OK, anyone want to talk more about 'advanced' DNA technology as used in slipper orchid breeding & conservation? We had a few posts back and forth on that a while back, but nothing more. There's lots non-technical stuff that can be explained to those interested, and technical stuff (ideas) that can be shared with those using (or wanting to use) the methods.

Best from Besk

I for one would be very interested in hearing more. While I don't have enough knowledge to contribute to the discussion myself, I enjoy reading and understanding what I can. :)

Got any particular question or fantasy?!

Besk

I for one would be very interested in hearing more. While I don't have enough knowledge to contribute to the discussion myself, I enjoy reading and understanding what I can. :)

Yeah, me too!
I don't know that I have enough knowledge to come up with a particular question either, but I'd love to hear more from those of you who do!

Again -- got any particular fancy? Exasperation? Are you an 'intelligent designer' (of orchid hybrids) and want to know more about what might be possible?

B from B

OK, I would like some speculation on a real breeding occurence. A breeder crossed White Knight, a large, complex white onto barbigerum to make Barbi's White Satin. The expectation was that the pod parent, barbigerum, being a miniature, would reduce the plant size. As happens quite often in complex white breeding, germination was poor. What has happened so far is both the flowers and the plant size of those seedlings that have bloomed so far have been very close in size to White Knight. There was been very little reduction in either plant or flower size.

Questions:
Can you speculate why the expected results have not occurred?
What would some of the expected results be of a back cross to barbigerum?
What might the results of a sib cross of the original cross, Barbi's White Satin?

Are these the type of questions that you want to address?

Q -1 - What is advanced DNA technlogy?
Q -2 - How advanced is advanced DNA tech? (the present day limits, etc)
Q -3 - What can it offer the orchid breeders?
Q -4- How soon can, say a blue slipper hit the market?
Mang

I'll be happy to try to ad lib some of these, and try to explain some others, but must go off soon to other duties!

I'll just start off with the question about a complex Paph crossed with barbigerum, and then about blue paphs. I'd guess that the complex was a high polyploid and therefore rather dominant over barbigerum due to the number of chromosomes inherited from barbigerum (n=13). I suspect a back-cross to barbigerum could be helpful, though I'd think that a selfing would run the risk of strongly asymmetrical sorting out of the parental forms in favor of the complex hybrid's phenotype. Re: blue paphs, Stock chimed in on this in another thread -- a hard knock; anthocyanin pigmentation (= blue through pink) genetics in orchids is not simple. Both Stock and rob Grisebach tried to generate blue Phal's with no (known!) success. Steve T seems to be much more optimistic, but I'd say he might not have been reading his pigment genetics papers too carefully! Of course, breakthroughs happen, but we don't even know enough about anthocyanin regulation in ornamentals like Gerbera -- the incredibly valuable cut-flower -- to make blue ones. And MUCH money has been spent to elucidate the Gerbera anthocyanin pathway... just imagine the profits.

Stock -- do you want to pipe in?

Best from Beskriver

RNAi technology might be interesting... Pick a gene, any gene, and kill it.

I'd also like to know what advanced DNA technology is... We do some really interesting things in the lab all the time. It would be kind of fun to try and apply some of that to orchids.

Sure I'll try; never quit when you are ahead? I am not sure of the ploidy of Paph White Knight. It may not be a polyploid but if it is then crossing is with Paph. barbigerum would yield triploid progeny. Not a good way to go. If Paph. White Knight is diploid then one would only have to contend with the chromosome differences between the two lines if any. If the progeny are fertile than back crosses could yield a spectrum of plants that might provide what the original cross was intended to do. As to blue Paphs, I believe that we are close in technology to being able to do this but the problem is the time and cost. In horticulture of orchids or other crop plants there are many other issues that gene work could focus on that produce more "bang for the buck" than a single blue line of Paphs would produce. I doubt that the resources will be put into blue clones for many year to come. I could be wrong, I was once!!!!!!!
Dean Stock

Hi!

