Hibiscus rosa-sinensis is an attractive ornamental shrub with large, brilliantly colored flowers, but it is unable to withstand the sporadically low temperatures of the mild winters of central California. H. syriacus is cold hardy but has smaller flowers with modest color intensity. Since the two species are cross incompatible, development of somatic hybrids combining the attractive flower characteristic with the cold tolerance is in progress. To provide a screening tool for callus derived from the somatic hybridization, RAPD banding patterns for the two Hibiscus species were developed. Genomic DNA extracted from callus of both species, and 40 arbitrarily selected 10-base-long primers were used to generate RAPD products in 45 amplification cycles. Several primers established reproducible RAPD markers characteristic of one or the other Hibiscus species investigated. The clearest polymorphic bands were obtained for OPD-2, OPD-4, OPD-16, OPF-1, OPF-3 and OPF-14 primers. It is expected that the markers will be suitable for identification of callus combining genomic DNA from both species hybridized.
Hibiscus syriacus L., commonly known as rose of Sharon or shrub althea, has been popularly cultivated in the north and south since colonial times (Photo 1). It can endure extreme heat and cold, poor soil environment, and its flowers are modest in size and color. H. rosa-sinensis, the tropical hibiscus, has glossy heavy foliage with large, brilliant and spectacular flowers but is not cold hardy (Photos 2,3). In California, H. rosa-sinensis is more suitable to coastal locations whereas in central California, H. rosa-sinensis suffers frost damage if not protected even during mild winters (Photo 4).
Crossing of the two species is not feasible; therefore, combining the attractive flower characteristic with cold hardiness through somatic hybridization of protoplast culture was undertaken.
Random amplified polymorphic DNA can provide simple and reproducible fingerprints of germplasm by employing single, arbitrary chosen primers (Welsh et al., 1990). RAPD markers can detect a large number of genetic polymorphism and when linked to major genes can be potentially useful in identifying morphological traits (Williams et at., 1990). They can also be used in monitoring diversity within plant populations (Dawson et al., 1993, Hu and Quiros 1991), for constructing linkage maps and for tracking hybrid species' origins (Crawford et al., 1993).
Some findings suggest caution in making conclusions regarding genetic relationships of cultivars or selections within a species (Levi and Rowland, 1997) and question the reproducibility of RAPD markers. However, in petunia (Petunia hybrida Vilm) and cyclamen (Cyclamen persicum Mill.), RAPDs were used successfully to test genetic purity of selected cultivars (Jianhua et al., 1997). Reliable and reproducible RAPD assays were also reported for cucumber (Cucumis sativus L., Staub et al., 1996) and for rose cultivar fingerprinting, where by the use of eight primers, five cultivars were distinguished (Torres et al., 1993).
In floricultural crops, morphological characteristics such as flower shape, size and color were used to discriminate cultivars. Often, long periods of vegetative growth elapse before such evaluation can take place. For example, genetic purity assessment of cyclamen is possible eight months after planting whereas RAPD technique provided a useful test of genetic purity which can be completed within 24 hours.
The objective of this study was to develop polymorphic RAPD markers that may help in distinguishing H. rosa-sinensis cv. 'Bhlliant Red' from H. syriacus cv. 'Aphrodite' in the callus stage or juvenile plant to avoid the expense of growing the plants from regeneration to full maturity for phenotype determination.
MATERIALS AND METHODS
Plant Material and DNA Extraction
Callus was derived from peduncles of H.rosa-sinensis 'Brilliant Red' and H. syriacus 'Aphrodite'; both shrubs were grown at the Plant Science Department nursery of California State University, Fresno. DNA was extracted from 6-8 month-old callus using the procedure of Doyle and Doyle (1987), and its concentration was determined spectrophotometrically.
RAPD Amplification Conditions
Amplification was carried out in a 20 ul volume with
- 10 to 30 ng of genomic DNA
- 250 uM each of dATP, dCTP, dGTP and dTTP (Promega, Madison, WI.)
- 2ul of buffer (InVitrogen, San Diego, CA., Table 1)
- 250 uM of decanucleotide (Operon, Almeda, CA., Table 2)
- 1 x reaction buffer (Promega, Madison, WI.)
- 1 unit of Taq DNA polymerase (Promega, Madison, WI.) overlayed with a drop of mineral oil.
A total of 40 decamer primers were tested in a thermal cycler (Thermolyne Amplitron). After an initial denaturation step of 94° C for 1 min; and 45 cycles of
- 94° C, 1 min. denaturation,
- 35° C, 1 min. annealing,
- 72° C, 2 min. extension,
the samples were analyzed by electrophoresis.
Ten ul of RAPD products were separated in 2% agarose gel (Promega) in 1 x TBE buffer, at about 1V/cm (constant voltage). Gels were stained for 30 min. in ethidium bromide and photographed under UV transilluminator. All reactions were repeated three times, and RAPD bands were scored as present or absent. The band size was estimated by comparing them to bands of 1 kb DNA Ladder (0.25 ug/lane; Gibco, BRL). Since the goal of this study intended to provide a screening tool for callus and plants derived via somatic hybridization for each Hibiscus species, only DNA extracted from the two shrubs was analyzed.
RESULTS AND DISCUSSION
Out of 40 primers examined, only 16 decamers (40%) produced scorable bands despite efforts to optimize MgCl2 concentration and pH for each primer tested. The remaining 24 decanucleotides either did not produce any bands or the bands were not clear enough to be evaluated. In evaluation of the band number, only those bands with enough intensity and difference in size from neighboring fragments were used. The size of RAPD bands produced was between 3500 to about 344 bp (Table 3).
The clearest banding patterns differentiating the two Hibiscus species studied were produced by 6 primers (15 % of all primers tested) OPD-2, OPD-4, OPD-16, OPF-1, OPF-13 and OPF-14 (Table 3, Photo 5). The highest number of RAPD bands observed for H. syriacus was 13 (OPD-2) and 12 for H. rosa-sinensis (OPD-8).
The usefulness of the RAPD banding pattern for identification of the fusion products combining genomic DNA from H. rosa-sinensis and H. syriacus from non-hybridized culture is yet to be tested. Although the RAPD results between labs may vary due to differing amplification conditions, DNA purity, or extraction method, reproducible results have been reported for multiple soybean [Glycine max (L) Merr.] DNA isolations (using 3 separate extraction procedures involving either multiple seed or single seed as a template source [Shatters et al., 1995]) and for vertebrates (Bielawski et al., 1995).
It is expected that the RAPD markers developed in this study will aid the screening procedure of the hybridized calli.
Polymorphic RAPD patterns distinguishing H. rosa-sinensis cv. 'Brilliant Red' from H. syriacus cv. 'Aphrodite' were generated using 16 primers. Based on these results, it is expected that this technique will be useful in identifying somatic hybrids of the two species.
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