Just a brief outline of the more sophisticated analytical techniques applied to orchids - as I understand them.

Early on allozymes were used to identify differences between species and populations. Allozymes of a particular enzyme have the same function, but changes to the DNA coding for them have altered the non-functional part of the protein, resulting in molecules of different sizes. Chromatography (inducing a purified extract to pass through a medium) will reveal which allozyme is present in a plant from the distance travelled.

Plastids are plant cell organelles such as chloroplasts. They have some maternal DNA present, much like animal mitochondria. This DNA can be isolated, subjected to enzyme cleaving into smallish fragments and again chromatogrpahy can be used to show the different sizes of the fragments. Choice of the enzyme is crucial in order to cleave the DNA at points where there are differences in the DNA. Plastid DNA not only helps to show differences within and between species or populations, but inform us of which is the parent species of a polyploid species

The Internal Transcribed Spacers (ITS) lie between small DNA sub-units and the DNA genes of ribsomal DNA. There can be thousands of theses in repeat sequences. They are easily amplified by DNA polymerase and again a chromatological technique is used to demonstrate which ITS are present.

Amplified Fragment Length Polymorphism is applied to nuclear DNA. Using enzymes to cleave purified DNA at certain points along its length, certain sequences of the DNA can be amplified using specific primers and DNA polymerase. Again the different lengths of DNA can be shown by chromatography. Any changes in the DNA code in different species or populations will yield quite different sequences of DNA.