Re: RNAi (or antisense if this didn't work), what gene would one knock out and why? Maybe dihydroflavonol reductase to make albas? Shots in the dark with RNAi could be a a waste of time, but a reasonable thought technologically if we knew the genetics of a pathway of interest. To go further, with 35S overexpression of an interesting gene, one might get nasty, unwanted pleiotropic effects throughout the plant...

Re: triploidy with a complex x barbigerum, I suppose it depends on the ploidy level of the complex.

Re: blue paphs, sure, one could engineer delphinidins into them, but the fine regulation may not be as simple as with chrysanthemum. For example, attempts with gerbera (also in the sunflower family) have not yielded 'true blue' (so far as I know). Of course, as you say, with increased technology and more money more becomes possible. Like the whole-genome seqs that could be very useful in this regard once the Curagen 454 sequencer (et al.) makes 1000 bucks a pop realistic.

best from Besk!

Also, on the blue Paph. issue - I think something to think about is the time to maturity. Gerberas are relatively fast growing plants from seed. Paphs, not so much, right?

That's going to slow down the process immensely.

Hi!

Re: RNAi (or antisense if this didn't work), what gene would one knock out and why? Maybe dihydroflavonol reductase to make albas? Shots in the dark with RNAi could be a a waste of time, but a reasonable thought technologically if we knew the genetics of a pathway of interest. To go further, with 35S overexpression of an interesting gene, one might get nasty, unwanted pleiotropic effects throughout the plant...

Re: triploidy with a complex x barbigerum, I suppose it depends on the ploidy level of the complex.

Re: blue paphs, sure, one could engineer delphinidins into them, but the fine regulation may not be as simple as with chrysanthemum. For example, attempts with gerbera (also in the sunflower family) have not yielded 'true blue' (so far as I know). Of course, as you say, with increased technology and more money more becomes possible. Like the whole-genome seqs that could be very useful in this regard once the Curagen 454 sequencer (et al.) makes 1000 bucks a pop realistic.

best from Besk!



Yep, I was thinking color pathways to begin with. The exact gene sequences wouldn't be known, of course, but the pathways are well conserved with other model plant species. And the genes well conserved too. Would be relatively trivial to pick a gene based on knowledge of a pathway, clone it out with degenerate PCR primers, and build the appropriate RNAi. RNAi has the advantage of knocking down all copies of a gene. Both chromosomes (or all four, for a tetraploid). Since plants are famous for gene duplication and coordinate expression as a way to increase protein levels (there are upwards of a hundred copies of some starch synthesis genes in arabidopsis, for example), RNAi might be a good way to achieve total knockouts. I doubt pigment synthesis is so important as to merit gene duplication, but the ploidy thing is always a problem if you try the traditional insertional knockout method.

I'm not sure we will ever get to 1000 bucks a genome sequence... Maybe for a SNP scan of a known genome (I know they are trying to create SNP maps for individual persons as a diagnostic tool). But I'm all for cheap sequencing. I will reiterate my standing offer to assist with any orchid genome project out there. Give me a genome, I can annotate it. That is what I do.

Hi Littlefrog --

Great comments! And BTW, I am not at all unfamiliar with gene annotation myself...

We would of course like to alter other developmental programs than pigment biosynthesis. Trouble is that we know very little about organ differentiation in plants compared to organ determination (via, e.g., MADS box genes). Downstream effectors are only just coming into 'analytical vogue' now that microarray analysis reproducibly permits a lot of gene fishing.

But we have no Paph EST/unigene library (that I am aware of), no genome maps from crosses betw. inbred lines, and no QTL results that might give us loci for warts, inrolled pouches, etc. Again, *that I am aware of*! We will need investment toward a significant floral EST library for anything to start.

ESTs are Expressed Sequence Tags -- random bits of DNAs from expressed genes that are sequenced and assembled into 'unigenes', which are presumed to be sigle genes. Once one has a significant EST/unigene 'library', one can start getting to business with genetics since the DNA sequences qill be for the most part comparable to Arabidopsis, the fruit fly of the plant world.

