The iMac User Guide (late 2009 models) indicates the two front slots (side-by-side, nearest the display) are to be filled first. Those are the slots your original RAM DIMMS should have been in. Presumably they are Bank 0. You can test this yourself by populating those positions, starting your Mac and checking System Information > Memory.
If you populate all 4 slots, technically it doesn't matter which DIMM is in which slot ... your Mac will work with whatever configuration you use.
Whether or not one configuration is "more optimal" than another is very hard to tell. Some years ago, with a couple of early iMac G5 models there was an optimal way to populate the slots; the Mac would work regardless but if the DIMMS were inserted in matched pairs memory speed was maximized. It was a consequence of the then-memory architecture of those iMacs. I have not heard of this being an issue in any Macs since that time.
In the absence of any definitive information to the contrary, I'd be inclined to insert the DIMMS ... in matched pairs ... with the 4GB DIMMS in Bank 0 ... and the 2GB DIMMS in Bank 1 ... "just in case" This is the same as what Crucial advised ... and ... they really know their stuff!
Thanks Martin, your reply was very helpful. You say, " If you populate all 4 slots, technically it doesn't matter which DIMM is in which slot ... your Mac will work with whatever configuration you use."
That is effectively what I have done, and yes the system does still work, as confirmed by the diagnostics. It is Crucial who are saying that the cofiguration, should be different and I obviously, want to optimise performance. However, I do not want to keep changing memory sticks.
You finish by saying insert the 4GB DIMM's in matched pairs, in BANK 0. As I am not a 'techie' can you confirm, that a BANK is 2 slots one above the other and that Bank 0 are the slots nearest to the On Button, i.e facing the screen, the left two slots? When you install the memory and put the screen face down, this reverses the situation and can be confusing, so clarification, would be a tremendous help.