A simple method of surveying plant populations for random amplified DNA polymorphisms.

Random amplified polymorphic DNA (RAPD) fragments can be separated on 8 cm long x 1 cm thick 5% polyacrylamide gels and silver stained to yield high quality data. This method requires far less technical expertise than other methods of separating RAPDs fragments, as gels can be purchased pre-made and complete equipment packages for such electrophoresis can be readily purchased. This method allows for analyses of RAPD variation under a much wider range of laboratory conditions than other such methods.

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There is little known of the genome of many plant species. However, there are many endangered plants for which management requires knowledge of levels of genetic diversity within and among populations. A simple method of surveying plant populations is needed to allow for surveys to be completed with a minimal amount of equipment and minimal levels of expertise.

Random amplified polymorphic DNAs (RAPDs) (Williams et al., 1990) is a simple method of generating large numbers of polymorphisms in species for which the genome is largely unknown. RAPDs are generated by using 10 bp arbitrary primers, which will anneal numerous times in any given genome. RAPD fragments that anneal within 5000 bp can produce a fragment using the polymerase chain reaction (PCR). Agarose gel electrophoresis, commonly used for RAPD surveys, often lacks the sensitivity to consistently detect RAPD fragments. To increase sensitivity, we have used 43 cm 5% polyacrylamide gels to separate DNA fragments and silver staining to detect DNA fragments as described by Antolin et al. (1996) to consistently and reliably detect RAPD fragments.

However, the large 43 cm long x 0.45 mm thick gels require a level of technical expertise that may not be available for all situations. Separation of the large plates, as required by the silver staining, with the gel adhering to only one plate, often fails. We thus developed a method for using 8 cm x 1 mm thick 5% polyacrylamide gels for electrophoresis and silver staining to detect DNAs to provide a reliable, sensitive method for detection of RAPD fragments.

RAPDs have been criticized as lacking sufficient rigor for molecular analyses, as fragments cannot always be reproduced (Brown and Kresovich, 1996). While this criticism is quite valid, many RAPD fragments can be consistently reproduced (e.g., Antolin et al., 1996). RAPDs are also very powerful as a means of assessing members of the same clone in clonal plants (Hamilton et al., 2002). RAPD fragments are generally short, less than 1000 bp, and are thus quite easily cloned. Once cloned, fragments can easily be sequenced, with sequences used to develop primers specific to a given sequence and used as sequence tagged sites (STSs) (Chaffin and Hamilton, 1998). STSs representing fragments that are shown to be most revealing of differences among individuals can be further investigated. No other method of surveying populations for genetic variation allows such a useful preliminary survey while facilitating such a powerful more in depth survey. RAPDs are thus a method well suited for investigations of clonal plant pop ulations for which there is little to no information regarding the genome.

MATERIALS AND METHODS

DNA samples were collected from daffodil leaves. DNA extractions were completed using the Qiagen DNAeasy plant kit. 20 mg of dry leaf tissue was shredded and placed into a 1.5 ml microcentrifuge tube with 106 micron glass beads added to each tube. Tissue was ground in 400 ml of Buffer A included in the kit preheated to 65[degrees]C, with 4 ml KNase A added following grinding. Instructions included with the kit were followed subsequent to the extraction step.

The "hot start" technique was used for the polymerase chain reaction. The hot start technique involves the separation of the primers and the template/polymerase until the temperature is above 36[degrees]C. We used a sterile DNase free reaction tube (Molecular Bioproducts) with bead of wax on the side of the tube. We prepared a lower reaction with primer in the reaction tube, heated the tube and added in an upper reaction mix with template/polymerase. The primer used for this example was Operon A-01 (5'-CAGGCCCTTC-3'). Primers were resuspended in 1 ml of TE buffer, pH 7.0, as per instructions from Operon, which results in about 5 pm/[micro]l of primer.

