total volume of 30 μL probe mixture per slide. Denaturation of the mixture was at 70°C for 10 min in a water bath and cooling was for 5 min in ice. Chromosome preparations were covered with the hybridization mixture and then plastic coverslips and denatured at 80°C for 5 min in an Omnislide humid chamber. The preparations were then incubated at 37°C for 12 h.
Preparations were washed after hybridization (for 5 min each time) twice with 2xSSC, twice with 20% formamide in 0.1xSSC (very stringent wash to allow sequences more than 85-90% homology to remain hybridized) and twice in 2xSSC. All the washing was at 42°C on a shaker. The final washing with 2xSSC was also twice, each for 5 min. This was followed by washing with 4xSSC/0.02% Tween 20 (detection buffer) on a shaker at room temperature.
In order to detect hybridization sites, preparations were first blocked by incubating with 100 μL per slide of 5% w/v bovine serum albumen (BSA) in 4xSSC/0.02% Tween 20, covered with plastic coverslips at room temperature for 5 min. Hybridization sites were detected by adding 100 μL of detection mixture per slide, which contained Cy3-streptavidin and sheep anti-digoxigenin conjugated to fluorescein isothiocyanate (anti-dig-FITC) in BSA and incubated for 1 h at 37°C. The chromosome preparations were washed twice, each for 5 min, in 4xSSC/Tween 20 and counterstained with 6 μg mL-1 4’-6-diamidino-2-phenylindole (DAPI) in McIlvaine’s citrate buffer (pH 7.0) solution under plastic cover slips for 10 min. Counterstained preparations were quickly washed in 4xSSC/Tween 20 and mounted in Antifade solution (Citifluor Glycerol/PBS solution AF1) for observation under Leitz epifluorescence UV microscope with Leitz filter sets (A for DAPI, 12/3 for Cy3, N2 for FITC and Omega Triple Bandpass). Photomicrographs were taken from good slides.
RESULTS
It was difficult to obtain good slides with clearly stained chromosomes from conventionally made cytological slides stained with FLP orcein. Low number of metaphase yield was observed in most of the slides. Instead, late prophase cells with uncondensed ends occurred more frequently (Fig. 2). The chromosomes were relatively small in size measuring about 3-4.5 μm long. A diploid chromosome number of 2n = 24 was counted for both cultivars of this taxon (Fig. 2a and b). There was slight cytoplasmic staining of mitotic tissue, which was deeper when lower strength of HCl was used to hydrolyze the root tips or when hydrolysis was abridged. Figure 2a shows non-uniform condensation of mitotic chromosomes. Spiralisation of the chromosomes tended to start from the centromere and extends to the telomeres of the chromosomes. A majority of the chromosomes were submetacentric and subacrocentric in structure. There was no observable cytological difference between the two cultivars.
The 18S-5.8S-25S rRNA gene: In situ hybridization showed the presence of 18S-5.8S-25S rRNA genes. Four chromosomes carrying signals representing sites of this gene were observed. The four signals comprised two major and two minor sites of the ribosomal gene. The chromosomes, which carried the copies of this gene, were all subacrocentric and were among the largest, measuring about 4-4.5 μm long. The sites were observable in both the condensing and condensed mitotic chromosomes as well as in the interphase nuclei (Fig. 3a-h). Signals on two of the sites on one pair of homologous chromosomes (Fig. 3a and g) were quite strong while the other two sites on another pair of homologous chromosomes were slightly less vivid. There was no difference in the number of this large ribosomal gene between the two cultivars.
| Fig. 2: | Mitotic mid and late prophase chromosomes of C. mannii showing small chromosomes and non-uniform chromatin condensation; a) mid
prophase and b) late prophase nuclei. Scale bar represents 5 μm |
| Fig. 3: | In situ hybridization sites of 5S and 18S-5.8S-25S
rDNA sequences on mitotic chromosomes of the two cultivars of C. mannii;
a) DAPI image of mitotic chromosomes of the cultivar with oblong-round fruit;
b) mitotic chromosomes showing two sites of 5S rDNA (arrows) and c) four
sites of 18S-5.8S-25S rDNA; d) transparent overlay of c on a; e) DAPI stained
chromosomes of the cultivar with cylindrical fruit; f) two sites of the
5S rDNA and g) four sites of the 18S-5.8S-25S rDNA comprising two major
and two minor (arrows) sites; h) transparent overlay of gone. Scale bar
represents 5 μm |
The 5S rRNA gene: Two homologous chromosomes had sites of the 5S rRNA gene (Fig. 3a-h). The chromosomes were mostly submetacentric and measured about 4 μm long. The sites were subterminal on the two chromosomes. The strength of the signals was considerably obvious though within the lower limits of sensitivity of detection. There was no double site in a location and no two sites on the same chromosome. No site of this gene was noticed on a chromosome carrying the larger 18S-2.5S-25S rRNA gene.
