Genetic Variation in Vegetative Propagation - Dr. Danelle Seymour

Hi everybody, and welcome to this week's Talking Biotech podcast. When we talk about tree crops and other types of vegetatively propagated plants, we make more trees and more plants typically by taking cuttings or scion material and making grafts, hoping to kind of replicate the good things that came out in that initial variety. And this is the way things are done. And for ages, since going back as far as I can remember, and certainly way back into history, this has been quite an art. I mean, we can think about Roman times when grapes and things were moved around by vegetative cuttings. And this has always been an exciting way to do it because it seemed to preserve the valuable genetics that people really liked. But there's been some new wrinkles that have come out in the age of genomics. And I saw a talk a few weeks ago at Plant Animal Genome out in San Diego, and I thought we have to touch more on this subject. Today we're speaking with Dr. Danelle Seymour. She's an assistant professor in the Department of Botany and Plant Sciences at the University of California, Riverside. So welcome to the podcast, Dr. Seymour.

Thanks so much for having me.

Yeah, this is fun. I thought this was one of the more interesting talks that I saw because it was one of those things that when I saw it, everything in the world made more sense. This explains so much of what I'm seeing. So let's start at the beginning just for the audience that may not be really into plants. Why do we bother vegetatively propagating plants?

So I think you already, you already touched on this a bit. So it allows us to essentially preserve some wonderful combination of genetics indefinitely. And so once a fantastic cultivar is identified, it can be propagated and grown throughout the world and be genetically identical to that original mother plant.

Yeah, genetically identical with an asterisk, right? And it all makes sense because folks who are close to plant breeding understand that, you know, a new citrus variety variety comes up what, once every, not very often. It takes many years to breed a new citrus variety. If you look at the apple varieties in the store, they're all really old in general with some exceptions. So it takes a long time to make it. And so because it's hard to develop and once you have it, you want to hang onto it. Well, why not just plant seeds? I mean, why not?

Yeah. So unlike some of our crop species, which are self-fertilizing, citrus and other tree crops have 2 sets of chromosomes, one from the mother, one from the father, that are not identical to one another. And so if you make seed from those plants, you'll have genetic segregation and shuffling of the maternal and paternal contributions. And so the seedling will be some unique genetic combination that is different from its parent. And the characteristics, of course, of that seedling can be quite different. Yeah.

And I, you know, I see so many examples, especially on Facebook. I do a lot of Facebook and Instagram extension, right? So I spend a lot of time talking to people who they'll say, I planted this apple tree or this citrus tree, and it's been in the ground for 5 years and I'm mad cuz it hasn't flowered. And I'll say, yeah, you probably grew it from a seed, right? And they'll say, yes, but when are my flowers coming? So why does it take so long?

Well, in, in, in tree crops, there's a juvenile period. So when you grow plants from seeds, it can take, you know, 5, 6, 7 years before flowers are produced. Whereas if you graft onto, if you take mature plant material and you graft onto a rootstock, you can get flowers much more quickly just in a couple years. Yeah.

So once you reach that reproductively, that reproductive competence, you now have the ability to produce flowers and that budwood remains determined, or at least turns the corner into a flowering competent state. Once it reaches that point, you can graft it and it retains that, right? Even if you put it on a juvenile rootstock. Or, well, I shouldn't say that. Most rootstocks come from mature tissue too, right?

It depends on the crop species. In citrus, it can come from seed. Yeah.

That's right. Citrus does do it from seed. But, but that's a great example. If you take a mature scion and put it on a juvenile rootstock, what happens?

You'll, you'll get flowering in just a couple years. You won't have to wait the full 7 years. So yeah, it maintains its non-juvenile state.

Yep.

Yeah.

So it's, so the mature tissue is what we propagate because it propagates and gets to flowering faster, even if you put it on a juvenile rootstock, which is tremendously powerful for industry. That's why I said citrus. When you said not all plants, I forgot you're a citrus person. That's where we use seedling rootstocks. I forgot. And we've been working with apples and things and peaches. We do seedling rootstocks too, I suppose. But anyway, so let's talk about, This tree that is a unique resource at University of California Riverside. So you guys are the citrus repository, or at least at University of California, or is there a USDA out there that's the repository?