Another thought -- there are some genes that code for dosage-dependent proteins that could VERY interesting:

The LEAFY gene of Arabidopsis is part of the switch from infloral to floral meristem identity (i.e., it is necessary to make flowers!). It is a mster transducer of external signals to the floral developmental program. It is known from experimental evidence that adding copies of LEAFY to the Arabidopsis genome leads to an **early flowering** phenotype. The effect is more pronounced the more LEAFY copies are transgenically inserted. If this were possible and phenotypically similar with the Paph copy of the LEAFY gene, then **one might be able to produce early-flowering paphs!!!**. However, one aspect of Arabidopsis plants with extra LEAFYs is that they form more compact plants that produce less flowers. Compact may be good, but fewer flowers probably not unless one were interested in fiddling with single/few-flowered species in the first place.

We can easily get the Paph LEAFY gene, but we'd have to prove that it works like the native LEAFY gene in Arabidopsis. LEAFYs from other plant species can play additional roles other than simply promoting flowering. If we can transform Paphs, the rest should be possible.

Would be happy to talk more!

Best, Besk

whew,
that was over my head.
I don't know how advanced this is, but I came up w/ a question!

http://www.slipperorchidforum.com/forum/viewtopic.php?p=24375#24375 :)

Hi Paphgirl --

There is no reason to think of exstaminodium or lindenii as more advanced since they self. Missing a staminode or a pouch could well be caused by mutations in single genes. In fact, I'd be surprised if that weren't the case. As such, exstaminodium and lindenii could have evolved 'immediately' from their normal progenitor phrags. The new changes would become fixed genetically and persist in nature since the plants are obligate selfers and don't need pollinators to make more of themselves. In terms of time frame, single genes mutate all the time -- it's all about whether or not the phenotypes that derive can survive and perpetuate -- or not. So with exstaminodium and lindenii, this could have occurred very recently.

Regarding Phrag popowii -- regardless of whether article 57 supports this or that, it is considered 'poor form' in the taxonomic community (in terms of ethical stylistics) to name a plant after yourself or a friend (or similar) if a name had already been suggested by someone else, perhaps long ago, but not validly published -- this older informal name could simply be officially validated. Popow had nothing to do with the wallisii issue. Take the case of Paph supardii, which could have been validated as devogelii.

Best wishes!

One more note about creating blue Paphs: As we pursued this in Phals it became very evident that adding a blue gene such as a gene for Delphindin, did not accomplish much. Some really nice red flowers are based on Delphinidin. You have to have the proper co-factor genes as well as the pigment genes and depending on the pigment, you also have to have metal complexing genes and pH control genes to make it all work. It gave us a real headache and we were happy to give it up. So trying to manipulate pigment genes to give specific flower colors is a real challange and although the technology is emerging to do this it it still a major project taking years and big bucks. In short, it is not going to happen any time soon. I suspect that it will take many years to obtain the genetic resources in orchids to do any meaningful genetic manipulations. If you could raise orchids by the acre and eat them, it would happen sooner.

Very educational thread...

A big question of mine is how best to transform paphs with genes of interest. Question for Dr. Stock: how do Phal folks insert and select for genes in Phals? Given the relative ease of cloning Phals, it would seem to me that inserting genes into Paphs would be much more difficult, given the low yield in cloning experiments.

RNAi sounds very promising, but again, how would we get the RNAi-expressing construct into Paphs? For that matter, how easy would it be to do that in Phals?

Here are some other assorted questions:
1) Anyone here had experience creating EST libraries from plant tissue? If so, please elaborate or provide references on RNA isolation techniques, cloning vectors, etc...
2) Anybody know of well-inbred lines that would be useful for use in future crosses for genetic analysis?
3) What are the best way(s) in plants to go after QTLs (Quantitative Trait Loci)? Has much of this been worked out in Arabidopsis?

I hope that's good fodder to keep the discussion going!

I have a question! pick me! pick me!

Could someone please explain fertile triploids (e.g. insigne 'Harefield Hall')? I don't get how some avoid the complications associated with aneuploidy, while most are pretty much sterile....

Thanks

--Stephen

I'd like to know what gene loci were used for differentiating species, and what was the rational for using particular loci.

Hi guys --

I think I'll let Stock explain the triploid issue so we don't cross!