For the Lower:

Primer 2.0: [micro]1

dATP (10 mM): 1.0 [micro]1

dCTP (10 mM): 1.0 [micro]1

dGTP(l0 mM): 1.0 [micro]1

dTTP (10 mM): 1.0 [micro]1

Stoffel Buffer (l0x) (100 mM,

Tris-HCL, pH 8.3, 100 mM, KCL): 2.5 [micro]1

25 mM Mg[Cl.sub.2]: 10.0 [micro]1

Sterile distilled water: 6.5 [micro]1

For the Upper:

Sterile distilled water: 19.5 [micro]1

DNA polymerase--Stoeffel

fragment(5 U/ml): 1.0 [micro]1

Template--from DNA prep: 2.0 [micro]1

Stoffel Buffer (l0x) (100 mM,

Tris-HCL, pH 8.3, 100 mM, KCl): 2.5 [micro]1

The reactions were amplified using the following cycling protocol:

1.95[degrees]C: 2 minutes

add lower, heat to 95[degrees]C for 30 seconds, remove reactions, pause program, then place reactions on ice, allow to cool, add upper.

2. 94[degrees]C: 1 minute

3. 37[degrees]C: 1 minute

4. 72[degrees]C: 2 minutes

5. Repeat steps 2-4...: a total of 45 times

6. 72[degrees]C: 7 minutes

Following PCR, 10 [micro]1 of DNA sample buffer was added to samples. Polyacrylamide gel electrophoresis (PAGE) was performed using 10 well and 15 well 5% BioRad Ready Gels and a mini protean PAGE system. 1.0 x TBE (tris-borate-EDTA, diluted from lOx stock, 0.89 M tris, 0.89 M boric acid and 0.02 M EDTA, pH 8.0) was used in the lower buffer tank and 1.5x TBE was used in the upper tank as running buffers. Power was set at 20 volts and the gel was run for 11 hours for a 10 well gel, with the running buffers changed after 4 and 8 hours of electrophoresis. We ran 15 well gels at 100 volts for 1 hour and 20 volts for 6 hours, changing the running buffer after 3 hours. We used a 100 bp ladder as marker DNA (100-1000 bp, in 100 bp increments), however any marker that will allow for size determination of fragments in the range of about 100-2000 bp will work well. We have been primarily interested in analyzing fragments less than 1000 bp and thus used the 100 bp ladder described. 25 [micro]1 of PCR product was loaded onto each sample lane. More sample can be added as the wells will hold at least 50 [micro]1.

Gels were stained using the following protocol:

1. Fix/Stop--200 ml 10% glacial acetic acid in ultrapure water.

Fixing the gel. Agitate the gel well for 20 minutes (an orbital shaker set at 60 RPM works well) or leave overnight in the fix/stop solution. The gel can be stored in the fix/stop solution for several days.

2. Rinse the gel 3 times (2 minutes each) in ultrapure water using agitation. Drain water from gel for about 20 seconds between rinses.

3. Staining solution--0.2 g silver nitrate, 0.3 ml 37% formaldehyde in 200 ml ultrapure water.

Stain the gel. Place the gel in the staining solution and agitate well for 30 minutes.

4. Rinse the gel for 5 seconds (no more than 10 seconds) in ultrapure water. The stain diffuses rapidly out of the gel during this step. If you rinse too long, restain. If you do not rinse at all, the excess stain will deplete the developing solution before the gel is fully developed.

5. Developing solution-- 6g sodium carbonate to 200 ml in ultrapure water. Chill to 1 0[degrees]C. Just before use, add 40 [micro]1 of 10 mg/ml sodium thiosulfate and 0.3 ml of 37% formaldehyde.

Develop the gel. Transfer the gel into half of the developing solution and agitate well by hand. When bands start to become visible, transfer gel to the other half of the developing solution. Continue with developing until maximal resolution is obtained. The decision of when to stop the developing reaction is somewhat subjective. You are looking for maximal contrast between the bands and the background. The reaction is stopped by adding the fix/stop solution. Staining will continue for a few seconds following the addition of the fix/stop solution.

Note: It is critical that ultrapure water be used during silver staining, and that all glassware and plasticware be rinsed as thoroughly as possible with deionized water before use. It is also critical to use sodium carbonate of sufficient quality. We used ACS grade sodium carbonate (Sigma S-2 148).

Following staining, gels were placed onto a sheet of thin acetate soaked in water. Another sheet of thin acetate was lain over the gels and air bubbles were removed from between the sheets of acetate. The gels were then dried in a BioRad gel dryer. Gels could be air dried in the absence of a gel dryer, however the acetate sheets must be fastened to each other so that they adhere stably to the gels and to each other during the drying process.

