Genetic Fingerprinting:

(1)'Kunming Institute of Botany, Heilongtan, Kunming, Yunnan, China.
(2)'Department of Plant Science, Massey University, Private Bag 11222, Palmerston North, New Zealand.

This paper introduces a new technique used in a study carried out in New Zealand to unravel some of the genetic mysteries in camellia plants. The DNA fingerprinting technique has been used to answer some interesting problems that have arisen where unprotected pollination has prevented the parents being identified with certainty and provides unique insights into the relationships between camellia species and their hybrids.

The current system of camellia cultivar identification is based predominately on the shape and form of the flowers and leaves. For many purposes this is fine as the grower will know the seed parent but if they want to know something more about the pollen parent then this may become exceedingly difficult. A very detailed physical description of the plant is required, this is time consuming and has become expensive in a user-pay environment. Furthermore, even the best botanical descriptions are subject to environmental influences that will only add to the confusion and uncertainty in any judgments concerning parentage.

In the early 1900's it became clear from studies of the thread-like structures observed during cell division and meiosis where gametes are formed, that these structures (now called chromosomes), contained the genes referred to in Mendel's laws of inheritance. From the 1920's it was known that chromosomes were composed of DNA and protein, but for many years it was thought that the DNA material was the same in each plant or animal and the inheritance was controlled by various proteins. It has been subsequently shown that the expression of the proteins was controlled by the genotype (or the DNA) and by environmental factors. While this was a useful aid to cultivar description it was not completely reliable as the growth conditions could influence the expression of the proteins or enzymes. When Watson and Crick discovered the structure of DNA they showed how it was possible to store biological information and how it could be copied accurately during cell division. We now understand that the DNA found in each cell is the basis for the inheritance of all the features seen in plants and animals.

The information stored on one set of the thirty chromosomes belonging to C. japonica or C. saluenensis would require over a million pages of print. Large portions of this information are the same in plants arising from the same genus but with important differences between species and cultivars. If these differences can be detected easily, then this could provide a useful method of describing each plant.

molecular biology allow many questions to be asked about the make-up of plants that were previously impossible to resolve. The widespread application of genetic engineering techniques has allowed the structure and function of DNA to be explored and has opened up opportunities to utilise this information to solve practical problems. New methods have been developed that detect and identify variation in the DNA between different individuals. The DNA fingerprinting techniques employed to solve forensic cases can also be used to solve paternity issues in the plant world.

In our camellia studies a polymerase chain reaction procedure was used to produce a randomly amplified series products (RAPD markers) that can be visualised as a series of bands that are characteristic of the plant and the DNA being examined. Thus DNA was extracted from the leaves or buds (flowers were of no use) by freezing the tissue with liquid nitrogen and then suspended in a buffer to protect the DNA from degradation. The DNA was removed after spinning in a centrifuge to settle all the solid material out of the suspension. The liquid left in the top of the solution now contained DNA which then precipitated out using cold ethanol.

This procedure was followed for every camellia plant examined. Small samples of the DNA to be tested were combined with several chemicals including a primer (a short piece of synthetic DNA) and an enzyme Taq polymerase. The reaction mixture was then heated to about 95°C for two minutes to melt the DNA causing it to break into a series of fragments. When the temperature was lowered briefly the primer and the DNA fragments paired up where they were complementary in their structure, then the Taq polymerase enzyme doubling the number of the copies present in the solution, This process was repeated automatically 35 times in a specially designed temperature cycling machine. As no pairing occurs when the sections don't match exactly, each cycle of the machine only doubles the copies of the DNA section selected by the primer. The composition of the primer and the ability to match a section of the DNA determines the number of fragments that are multiplied. Sometimes there will be few fragments produced while other primers may produce many fragments by the time the machine has finished cycling. The fragments are separated by size by running on a chromatogram, and in this way the DNA is able to produce a characteristic series of bands that can be visualised after staining to produce a fingerprint for each plant. When several species or cultivars are chromatographed at the same time the similarities and differences between each plant are immediately obvious when the banding patterns are compared.

This study indicates that RAPD markers could be very useful aid to the identification of camellia species, cultivars and their parents. These topics will be developed in a series of additional articles in the Bulletin.

Acknowledgements

This work has been made possible by substantial grants from the Camellia Memorial Trust and the Pukeiti Rhododendron Trust in New Zealand. Camellia and Rhododendron enthusiasts have also contributed generously by supplying plant material and funds for this project. The authors wish to thank Mr Hugh Neilson and Dr George Ionas at Massey University for valuable advice and assistance.

RAPD banding patterns produced by camellia species leaf extracts with a single primer. The lanes from left to right are: (M) the standard marker (with bands at 100 base pair intervals), (1) C. chekiangoleosa (2) C. wabisuki (4) C. polyodonta (5) C. vietnamensis (31) C. reticulata 'Crimson Robe' (17) C. reticulata 'Captain Rawes' (18) C. x 'E. G. Waterhouse' (22) C. sasanqua (29) C. saluenensis (30) C. pitardii var. pitardii (53) C. saluenensis 'Sunnybank' (54) C. vernalis 'Star above Star' and (55) C. vernalis 'Ginryu'.