There's both, and the resources are integrated between the two. So there's the repository for, the clonal repository for citrus and dates is from the USDA ARS. And then there's what's called the Givaudan Citrus Variety Collection. So this is Among one of the largest germplasm collections of citrus and its relatives in the world. Um, it is maintained by Dr. Tracy Kahn here in our department, and it includes over 1,000 different varieties that are maintained as living trees, um, that we can utilize for breeding and also for preserving important germplasm for, for other uses.

It's a little bit of a tangent. We have this problem here in Florida of HLB, the citrus greening disease. And when you have a valuable resource like you have out at UC Riverside, how do you protect it from the disease?

Yeah, so a great question. There's quite a lot of effort that goes into ensuring that this germplasm doesn't become diseased. So of course we have field trees. But, um, through donations, particularly from Givaudan, which is, uh, the collection is named after, we are creating a, it's called Citrus Under Protective Screen. So it's a 1-acre structure where a copy of each accession will be maintained. And then of course there's a backup in a greenhouse in addition that is protected from insect ingress to prevent the spread of the disease.

They do that out here too. Um, just north of where I live in a town called Archer, but north in a town called La Crosse, middle of nowhere in Florida. There's not a citrus tree for miles, and they have a big repository there from Department of Plant Industry, from DPI here in Florida.

Yeah.

And they maintain all the germplasm. It's really quite an operation. But out in Riverside, you have this under the protective screen, which is another thing they do here in Florida now. Works pretty good. But you have one tree in particular that is of special interest. So what's up with the Washington navel?

Yeah, so I will say that it's also under a protective screen because of the spread of HLB in California. So this is a tree that's more than 150 years old. It's actually on land that's, I think, owned and maintained by the city in collaboration with UC Riverside. And so this is one of a few trees, the first navel orange trees that arrived in California in the 1870s. And a few of those trees have not lived so long, but there is one remaining survivor. And so this is called the Parent Washington Navel Orange. It's a historic landmark. Um, and a lot of effort has gone through, gone to protect it over the years, including inarching with numerous rootstocks that provide resistance to various diseases that are spreading in the state, and then now, um, enclosure in the canopy. So if you go up to this tree and you look at the root structure or the base of the tree where it meets the soil, You can see clear through to the other side because so many rootstocks have been inarched to, to, to protect it. And then the original rootstock has decayed.

No, no. And I do some of that intergrafting or bridge grafting where you actually use, and just so folks can understand what this is, is that when that original rootstock starts dying, you can put it, or even when the tree starts dying, you can put in a better rootstock and connect it to the top, to the whatever, I guess, kind of big for call it a scion, but to connect it to the top of the tree. And as long as you connect the vascular plumbing, it will eventually grow into it and provide multiple ways of connecting the top of the tree to the earth, even if the original roots die and decay.

Yeah, it's amazing. And the, the, I've seen this ongoing for another historic tree that's on site and they essentially just, you know, wound it, make the connection and zip tie it to ensure that it doesn't come apart. And, and with enough time passing, they'll, they'll become, you know, healed and connected.

Well, zip tie is a great idea. I even, I use electrical tape because I like that.

That works too.

But I think zip ties, there's something about that. Oh, this is really cool. So with this Washington navel, and it may not be that tree, but Washington navel in general, what is, what is the evidence that it creates a lot of vegetative sports, you know, genetic variants that come from the original tree?

Yeah, so, uh, I mentioned the germplasm collection, the Givaudan Citrus Variety Collection earlier. And so this is where my interest in this historic tree and in sweet oranges in general arose. So of those 1,000 or so accessions, there's over 70 or 80 entries that are navel oranges. So these are, um, accessions are cultivars that have been preserved because they have some unique characteristic. So that can be like the pigment accumulation in the flesh of the fruit. So like Cara Cara is a variety that's lycopene accumulating. So this is a sport of Washington Navel, and there's many other sports with unique characteristics. Often it has to do with seasonality, so the, they will produce fruit a little bit earlier in the season or a little bit later in the season, and it allows growers to, you know, have a larger share of the market. And so, um, I became interested in these 70 or so accessions that are all supposed to be genetically identical to one another. So they're clonally related, but they have this amazing range of phenotypic characteristics. And so me and folks in my lab became curious of, you know, what's going on to cause these, um, phenotypic differences.