DISCUSSION
One major problem with cucurbits, especially C. mannii is the tendency of dividing cells to relapse to interphase if disturbed during harvest of root tips for chromosomal investigations. This is apparently the cause of the regular low yield of metaphase cells in this species and other cucurbits. The problem of small chromosome size and non amenability to cytological treatments (Whitaker, 1974; Jeffrey, 1980; Okoli, 1984) in the cucurbit family has constituted a serious obstacle to karyological and genomic understanding and characterization of members of the family. The relatively small size of the chromosomes compared with those of the Brassicaceae (Maluszynska and Heslop-Harrison, 1993), Araceae (personal observation), Poaceae (Leitch and Heslop-Harrison, 1993) constitutes a major handicap to its cytology. These problems may have contributed to the incorrect count of 2n = 22 reported by Jeffrey (1980). The authentic chromosome count of 2n = 24 established in this work for both cultivars is therefore the correct somatic (root tip) chromosome number for C. mannii. The chromosomes were about similar size range with chromosomes of Musa spp. though slightly larger (Osuji et al., 1998), the only variation being that the difference in length between the largest and smallest chromosomes is less in C. mannii.
The occurrence of rDNA in the genome of C. mannii shows that these sequences are ubiquitous among the angiosperms. The 18S-5.8S-25S rDNA is often associated with chromosomes which have secondary constrictions and attendant nucleolar organizers (Osuji et al., 1998). Hence occurrence of four sites of 18S-5.8S-25S rDNA indicates the presence of four chromosomes with secondary constrictions and nucleolar organizers. The large signal sizes at the sites of this gene indicate a large number of tandem insertions (i.e., multiple copies) of the individual gene at the fluorescent sites. The variation in signal strength of the 5S rRNA gene is an indication that the number of copies per site and their degree of expression may differ in the same genome.
The number of sites of the 18S-5.8S-25S rDNA does not have direct obvious relationship with ploidy or with the genome size. For instance, rye, with 2n = 14 has only a single pair of sites while the related barley, which also has 2n = 14, has six pairs of sites comprising two major and two minor sites (Leitch and Heslop-Harrison, 1993). Diploid Brassica species have variable number of 18S-5.8S-25S rDNA sites (Maluszynska and Heslop-Harrison, 1993). In the Fabaceae, Vigna unguiculata, 2n = 2x = 22, had four major and one minor (Galasso et al., 1995) while Vicia faba, 2n = 2x = 12, has only one major pair (Pearce et al., 1996) of sites of 18S-5.8S-25S rDNA. But in Musa, the number of sites of the 18S-5.8S-25S rDNA reflects ploidy while the number of sites of the 5 S rDNA varies within cultivars at similar ploidy level (Osuji et al., 1998). In Arabidopsis thaliana, sequences coding for 5S rDNA have been mapped to the short arm of chromosome 4 (Murata, Heslop-Harrison and Motoyoshi, 1997) while sequences coding for 18S-5.8S-25S were not detected on this chromosome (Thompson et al., 1996a,b).
In the tropics and subtropics, curcurbits constitute a major source of fruits and vegetables for healthy dieting. Whereas some species have evolved as major crops, some others are rather diminishing in importance and relevance owing to poverty and other economic factors. Cucumeropsis mannii is one of the cucurbits that have been selected against due to their inability to compete favourably with rival species and cultivars. For instance, Citrullus lanatus (commonly called egusi) is an alternative crop as an item for soup thickening but obviously yields more fruits and seeds than C. mannii. Cucurbita moschata (pumpkin) is eaten as porridge with yam in just the same way C. manni is eaten with yam. But C. moschata yields edible fruits, seeds and leaves, which altogether make it more favoured. For these reasons, a combination of C. lanatus and C. moschata out compete C. Mannii. Hence the very existence of C. mannii is threatened despite its food and medicinal values. This crop species might get extinct in the next ten years if nothing is done to conserve, characterize and exploit the benefits of its gene pool. The species could serve as a source of novel gene(s) for future plant improvement programme(s).