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DNA Testing Peppers

AJ Drew

AJ Drew

SHU testing is now within the reach of many hobbyists thanks to South West Biolabs.  Decades ago, my half sister found me and we each paid $200.00 for dna testing to see if we were right about being related.  Now an ancestry site offers the service.  I know a breeder who uses dna testing to prove his dogs lineages.

So why does it seem dna testing of plant material is still out of reach?  I understand it costs thousands.  Not that I am high tech enough to use such a tool, but I'll bet some of you smarter cookies that do a lot of crosses could really use such a service to see which parent contributed to various aspects of your cross.

Thoughts?
 
I don't know much about genetics, but from what I understand, most superhots are so closely related, it would be difficult to pinpoint differences in the genome. They are certainly all the same species. Anyway, I might be wrong. That's just what I was thinking about
 
ajdrew said:
SHU testing is now within the reach of many hobbyists thanks to South West Biolabs.  Decades ago, my half sister found me and we each paid $200.00 for dna testing to see if we were right about being related.  Now an ancestry site offers the service.  I know a breeder who uses dna testing to prove his dogs lineages.

So why does it seem dna testing of plant material is still out of reach?  I understand it costs thousands.  Not that I am high tech enough to use such a tool, but I'll bet some of you smarter cookies that do a lot of crosses could really use such a service to see which parent contributed to various aspects of your cross.

Thoughts?
Click to expand...
 
You can get it done for plants relatively inexpensively these days. There are numerous places, below is just one I picked at random. Costs are basically associated with tissue preparation, which in plants is a bit more tricky, but the rest is identical to testing any DNA. Market forces dictate prices. If there`s only a small demand it`s a special order and you need a few different reagents to extract DNA from plants. It also depends a great deal on what you want to do, so you have to have a specific question to ask. Do you want to compare 2 plants and see if they are related? Profile the DNA into some sort of type fingerprint? There are roughly 4 billion base pairs in the Capsicum genomes, so with such a gigantic amount of information you must have a very specific question to ask, or it`s pretty much uninterpretable and worthless.
 
Complete genomes are being sequenced for around the $5000 mark now and the C.annuum and C.chinense genomes are slightly larger than the Human one. I have a friend who pioneered genome sequencing in crop plants - Rice, Wheat, Barley etc and his lab does it all the time.
 
http://www.biogeneticslab.com/category/plant-dna-testing/
 
Can't wait for the new season.
 
 
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Mene said:
I don't know much about genetics, but from what I understand, most superhots are so closely related, it would be difficult to pinpoint differences in the genome. They are certainly all the same species. Anyway, I might be wrong. That's just what I was thinking about
Click to expand...
 
 
     Humans are all the same species, though and DNA testing works for us. Dogs, too. 
     Some parts of our genomes (genes) are pretty similar from one individual to another - differences capable of identifying familial relationships would be difficult to find here. Other areas (genetic polymorphisms) are kind of like fingerprints. They are unique to each individual and can be used to find out who a pepper's individual parents were.
     I don't think this is what aj was getting at here, though. In order to find which parents contributed which genes to offspring is more difficult. You'd need to identify genomic markers in close proximity on the DNA strand to the genes in question and use those to find out which ones were passed on to the next generation plants. Not as easy as just finding out simple familial relationship.
 
Coldsmoke, the topics on particular peppers being compared and contrasted are what got me thinking on the topic.  But wanted to talk about the science, so thought it best to start a topic void of any particular pepper.  Hoping to stimulate conversation on the science.

Mene, I am so very not high tech.  But I imagine if you had a chart of dna from a Trinidad Sweet and a Trinidad Moruga they would be very common in many areas but in what ever it is that produces the heat, we would see a huge difference.  I imagine same for color differences or other things very distinct.  No real clue, just guessing.

Nigel, ah... so when they were looking to see if my sister and I were really related they knew one specific area to look for, so it was cheaper.  I read somewhere that because all humans carry so much of the same dna that all the dna that would make a person different could fit on a single old floppy disk.  I wonder if plants are the same way.  I am sure the bio engineers at the big seed companies already know these things.
 
 
Hybrid said: " I don't think this is what aj was getting at here, though"

Mainly just trying to stir conversation.  Was thinking that dna could help folk steer their crosses for certain traits.  I am absolutely sure the big boys in seed development look at the dna in their efforts.  Best example I can think of, Monsanto came out with a drought resistant gmo corn.  Tests show it is about as good but no better than an existing hybrid.  I figure they looked at the dna of the drought resistant hybrid to come up with their own version.

So I guess where my head is at is wondering why it seems we hear so much about dna manipulation in gmo crops, but not so much in hybrid / crosses.  Seems like there is tons of science without going in to gmo but we dont usually think about it.  Kind of like when I tell folk I am interested in aquaponics and they tell me they prefer organic as if fish shit is not organic.

Probably rambling... too much coffee.
 