Well, this is the first part of Talking Biotech today. We're gonna take a break here. We're speaking with Dr. Danelle Seymour. She's an assistant professor at University of California Riverside. And we're talking about genetic variation that happens in tree crops, or maybe in other vegetatively propagated plants. We'll learn about that on the other side. But this is the Talking Biotech Podcast, and we'll be back in just a moment. And now we're back on the Talking Biotech Podcast. We're speaking with Dr. Danelle Seymour. She's an assistant professor at the University of California Riverside, and we're talking about mutation rates and how these happen in places they're not so supposed to in vegetatively propagated material. And before the break, we were talking a little bit about the Washington navel and its offspring. But before we get to that, you know, you can— you and I share kind of a common thread in that you were an Arabidopsis person before citrus, right? So how did you get to citrus from Arabidopsis?

Yeah, so I am a geneticist, and so I am interested in so many questions related to inheritance and the relationship between genetic variation and phenotypic variation. And so much of my career I studied that relationship in Arabidopsis and some grass species and other plants or crops that were suitable for addressing specific questions. And then I, I transitioned to citrus just 6 years ago. UC Riverside has an enormous citrus research community, and there was an opportunity for a geneticist to join that community, and so I snatched it up. And so it was just a sort of good timing.

Well, it's also a good lesson for students who listen to this or postdocs who think that the, we figured everything out and the world is saturated and what am I gonna do? But I think I had the same thing when I came to Florida and interviewed. I was in a Arabidopsis dude who had a pretty heavy toolbox and they said, how would you like to work on strawberries? And I said, okay, that's great, but I'm gonna need some strawberry trees. I mean, I knew absolutely nothing about horticultural crops and coming to a horticulture department, they were interested just in somebody who maybe has a dusting of ADHD who might be willing to do a lot of stuff with clever solutions. And so that's where, that was my kind of story. But citrus is the same thing, much more interesting than Arabidopsis though.

I mean, you can, like you said, it's the same toolbox and you identify interesting questions in the crop or species that you're working on. So there's some cool questions that you can address in citrus that are more challenging in other species. I think the only real difference is a lot more patience is involved just because it's quite a long time to develop populations and advance generations.

Yeah, it's definitely true. Now, correct me if I'm wrong on this one. So I remember, so I was a department chair here for almost 6 years, and before me, there were a number of other department chairs. One of them, his name was Al Kresdorn, and Professor Kresdorn allegedly brought the Cara Cara orange to Florida from Venezuela, from a Washington navel. Does that seem correct?

I don't know the history of Cara Cara quite so well, but I do remember that it came in the, in the '70s from Venezuela. So I think that part is pinging, pinging my memory, but I don't know much more than that.

And so I think that fits. I was kind of wondering if it came through, you know, Riverside or through that famous Washington Naval. So it came to the US in this other way, probably in a suitcase with greening disease, right? Or one of those things. But all right, so let's go back to the main topic. So you have all this phenotypic variation, which is interesting because these are the visible traits that made somebody say, we're going to take this piece aside because it's of interest. But when you start to take those very, very visible traits, how many other traits are coming along? Or I should say, how many other genetic changes are coming along with that? And how do you go about sorting that out?

Yeah, so maybe I can first just give a little bit of background on sweet oranges in general to talk about.

Yeah, very good.

So all sweet oranges are clonally related to one another. So that includes Valencias, which are commonly grown in Florida, blood oranges, the oranges that have the anthocyanin accumulation, and also navel oranges, which is the orange that's most widely grown in California. So these have all been related. They come from some common ancestor at some point, and then have sort of diverged in their own lineages over time. And those oranges have clear Visible differences. So the timing of fruit maturation, seediness, juice characteristics, et cetera. And so, you know, with the decreased cost in sequencing, we can begin to understand how these different lineages are related to one another, how much variation there is between them, and then also within them. And so we really focus on navel oranges because that's what's most important in California, and we have access to all this important material.

Mm-hmm.