Regarding loci used for IDing species, first off, let me say that IDing 'species' is somthing of a different issu from establishing phylogenetic relationships between species. For the latter, so far, non-protein-coding DNA sequences have been used. These are spacer DNAs that lie in between 2 of the major genes that code for ribosomal RNA. Ribosomes are the guys that make translate RNAs into proteins; in turn, RNAs are the nucleic acids that are transcribed from the original DNA. Try googling some of these terms if the above sounds too obscure. Importantly, the spacers maentioned have nothing to do with the production of any protein let alone any given protein, so the pattern of slipper relationships one gets is detached from their morphology and only represents descent with modification (= evolutionary history). Now we could add lots of other genes to strngthen the story, but this hasn't been done yet for a broad number of slippers, only a few.


As to species IDing, Norris Williams and gang have made a contribution with establishing certain motifs in various DNA loci that mark off Phrag kovachii from the rest. Certainly the same can be done with other slippers. Other possible means of IDing that are under active development are using straight DNA fingerprinting or fingerprinting employing special repeated elements within the slipper genome. But the slipper genomes are really big, which puts a limit on these more standard fingerprinting approaches -- cause there's more DNA to fish through (short and sweet explanation). Other techniques can be used that should overcome this.

There's clearly lots of room for more work in the slipper DNA area, not the least of which is to identify the genes responsible for certain morphological traits. This would be exciting not only for those evolutionarily inclined, but also possible for breeders.

Best from Besk

Read through the posts - most of which flew way too high - & from the little that I can comprehend it appears that 'Advanced' DNA tech wrt orchids isn't really advance! Maybe wrong, just my dumb thoughts!
Mang

No, we are pretty advanced, just need to advance more. Quite a lot is possible now.

No, we are pretty advanced, just need to advance more. Quite a lot is possible now.

If it's not too much of a problem will you kindly elaborate on some of the possibilities.
Regards Mang

Well, we can do a lot more than Cox et al. did to ascertain the evolutionary relationships among slipper species. We can also do a lot better with DNA fingerprinting methods to ID species than have so far been used. Such technologies stem, e.g., from work on plants of agronomic importance. We only need a good bit of money. We can use the markers obtained to map (on chromosomes) genes of interest having to do with things such as color, shape, warts, etc. This would establish patterns of genetic linkage that could in turn be directed toward breeding programs. Once one knows that purple color, big petals, long inflorescence stems and warts are linked (e.g.), then one can direct breeding accordingly -- or even try to isolate the genes involved and use them for genetic alteration (see below). Only money is involved -- the technology has been there for a number of years. Given that we can very likely develop protocols to genetically transform slippers with some gene constructs of interest, we could for example artificially promote early flowering. We only need to do a little research (with a lot of money) to establish a stable transformation protocol. We can probably also transform with constructs that would block anthocyanin pigemnt production to produce an artificial alba. Blue slippers will require much more work on the anthocyanin biosynthetic pathway in slippers, but with enough money, this could be a reasonable goal in just a few years. One may question, however, whether blue plants would be marketable enough to warrant all this expenditure. I would guess that early flowering would be a much more interesting goal.

Thanks Besk! The picture you have painted is exciting and kinda adventurous. Now if we only have the money.....
Regards Mang

Are we dead with this thread?

fertile triploids?

Regarding "fertile triploids", I am not familiar with P. insigne 'Harefield Hall' but fertility in triploids is usually restricted to being a pod parent. If this clone has good pollen, it may not be a triploid. A few tetraploid plants show up in otherwise triploid grexs due to unreduced gametes in the diploid parent. In those triploids that do breed as pod parent (cross really should not have been made) the resulting progeny are aneuploid with varying chromosome counts and should not be used for breeding even if they will form a pod. The fact that they should not be used has not stopped many hybridizers. To bridge the gap between diploids and tetrapoids, diploid protocorms should be transformed to 4n with colchicine.
Dean Stock

Is there spieces with 4n in the Slipper family? Not hybrids or treated spieces, but "naturally accuring"?
If so how common is it?
Has it been seen in Cyps aswell? Could that be part of incompatible problems between species within the Cyp family?

Nice discussion with lot of valuable information

Joakim