Many Capsicum traits are already liked to specific genes. The classic example is the PUN1 gene (pungency 1). Cultivated Capsicums with no heat almost always have a deletion in that gene, making the gene product unable to function and the plants unable to make capsaicin. Here is an older manuscript detailing some of the genes and their proposed functions.
 
http://www.chilepepperinstitute.org/content/files/Genes%20of%20Capsicum(1).pdf
 
Gene manipulation in GMO crops is basically a one shot and done thing. Blast the plants with DNA and hope a small percentage take it up stably. It enters the genome at random, often in more than one place and can therefore interrupt pre-existing genes in a random way. The way they do it, the genes are linked with what`s called a promoter, which is basically an on switch for gene transcription. Most often they use one from Cauliflower mosaic virus that is very powerful. They also add an antibiotic resistance gene, as if you take all the cells you blast with DNA you can treat with lethal doses of antibiotic and kill all but the ones that have the DNA you want to put in. It all goes in as a single cassette of DNA. Then you grow and cross and hope the DNA stays put. You can basically put anything non-lethal in - herbicide resistance, jellyfish green fluorescent protein, spider silk, elephant muscle, you name it. Every single one has the viral DNA and the antibiotic resistance DNA in addition to what you add. Time frame is maybe 1-5 years, depending on what the plant is.
 
With regular breeding you select for specific traits and breed many generations, selecting at every step. No non-plant DNA is added, only the DNA of compatible plants, so you can`t add a cactus DNA sequence to a Pepper, for instance. Time frame is maybe 4-10 years, depending on what the plant is. 
 
The first C.chinense to be sequences was PI159236, which I thought an odd choice at the time. However, it`s known to have specific resistance genes and they wanted the sequences of those. 
 
ajdrew said:
Mene, I am so very not high tech.  But I imagine if you had a chart of dna from a Trinidad Sweet and a Trinidad Moruga they would be very common in many areas but in what ever it is that produces the heat, we would see a huge difference.  I imagine same for color differences or other things very distinct.  No real clue, just guessing.

 
Click to expand...
 
 
     It's probably not that simple, though. As is the case with biochemicals like sugars, steroids, alkaloids and stuff like that, there's a whole concert of enzymes needed to turn CO2, H2O and other nutrients into capsaicin. Think of it like a Rube Goldberg contraption. All those individual steps have to be taken to achieve the end product. Maybe one pepper has an ability to produce extra enzyme X that is responsible for turning a precursor chemical into capsaicin. Maybe another has a mutation in enzyme Y that inhibits a plant's ability to turn one of the precursors into another chemical besides capsaicin. Both of these deviations may lead to similar ends but through entirely different means.
     There may be dozens or hundreds of enzymes that all play a part in capsaicin synthesis in a pepper plant. Enzyme overproduction, deleterious mutations in enzyme genes, even mutations in other functional proteins that help regulate these enzymes  could all potentially alter significantly the capsaicin production of a given plant. And the genes in question here might be physically located all over the plant's genome. 
     Take home message: which part of your car is essential for it to go fast and where is it located? Engine? Transmission? Fuel pump? Wheels? They all need to work together. It's difficult to predict a car's speed based on one specific part.
 
 
 
edit: cuz it's been a long time since I studied this stuff.
 
Hybrid Mode 01 said:
 
 
     Humans are all the same species, though and DNA testing works for us. Dogs, too. 
     Some parts of our genomes (genes) are pretty similar from one individual to another - differences capable of identifying familial relationships would be difficult to find here. Other areas (genetic polymorphisms) are kind of like fingerprints. They are unique to each individual and can be used to find out who a pepper's individual parents were.
     I don't think this is what aj was getting at here, though. In order to find which parents contributed which genes to offspring is more difficult. You'd need to identify genomic markers in close proximity on the DNA strand to the genes in question and use those to find out which ones were passed on to the next generation plants. Not as easy as just finding out simple familial relationship.
Click to expand...
 
I guess that's kinda what I was getting at. Humans are the subject of an enormous amount of DNA testing, so the human genome is pretty dialed in at this point. Pepper, on the other hand, probably have a long way to go. So it would not be so easy to see the differences right away, or at least know which traits come from where. Again, I have a fairly small understanding of this (just a few high school and college classes with a segment about genetics) 
 
Hybrid Mode 01 said:
 
 
     It's probably not that simple, though. As is the case with biochemicals like sugars, steroids, alkaloids and stuff like that, there's a whole concert of enzymes needed to turn CO2, H2O and other nutrients into capsaicin. Think of it like a Rube Goldberg contraption. All those individual steps have to be taken to achieve the end product. Maybe one pepper has an ability to produce extra enzyme X that is responsible for turning a precursor chemical into capsaicin. Maybe another has a mutation in enzyme Y that inhibits a plant's ability to turn one of the precursors into another chemical besides capsaicin. Both of these deviations may lead to similar ends but through entirely different means.
     There may be dozens or hundreds of enzymes that all play a part in capsaicin synthesis in a pepper plant. Enzyme overproduction, deleterious mutations in enzyme genes, even mutations in other functional proteins that help regulate these enzymes  could all potentially alter significantly the capsaicin production of a given plant. And the genes in question here might be physically located all over the plant's genome. 
 