And so one challenge with studying the mutations that are occurring in the somatic or vegetative tissue of the plant is that they may not be found in all the cells. So they can occur in just a portion of the cells. And so the way that you go about doing the sequencing is really important for identifying these mutations. And so we essentially Oversequenced a bunch of branches and different cultivars of navel orange. And we found that there are thousands of mutations that are found that distinguish these different lineages and also the sports or the varieties within lineage.

Yeah. So when you mentioned that, you know, there's maybe a population of cells in the plant that have it, is this from some sort of mosaicism that happens in the initial bud or where does that come from?

Yeah, so, you know, mutations can occur in any cell, right? And if they occur in a cell that's at a sort of developmental dead end, they will be harder to transmit to, through propagation. But if they occur in a cell that is transmitted through the meristems, they can increase in frequency in the plant and be more likely to be transmitted via asexual propagation or grafting, essentially. And so it's not that mutations are necessarily happening in some types of tissue more than others. I mean, possibly, I don't know exactly, but there are like sort of characteristics of different tissues that will make it more likely to be transmitted.

And along that same line, just for folks who maybe aren't so familiar with this, how are mutations occurring? I mean, it seems like biology likes to constrain mutations because certainly they can lead to disease in humans when we have errors, things like that. So how does it happen in plants and is there some sort of maybe adaptive significance to it?

Yeah. So every time a cell divides, the DNA has to be copied. And although the machinery that does the copying has incredibly high fidelity, it does make mistakes. Some of those mistakes can be repaired and some are not. And so it's just sort of a chance occurrence that gives rise to these differences. Thinking about somatic mutations in plants is very similar to thinking about somatic mutations in humans. So like the types of mutations that give rise to cancer are occurring in somatic tissues similar to what we're talking about in plants.

So in all of your genomic analyses, when you sequence, you know, trillions of Bases of DNA. It is funny how cheap it's gotten, by the way. I mean, we're sending off 20 genomes this week and, you know, at one time it took us years to do one, you know? But, so what are you finding when you do lots of deep sequencing? How frequent is it to have mutations?

Yeah, so we focused on this 150-year-old tree with the thought being that More cell division has occurred because the tree is older, so it gives us more opportunity to identify these mutations or errors. And, um, I think that, that bet worked out for us. So in a single tree, if you sequence different places across the canopy, you can identify more than 10,000 mutations. Most of those mutations are not found in all the cells, so they're, they're occurring in just a fraction of the cells. And I think the decreased cost in sequencing allows us to identify those like lower frequency groups, which wouldn't have been possible even, you know, 5+ years ago.

Yeah, I remember this image on your slide from your talk that you have this really old tree and that if you can kind of picture in your mind's eye, you know, a tree that then you're able to sample from the inside out and be able to essentially walk across a timeline. Right. You could actually say this was the first branch that came off this thing, and here's the most recent twigs that came off this thing. And you can actually see that progression. And do you see like an accumulation of mutations the further you go away from the center?

Yeah. So it's, it's for the types of mutations that we can be confident that are transmitted through these branching events, we see that pattern. So the, the newer parts of the tree, which have undergone more cell division, have more mutations. And this is consistent across There's 7 branches of this historic tree. So that was consistent across all those 7 branches.

Is there any kind of pattern as to where these mutations occur, or at least are allowed to stick around, like, uh, in, uh, in genic regions, or are they, you know, encoding regions of genes or just kind of random?

Yeah. So that was a question we were really interested in because, um, it's not clear. It wasn't, it's, you know, we're still in the early stages of understanding whether or not there are Is selection, for example, acting within the tree? Of course, we know that selection can act when you go from parent to progeny, like via seedlings. And so one expectation is that if there's some force that's shaping the pattern of mutations that we discover, we would expect to see fewer mutations in genes or fewer number of certain types of mutations in genes. And that's indeed what we found. So we did find a signature. some signatures, especially of purifying selection, where certain mutations that you would expect to have damaging effects in genes are, are not found as frequently as one might expect.

And, uh, what's next in the project when you start, uh, thinking about the next steps?

