Different cultivars have different characteristics so correct cultivar identification is important.
An Australian report relating to olive cultivar identification, ‘Olive Oil Yield, Quality and Cultivar Identification’ by Kevin Robards and Rod Mailer, can be found at https://rirdc.infoservices.com.au/items/01-023. The research described is the start to ongoing work with the aim to develop DNA testing to identify olive tree cultivars (clones/ near clones) and to determine relatedness of different olive cultivars (see dendrogram Fig 5). Cultivars from the Wagga Wagga Olive Grove (WWOG), situated on the campus of Charles Sturt University and a grove at Yanco were used in the study. The findings were especially interesting considering that many trees growing in commercial orchards have been developed from cuttings taken from WWOG. The study found that some trees had been incorrectly identified and also that there appears to be considerable DNA variation within some of the trees previously identified as like-type cultivars. To fully understand the report we need to understand how the trees were ‘fingerprinted’. The DNA analysis procedure used in the study is called RAPD (Random Amplified Polymorphic DNA).
DNA terms explained (background info only)
Deoxyribonucleic acid (DNA) is a molecule that contains the instructions for the development and functioning of living organisms (animals, plants, bacteria etc). The DNA molecule is a sugar/ phosphate strand (backbone) that has atom groups called bases attached (see Figure 1). The order of the bases is the DNA code (Figure 1 and 2). Adenine (base) attracts Thymine (base) and Cytosine (base) attracts Guanine. In this way two DNA strands (the helix) are held together by molecular attraction.
A gene is a region of DNA that influences a particular characteristic in an organism. It contains the DNA code used to assemble proteins. Only about 1.5% of the human genome consists of protein-coding areas.
RAPD DNA testing (fingerprinting)
Related organisms have similar DNA. DNA testing determines relatedness by comparing segments of DNA. The RAPD method does not compare all of the organisms DNA. It does not compare genes. It only compares bits of DNA that might well not code for anything in particular (protein or otherwise).
What is compared?
The short segments (pieces or bits) of DNA that are compared are defined by two segments that contain specific codes within the DNA. It is not the codes that are compared but the distance between them and how many times they occur. The short specific DNA coded sequences define the beginning and the end of a segment that is compared. The amount (length) of DNA between the beginning (specific coded region) and end (specific coded region) varies from organism to organism. The number of times these codes occur in the DNA also varies organism to organism. The more often these specific coded areas occur the more segments there will be. Closely related organisms will have similar numbers of segments of identical length. Clones (identical organisms) have the same number of identical length segments.
How do we find these specific codes?
Small DNA molecules called primers locate the specific codes. Primers are designed to bind the specific coding regions. The primers act as a starting point and facilitate ‘*copying’ of DNA material.
How do we find out how many segments there are and how long they are?
To see the DNA segments with the naked eye you need an awful lot of it. To make a lot of DNA, the segments are ‘*copied’ many times. This is called amplification.
* Note: An exact ‘copy’ is not made but a ‘matching copy’. When the ‘copy’ is copied it will be the same as the first.
Equipment required to amplify the DNA segments?
The DNA amplification process is called a PCR (Polymerase Chain Reaction) and it is done in a PCR machine (Figure 3). The PCR machine is an automated cycler that can quickly heat and cool the reaction mixture. Each ‘cycle’ doubles the number of DNA segments in the mix. After the first cycle there are two copies of each segment. After the second cycle there are four copies of each segment and so on until after 30 cycles you have more than a billion. Each cycle involves three major steps that are repeated 30-40 times:
Step 1 The mixture is heated so that the DNA helix strands separate.
Step 2 Cooling allows the primer to bind single DNA strands.
Step 3 Warming encourages the enzyme (polymerase) to make DNA using a primer as a starting point.
Separation of segments and measurement
The amplified DNA is placed in a gel and an electrical charge applied. DNA contains many phosphorus atoms, which are negatively charged causing the DNA to be drawn through the gel by the current. Long pieces of DNA move more slowly than the shorter pieces. The DNA segments are inserted into the gel together (one lane per organism). As time passes the shorter strands separate from the longer strands (see Figure 4).
Setting up a lab
Thinking of setting up a lab in order to test your own trees? Table 1 contains the prices of some items you will probably require.
Can I have my olive tree DNA tested today?
It appears that olive DNA fingerprinting is not at this time available in Australia. Previously olive testing has been done at Wagga Wagga as per the ‘Olive Oil Yield, Quality and Cultivar Identification’ (OOYQCI) report (3) and at South Australia’s University of Adelaide’s Waite Agricultural Research Institute/ South Australian Research and Development Institute (SARDI). The good news is that Rod Mailer Principal Research Scientist DPI (Department of Primary Industries) Adjunct Associate Professor Charles Sturt University at Wagga is expecting to start another olive tree DNA testing project in the not too distant future (2007). Rod thinks they will be able to DNA fingerprint any olive tree for approximately $50 per tree and once they are “rolling” it should only take a couple of days to process the DNA and obtain a result (see footnote). As far as I’m aware ‘simple on-site’ DNA testing of olives and other fruit trees by an average gardener’ is not available yet. One day (who knows when or how) DNA field testing using a simple handheld machine will probably be possible. Don’t hold your breath.
The RAPD technique doesn’t provide information regarding the exact characteristic differences between the trees tested just whether there are differences. This is because only random areas of selected DNA are compared not actual genes. Those conducting the tests do need to have access to the ‘true variety’ ‘markers’ in terms of the lengths and number of different segments that will be amplified using specific primers. If you have two unknown plants and want to know if they are clones, no information is required. RAPD can do the job by comparing the segments amplified from the two DNA samples (eg. young leaves) you provide. Any organism (plant, animal etc) that has DNA can be tested using the RAPD technique.
Just before this article went to print we contacted Rod Mailer. He provided us with Ref 4 and advised us that this year the ‘microsatelite’ technique would be used to fingerprint trees. It should be more useful than RAPD. The dendrogram (Fig 5 from Reference 4) is a fine example of how important and useful the research (Ref 3 and 4) has been in terms of olive cultivar identification and relatedness determination. Trees on the same branch are similar. You can see for example that all of the Sevillano trees are on the same branch. The closer the branch is to the right of the page the more related the two cultivars. Apparently other publications have not produced a dendrogram as successful as that in Fig 5.
4. Mailer, R.J. and May, C.E. 2002. Variability and interrelationships of olive trees and cultivars using RAPD analysis. Advances in Horticultural Sciences. (16)3-4: 192-197.