NSW Agriculture, WWAI, Private Mail Bag, Wagga Wagga, NSW 2650
Randomly amplified polymorphic DNA (RAPD) has been utilised to discriminate between cultivars of Australian canola (B. napus) cultivars. Canola is a predominantly self-pollinating species although it has been estimated that 20% of seed is a result of outcrossing. Because of this Canola cultivars consist of a population of individuals but can be discriminated by utilising a bulk DNA sample from 40 or more plants. These DNA fingerprints have been useful to show differences in the various breeding programs and how cultivars from each program group together.
This study has shown the variation between individual plants within a cultivar and its effects on application of DNA fingerprinting for canola cultivars.
With commercial interest in Plant breeders Rights (PBR), cultivar registration, and the necessity to illustrate distinctness, uniformity and stability (DUS), the unambiguous identification of crop cultivars has become increasingly important.
Recently, identification methods have focussed on the application of DNA markers. DNA based markers have a number of advantages over other tests for cultivar discrimination in that DNA is unaffected by environmental factors or the developmental stage of the organism. Further, the RAPD methodology has an advantage over other DNA fingerprinting methods in that it is fast, requires no radioactive handling facilities, and the costs are relatively minimal.
RAPD analysis using the bulked DNA from 40 or more rapeseed plants is commonly used in our laboratory (Mailer et al., 1997) for cultivar registration, especially when morphological characteristics are not sufficiently clear (eg. cv. Oscar, Australian Plant Breeders Rights Certificate Number 589, 1996). However, it is still important to investigate genetic variation within a cultivar to determine the number of plants that are necessary to obtain a representative sample of the population. Consequently, we have investigated the variation that occurs within canola.
Plant material and DNA extraction
Fifty seeds of each of the canola cultivars Oscar, Dunkeld, Narendra and Rainbow, were germinated and grown to the four-leaf stage. The seedlings were then freeze-dried and stored in a desiccator until required. To extract DNA from these samples, the leaves from the freeze-dried plant material were placed in 2 ml microcentrifuge tubes & macerated with micropestles. The DNA was extracted and precipitated from the pulverised leaf tissue (Mailer et al., 1994), rinsed three times in 75% ethanol/10mM ammonium acetate, dried under vacuum, and redissolved in 500 μl of Tris/EDTA (TE) buffer. Ribonuclease (1.5 μl, 10 mg/ml) was added and the solution incubated at 37oC for 3 hours. The DNA was re-precipitated by the addition of 50 μl of 3M sodium acetate and 500 μl of
-20oC isopropanol. It was stored at -20oC for 2 hours, centrifuged at 5,000g for 30 min., washed 3 times with 70% ethanol (-20oC), and dissolved and stored in 500μl TE buffer. The DNA concentration of the final samples was measured by ultraviolet (UV) spectrophotometer at 260nm. The integrity of the DNA was checked by electrophoresis in a 1.2% agarose gel in TAE buffer (Tris/NaAc/EDTA, pH 7.8).
Three commercial sets of 100 x 10bp oligonucleotide primers were obtained from J. Hobbs (University of British Columbia) and diluted in 500 μl of TE buffer. The RAPD reaction mixture comprised of 1 x Promega reaction buffer, 1.5 mM MgCl2, 0.2mM dNTP (deoxyribonucleoside 5’ -phosphates), 0.25 μM primer, 1.5 units Taq polymerase (Promega M186A), and 100 ng of genomic DNA, made to a final volume of 25μl with sterilised, distilled water. Amplification of the DNA was carried out in microtitre plates using a Corbett FTS-960 thermal sequencer. The sequencer was initially programmed for 1 min. at 94oC, followed by 45 cycles of 1 min. at 94oC (denaturation), 1 min. at 37oC (annealing), and 2 min. at 72oC (elongation), completed by a final stage of 10 min. at 72oC. The microtitre plate was removed from the sequencer and the reaction products mixed with 5μl of blue stop buffer. The RAPD sequences were then separated by electrophoresis in a 1.2% agarose gel and 1 x TAE buffer. A 1Kb DNA standard ladder (Gibco BRL) was included as a size marker, and the DNA bands detected by UV transillumination after staining with 2.5μl ethidium bromide (10mg/ml) per 100ml TAE buffer.
The RAPD profiles produced by four of the primers, UBC # 106, 127, 147, and 250, were analysed. The seedling DNA samples were treated individually, and as bulked samples prepared by mixing 5μl samples from all of the seedlings of the separate cultivars. We also investigated the results of mixing the DNA from two different individuals of one cultivar in various proportions, 100%, 20:80, 40:60, 60:40, 80:20, and 100%.
Photographic manipulation and analysis.
The electrophoresis gels, obtained from RAPD analysis, were photographed using a CCD 800/1600 image processor. Fragment sizes of RAPD were estimated from the gel by comparison with the 1 kb ladder marker. The bands were recorded as either present or absent into a database of “0”and “1”’s
1. Admixtures of samples
The DNA banding patterns shown when DNA from two different specimens were mixed in differing proportions and processed with a primer. It was apparent that even at 20% conc., some bands were still obvious.
S1 100 80 60 40 20 0
S2 0 20 40 60 80 100
Figure 1. Electrophoresis gel showing the varying proportions of each of two individual specimens from one cultivar. S1 being specimen 1 and S2 the second specimen. At 20% the banding pattern is the same as a bulked sample.
2. RAPD variation within cultivars
An example of the variation of RAPD fragments expressed by individual samples of DNA from seedlings of a single cultivar, the RAPD banding produced by UBC # 127 with 15 seedlings and a bulk sample are shown in Fig. 2.
M b 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Figure 2. Electrophoresis gel of RAPD bands of 15 individual samples of one cultivar of canola. M, marker; b , bulk of 40 individual plants.
3. RAPD variation between cultivars
To illustrate the RAPD variation present within and between cultivars, another cultivar using the same UBC # 127 primer and the same number of individual samples show different variation of RAPD fragments to Fig. 2.
M b 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M
Figure3. Electrophoresis gel of RAPD bands of 15 individual specimens of a different cultivar to Fig. 2. M, marker: b is bulk of 40 individual plants.
When two individual samples of the one cultivar were mixed in differing proportions from 0% to 100% (Fig. 1), it can be seen that at only 20% of the second sample, a band atypical of the original sample becomes obvious. This mixing of the two individuals at 20% of one to the other then giving the banding pattern of a bulk of the cultivar which has been consistently repeated.
Fig.2 shows inconsistency within cultivars. When using bulk samples of 40 seedlings, results were consistent for a variety. Fig. 3 showing similar inconsistency but different banding patterns showing differences between cultivars.
Despite many bands being similar between cultivars significant differences were identified between cultivars to be able to discriminate. Repeated analysis indicate consistency for each cultivar.
1. Mailer, R.J., Scarth, R., and Fristensky, B. (1994). Discrimination among cultivars of rapeseed (Brassica napus L.) using DNA polymorphisms amplified from arbitrary primers. Theoretical and Applied Genetics 87, 697-704.
2. Mailer, R. J., Wratten, N., and Vonarx, M. (1997). Genetic diversity amongst Australian canola cultivars determined by randomly amplified polymorphic DNA. Australian Journal of Experimental Agriculture 37, 793-800.