Yeah. So I think one thing that is clear, not only from our work, but there's many other folks that are doing wonderful jobs studying somatic mutations in tree crops and other clonally propagated crops is A lot of these mutations are not found in all the cells, but they're found in specific layers of cells that make up different parts of the plant. And so I think it, so essentially we have a bunch of chimeras, right? And so I think it's important to try and understand how being genetically mosaic or chimeric affects You know, the folks that are producing these trees. And so we're going to dig in a little bit and try and understand, you know, how phenotypes are related to chimerism and what that means for asexual propagation and also for the industry. So some of those interesting characteristics can be lost because the mutations aren't found in all the cells.

And kind of moving away from citrus a little bit, what are some other trees which are well known to produce these kinds of vegetative mutants?

So thinking just of crop species to begin with, I mean, grapevine, right? So there's many important sports of grapevine that lead to, or are the basis of specific wines that are produced. I would say almost any tree crop, right? So I can't think of— grapes are the first that come to mind. Yeah. Right.

That's a really good one. That's what I always talk about because, you know, I always talk about 1960s 62 in Bruce's sport from Sultana, which turned out to give rise to the golden raisins, you know, in a mutation in polyphenol oxidase or something. But yeah, I only asked that question because someone meant the other day, since I saw your talk, I've been watching the buds of trees a lot with a very different eyeball.

Yeah.

And I saw a branch or piece, newly emerged tissue on a certain variety of peach that on Tropic Beauty peach, which was vegetative. It was growing without a terminal bud. And I thought, this is really cool. And I asked the peach breeder about it. He says, oh yeah, that thing throws mutations all the time. So I thought this was something really cool. It turns out it's really not. And folks who do apples, I mean, there's a mountain of, if you go Gala, there's a whole bunch of different Galas. So it seems to be like you're in a very interesting area at the front edge of a very interesting area that people are just really starting to dig into meaningfully. So very cool. Cool stuff. Is there any other question I should ask you?

I mean, I think we covered the basic interesting aspects of the history of sweet oranges and some of the, some of the main results from, from that talk. Yeah.

Is there, is there, um, like a utility of this? Maybe I can ask you about future breeding efforts. Is there a utility in, uh, can you induce mutations? I don't know what, let me, let me just ask you about that real quick. So in both of our universities are very interested in pushing, uh, breeding of citrus and how can genetic variants actually speed this process? I mean, the tree is already a good producer of a good quality product. So is there an effort to look at sports more carefully now, or even possibly induce mutations in sports?

Yeah. So citrus scions are typically bred in one of two ways so that you can use traditional breeding by hybridization to make new genetic combinations. But often we have some favored cultivar that has some deficits, often seeds, or it's common to use radiation or other mutagens to induce mutations. So we just talked about natural mutations that can arise, but we use mutation induction to develop more quickly sports that have some characteristics that are of interest. But one application, I think, of understanding the levels and types of mutations that occur naturally is that In order to ensure that we're not spreading diseases in citrus and other tree crops, we have to go through an important stage where we do what's called shoot tip grafting. So this is taking a microscopic meristem and grafting it onto a rootstock in tissue culture. And the cells that exist in that meristem will affect what results. The clean material that will essentially be disseminated to, for commercial production. And there's a lot, so if you have genetic mosaicism or chimeras, you can lose some of the mutations at that step. And it's very common to have a tree that doesn't have seeds, for example, and you go through the shoot tip grafting and then you end up with something that's seedy. And so understanding what's happening during that process and what mutations remain and which ones are lost can help us to make that more efficient in the future.

It's a really cool process and a really, really good point. You know, I, uh, you know, wish you all the best in the future. And when something exciting happens, give us a shout and we'll talk about this more. So thank you very much for joining me today.

No problem. My pleasure.

And if you have fruit trees at home, look carefully at those buds and look at the variation that comes off of the branches and twigs. that come off of your big scaffold branches, because you might have something there that could be the next revolutionary plant, the next, the next variety that revolutionizes an industry. I mean, I mentioned the golden raisins that came from one funny little variegated sport that came off of a popular variety. So it may happen to you. So watch your plants carefully. Talk to them. Tell them I said hi. This is the Talking Biotech Podcast, and we'll talk to you again next week.