Following the drying process, gels were cut out from the acetate sheets. Copies of gels were made onto thicker acetate sheets but placing the gel on a white light box and tracing the gels onto another sheet of acetate. A line running across the bottom of the wells was also traced onto the sheets as the origin of the DNA fragments during PAGE. The distance of each fragment from the origin was measured and recorded. The 100 bp ladder marker DNA was used to create a standard graph of distance migrated vs fragment size on semi log graph paper. The standard curve was then used to determine the lengths of fragments in samples.

RESULTS

Figure 1 shows the 10 well gel run at 20 volts for 11 hours. Figure 2A shows a 15 well gel run at 20 volts for 6 hours and figure 2B shows a 15 well gel run at 100 volts for 1 hour. Data are much easier to visualize using the 10 well gel (fig. 2). Separating fragments at high voltage for a short time (fig. 2B) led to the poorest quality gel. Electrophoresis for 11 hours produced the best quality of results for the primer used. If it is necessary to observe shorter fragments, a shorter running time would be needed as fragments less than 450 or so base pairs were run off the bottom of the gel when gels were electrophoresed for 11 hours. However, electrophoresis for 6 hours did not get the 100 bp marker to the bottom of the gel (fig. 2A), and thus running times of over 6 but less than 11 hours are probably most optimal, and would have to be determined empirically for any system on a case by case basis.

CONCLUSIONS

This method can be completed easily in a minimally equipped laboratory. A microcentrifuge is necessary for DNA extractions. A thermal cycler and BioRad mini protean electrophoresis system (or a similar system) are necessary for PCR and electrophoresis. The 10 well gels produced data that were much easier to observe than the 15 well gels (compare figures 1 and 2). The 10 well gels also hold more sample, and thus are more flexible with respect to amount of sample leaded onto a gel. The quality of the data are identical to that of the 43 cm gels in the range within which fragments can be observed, with the limitation of the smaller gels being a narrowing of the range within which one can make quality observations.

This method can generate a great deal of data for assays of genetic homogeneity within populations with little preparation time (as the gels can be purchased pre-made and the DNA extraction is performed using a kit). While RAPDs are difficult to use as a means of resolving degrees of relatedness among organisms, identical or highly similar RAPD fingerprints are possibly the best indicators of genetic homogeneity available. As homogeneity is the parameter of interest to ecologists, the method outlined in this paper would be useful in molecular ecological studies; there is no need for prior information about the genome of subjects, methods are simple to perform, data can be extended by cloning and sequencing of products and large amounts of data can be collected in a short period of time.

Data can be analyzed with a simple measure of diversity, such as the Shannon-Weaver index (Shannon and Weaver, 1949), where diversity is estimated using a measure of information content, H' where H' = -[SIGMA][p.sub.i]log [p.sub.i], where [p.sub.i] is the frequency of a fragment within a population sampled. Gustafson et al. (2002) describe more elaborate comparisons that can be made of RAPD fragment polymorphisms within and among populations.

LITERATURE CITED

Antolin, M.F., C. Bosio, J. Cotton, W. Sweeney, M. Strand, and W. Black. 1996. Intensive linkage mapping in a wasp (Bracon hebetor) and a mosquito (Aedes aegypti) with single-strand conformation polymorphism analysis of random amplified polymorphic DNA markers. Genetics 143:1727-1738.

Brown, S., and S. Kresovich. 1996. Molecular characterization for plant genetic resources conservation. Pages 86-93 in Patterson, ed. Genome Mapping in Plants. Academic Press. San Diego, CA.

Chaffin, C., and R. Hamilton. 1998. A method of producing STS (Sequence Tagged Site) markers from RAPDs (Random Amplified Polymorphic DNAs) in Ceratopteris richardii. C-fern web journal. http://cfern.bio.utk.edu/journal.html.

Gustafson, D., D. Gibson, and D. Nickrent. 2002. Random amplified polymorphic DNA variation among remnant big bluestem (Andropogon gerardii Vitman) populations from Arkansas' grand prairie. Molecular Ecology 8:1693-1701.

Hamilton, R.T., T. Scarf, K. McGehee, and D. Morgan. 2002. Resolving variation within Narcissus cultivars. J. Miss. Acad. Sci. 47:14.

Shannon C.E., and W Weaver. 1949. The mathematical theory of communication. University of Illinois Press, Champaign, IL.

Williams, J., A. Kubelick, K. Livak, J. Rafalski, and S. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful genetic markers. Nuclic Acids Res. 18: 6531-6535.

Janet Marie Gibson McDaniel, Robert G. Hamilton (1)

(1.) Author for correspondence