Thoughts off the top of my head: August 2012
Since the big switchover to digital TV a few years ago, we got less and less channels over the airwaves until finally we had none. Some improvement, huh? So I have been recently watching old CSI episodes on DVDs. When the evidence finally “talks”, it seems that usually it is the DNA evidence that shouts the loudest. It seems that DNA testing can solve almost any mystery today.
I hear this in the hosta world also. If we just did a DNA test we could tell if these two hostas are the same or different. With a big enough data base, I guess at some time we could identify any hosta you might have lost the label for by comparing its DNA to that on file with the Registrar. DNA would solve everything because DNA is the final answer.
For me the most interesting application of DNA sequencing is in the study of hosta species. It, like protein analysis, can show us which hostas in the wild are the most closely related based not on their outward appearance but on a chemical level inside the plant. The theory goes that over thousands of years DNA mutates by making copying mistakes when it replicates. Other external factors like radiation can also cause mutations but mutations are a natural part of DNA replication. Most mistakes are bad, if they were good we would call them something else, but a few make a better plant or help it adapt to changing environments. Individuals with a good mutation will survive and have a competitive advantage. Those with a bad mutation will at best be relatively unchanged or in the worst case die. Mutations that occur in the formation of sex cells, eggs and pollen, have the biggest impact on species changes.
Most mutations in genes, because they are bad, are not then preserved from generation to generation. They are lost to the gene pool of the species. DNA is not however just a pearl necklace of genes on a spiral string. There are spaces between the genes called obviously intergenic spaces. Mutations that occur in these spaces do not affect the genes or the individual and are passed along without harm from generation to generation. It is the differences in the DNA code in these spaces between the genes, in the order of the bases, adenine, thymine, cytosine, guanine, (A,T,C,G), that can help us follow the evolution of species and compare cultivars for parentage.
Find two similar fragments of intergenic DNA after extraction and amplification, from two different hostas, and see how the DNA sequence is varied. Study enough species or cultivars and a family tree begins to appear based on how many bases are in common or different. In hostas this can be done with chloroplast DNA also which is maternally inherited and again a family tree of grandmothers and great grandmothers can be assembled. For a free peak at what this research looks like check out hortsci.ashspublications.org website and search for the article HortScience 40(5) 1243-1245 2005. ( Search for an article on the home page. 2005 is the date, 40 the volume number and 1243 the first page, press submit.) There are photos of the bands and a family tree of hosta species.
This all seems foolproof enough. But…in this study, many of the species that we thought were closely related based on geography and morphology, the way they look, are far apart on the family tree and some that are very different like sieboldiana and laevigata, tibae and montana are shown as closely related. It turns out that not all DNA sequencing uses the same methods and there is some discussion as to which is the most accurate in portraying the true picture of species relationships. I am in no position to judge but I am sure eventually there will be better and better methods developed over time and hopefully what we see with our eyes will mirror what the DNA testing tells us.
While we are on the subject, let’s talk triploids. As most of you know we have been fortunate to have Ben Zonneveld in Holland study the polyploid issue in hostas. Ben uses yet another type of DNA testing, flow cytometry, that measures the total weight of DNA in a sample and does not try to sequence the bases. This test is very good for measuring the amount of DNA present and thus whether two sets of chromosomes are present, diploidy or three, triploidy, (four sets, tetraploidy also, but triploids are the issue today), but cannot tell us much about the nature of the DNA. It doesn’t see genes or chromosomes, it only sees DNA.
Ben has tested many of the ‘Sum and Substance’ family of hosta sports. If you wish to see the paper he published in June, (Plant Syst & Evol (2012) 298: 1037-1043), google Plant Systematics and Evolution. On the home page click “Read Online”, then at the left, Number 6/ June 2012. Find the article and click on Download PDF. The tables are easy to read. A cultivar that is 3-3-3 is totally triploid like ‘Sum and Substance’. Numbers less than 3 like 2.8 indicate a loss of part of a set of chromosomes. Numbers greater than 3 indicate excess chromosomes. Hostas that have more or less than a complete set of chromosomes are said to be aneuploid.
I wanted Ben to test ‘Final Summation’ for me as well as the green sport of it ‘Zero Sum’. As you know usually the all green sport of a yellow variegated hosta grows faster and bigger than it. In this case, ‘Zero Sum’ grows slower, an indication that it might be of a different ploidy than ‘Final Summation’. I assumed that ‘Final Summation’ might be triploid in the tissue of its yellow center and maybe something else in the green portion, maybe hexaploid, (six sets of chromosomes)?
Well, it turns out that chromosome doubling does not occur with triploid ‘Sum and Substance’ as you can see in the tables. This is not a diploid, tetraploid situation. In fact hexaploid plants are very rare. ‘Final Summation’ in fact was not triploid but aneuploid, 2.9 in all layers, yellow and green. ‘Zero Sum’, derived from the green marginal tissue of ‘Final Summation’, was 2.9 in all layers also. If the ploidys are equal then shouldn’t the green plant outgrow or at least grow as well as the variegated one?
Again, flow cytometry only measures the total weight of DNA. Yes, both plants are a “quart low” when it comes to three sets of chromosomes but which chromosomes are missing and more importantly which genes? Evidently, ‘Final Summation’ and ‘Zero Sum’ are missing different chromosomes and or genes. The green tissue and the yellow tissue in ‘Final Summation’ while they have the same weight of DNA are genetically different, different enough to cause them to grow at different rates. The DNA evidence is not wrong it is just incomplete. I have learned a lot but there is so much more to know!
One other thing about ploidy, converting a hosta to a tetraploid does change some things about its structure, thicker leaves, shorter scapes, bigger flowers, but it doesn’t fix everything. If slugs like a diploid hosta, then they will like the tetraploid one too. Thicker leaves do not intimidate them. If the leaves are subject to fungal diseases, tetraploidy will not fix that either. In fact the shorter roots of tetraploids may actually make them less drought tolerant.
Finally, frequently several mutations may occur in a hosta sport before we can see a change in color. Changes in variegation are in no way linked to ploidy but we often recognize more readily changes in ploidy if they are accompanied by color changes. I assume that there are solid colored hostas that are polyploidy chimeras also. We just do not recognize them as easily. They are not different enough to select for in the lab and are shipped out with their diploid cousins. That is how H. plantaginea ‘Doubled Up’ ended up at my nursery, easily overlooked in a flat of small liners of H. plantaginea. Keep your eyes open!