Click to expand...
 
That`s all absolutely right. In practice, biosynthetic pathways have extra control steps built in, too. Things like turning enzymes on or off at a specific stage. Most also have what is called a rate-limiting step. That`s one step that is like a bottleneck. The size of the bottle neck regulates the rate at which the entire process works, so is rate-limiting for the production of Capsaicin, in this case. This step is thought to be Capsaicin Synthase (CS), which is the PUN1 locus. So if you do not have a working CS (as in PUN1 mutants), you get no capsaicin, but the rest of the pathway is more or less OK.
 
See Figure 1 in this paper for the pathway. Every arrow represents a different enzyme-catalysed step.
 
http://scialert.net/fulltext/?doi=ijpbg.2011.99.110&org=10
 
 
 
 
 

Mene said:
 
I guess that's kinda what I was getting at. Humans are the subject of an enormous amount of DNA testing, so the human genome is pretty dialed in at this point. Pepper, on the other hand, probably have a long way to go. So it would not be so easy to see the differences right away, or at least know which traits come from where. Again, I have a fairly small understanding of this (just a few high school and college classes with a segment about genetics) 
Click to expand...
You are right Peppers are not as dialled in yet, but there is plenty of work on them at this level and things are becoming a little clearer. Here is a paper looking at just C.baccatum varieties and finding plenty of measurable differences in their DNA.
 
http://link.springer.com/article/10.1007/s10722-011-9700-y
 
Nigel said:
 
 
See Figure 1 in this paper for the pathway. Every arrow represents a different enzyme-catalysed step.
 
http://scialert.net/fulltext/?doi=ijpbg.2011.99.110&org=10
 
 
Click to expand...
 
 
     Neat, it's been a few years since I've looked at one of those. I remember seeing synthesis pathways like that when I closed my eyes trying to sleep after taking morning biochem tests after all-nighters.
     But what happens if you have an upstream bottleneck in amino acid synthesis? Are those pretty much fatal for a plant? I'm pretty sure all aa's are essential for plants (maybe not in carnivorous plants... I dunno) but if valine or phenylalanine is just reduced (not absent altogether) could a plant be healthy, but with less capsaicin?
 
 
 
     
 
Hybrid Mode 01 said:
 
 
     Neat, it's been a few years since I've looked at one of those. I remember seeing synthesis pathways like that when I closed my eyes trying to sleep after taking morning biochem tests after all-nighters.
     But what happens if you have an upstream bottleneck in amino acid synthesis? Are those pretty much fatal for a plant? I'm pretty sure all aa's are essential for plants (maybe not in carnivorous plants... I dunno) but if valine or phenylalanine is just reduced (not absent altogether) could a plant be healthy, but with less capsaicin?
 
 
 
     
Click to expand...
Only a tiny proportion of the Valine/Phenylalanine are funnelled into this pathway, so on a plant-wide scale it`s not important.
If you mess up specific amino acid biosynthesis, then yes, you could kill the plant, assuming it isn`t getting them from exogenous sources, such as fungi or bacteria around the roots. In cases like that, if you assume it`s caused by a mutation, then the mutations would be likely to be lethal and you`d never get a plant in the first place. 
In fact, numerous herbicides are amino acid synthesis inhibitors, such as sulphonylurea. Glyphosphate, Round-up to us, is actually an amino acid derivative and messes up amino acid biosynthesis.
 
     Cool. That all makes sense. Do you happen to know if theres some kind of feedback loop in play that shuts down CS? Like allosteric hindrance? ( I think that's what it's called...)
 
Hybrid Mode 01 said:
     Cool. That all makes sense. Do you happen to know if theres some kind of feedback loop in play that shuts down CS? Like allosteric hindrance? ( I think that's what it's called...)
Click to expand...
 
It`s a very good question. I don`t know if there are any feedback loops, but I`d be very surprised if there weren`t.
Allosteric hinderance is more a spatial thing, where the binding of something to something else prevents access to that binding site. That`s one way that feedback loops can work, the product upstream then binds to an enzyme producing a precursor and switches it`s production off (usually). Let`s say, in this case, Capsaicin binds to one of the enzymes that turns Phenylalanine into Cinnamic Acid early in the pathway and prevents the binding of Phenylalanine, so the enzyme can`t work any longer. That would be allosteric inhibition via a feedback loop. 
 
Appreciate the time Nigel and Hybid took.  But gotta say that with what I got upstairs, what Hybrid said makes more sense.  I think I gotta stick with what looks and tastes good because the science you guys are sharing is so far over my head.
 
 

     Take home message: which part of your car is essential for it to go fast and where is it located? Engine? Transmission? Fuel pump? Wheels? They all need to work together. It's difficult to predict a car's speed based on one specific part.
 
 
 
edit: cuz it's been a long time since I studied this stuff.
 
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