Breeding the Next Amazing Apple
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Kevin Folta: [00:00:00] Hi everybody. And welcome to today's podcast. Today. We're going to talk about genetic improvement of an important fruit crop that we all recognize a one a day. Keeps the doctor away. You give one to your teacher. If he or she is doing a reasonable job. Yeah. That's the apple right? Apples. We think of so many varieties in the grocery store that, okay, you've got your red delicious, your golden delicious, your Fuji's, your galas, your whatever you got.

But that big selection represents only a tiny sliver of the number of varieties are out that are out there, which experts say is more than 8,000 different kinds of apples that are. So why so many, but yet we see so few and really what it is is the few that were selected for wide commercial growth are those that can be stored or those that can be shipped.

Those that can survive disease because apple has many different challenges to be able to get. So today we're going to speak [00:01:00] with Dr. . He's in, he's an associate professor in the school of integrated plant science at Cornell university, and he's an expert in apple disease and apple breeding. So welcome to the podcast, Dr.

Khan. 

Awais Khan: Thank you, Kevin. Thank you for having me. 

Kevin Folta: Yeah, this is really, really neat. I, I really was excited that you were able to join us because we've talked about apples on the podcast before it's been a while. And really it was about time. We started. Come back to speed with this particular crop. So let's start at the beginning.

If I'm an average listener, as I mentioned in the introduction, there's so many different kinds of apples apples aren't broken, right. So why our science is trying to improve them. 

Awais Khan: Yeah. So it's a very good question. And as you said, there are so many. Pathogens out there. There are so many biotech and air biotic stresses.

What apples for any crop out there and pathogen populations are also changing over [00:02:00] time. But in addition to that also consumer demand and preference for different traits is changing with the times. So to meet all these challenges for apple production, which is a bacterial pathogens, fungal diseases, viral diseases, as well as insect and pest pressure and air biotic stresses.

Drought, huge stress and so on and consumer preferences, we need to really come up with new varieties, which can perform in this stressful environment. 

Kevin Folta: Okay. So new varieties. So how long does it take to make a new variety? I mean, it must just be what a couple of months. 

Awais Khan: Ah, that's a good question. Yeah. So we are talking about apples and let's step back and think about how apple breeding could be done.

I think to do apple breeding, we have to think about biology and genetics of apples. [00:03:00] So apples are heterozygous. They are highly heterozygous. I will say that at each part of their genome, there's a lot of diversity there as well as they are. They have a very long juvenile phase. So if you grow apples from seed, until you get first flower, it can be four to six years or seven years, depending on.

Which a variety of apples you are growing. So if you take these two factors in account, the high level of factors, the gusty, as well as the juvenile phase. And if you think that an apple breeder shoe needs to be. Four to five crosses. So you can multiply five years of journal phase with five crosses.

It will be 25 years. So it's a long process until you can get some reasonable [00:04:00] quality and some reasonable traits in a new background. 

Kevin Folta: And so when you're talking about a juvenile phase, it really is a juvenile phase that really is characterized by the inability to reproduce. Right? You can't flower during that phase, you're growing just kind of like a juvenile human.

You're not competent for reproduction. So you have this long time. You have to wait before you can make a cross. Then you make the cross. The next generation has a long juvenile faith it's just takes forever. How much of a problem is it? You mentioned this idea of heterozygosity, which means that you got a chromosome from mom and a chromosome from dad, and a high degree of heterozygosity means that at any one point on those two chromosomes, they're not, probably not the same good mixture of genetic variation.

How much of a problem is this idea of self and compatibility? So in other words most apples can't cross with themselves. You have to cross them with yet another [00:05:00] completely different genome. So how much does that mix mix? The whole situation? 

Awais Khan: Absolutely. We have different self incompatibility in apples.

And when a breeder is making choice of parents and making a dis designing a breeding program or making plan for crossing, they have to really account for that because they cannot cross apples, which are which have similar SLE yields because those apples will not be. Th there will be incompatible.

And also if you want to create homozygosity at certain regions of the genome, like certain gene , which controls specific traits like you need to make. Something, and that is also not possible as you said. So therefore we have to work with heterozygous genomes, which create a lot of variation at each locus.

One example could be that if you take an apple, [00:06:00] it has. Chromosomes from father and mother and at each locus of one gene, which controls, for example, fire, blood resistance. You can have two versions of that gene in one parent, and then you have a chromosome from another parent. It can have another two different.

So at the end, in the progeny, in the first generation, you might have for a leader of the same trade for the same gene segregating in the progeny. So a lot of variation in a given cross. 

Kevin Folta: And you mentioned a Fireblade is one of the low side, for instance, resistance to Fireblade. What are the major problems to apple?

What are the major challenges in getting it to grow and be productive? 

Awais Khan: So there are a lot of challenges for apple production and any [00:07:00] crop you, you, you talk about. So if we have to really think about the whole value chain from. You know, start to the and until a product goes to the consumer.

So I can talk more specifically about breeding challenges are genetic challenges. So in terms of production system and in the production system, you can have a lot of diseases. And I mentioned previously, you can arrange a fungal diseases out there. You can have Rangers. Bacterial diseases, you can have range of viral diseases and insect pest pressures as well as air biotic stresses out there.

So. An apple grower has to deal with all this biotech antibiotic stresses in the field. But at the same time, I think there are also economic pressures, as well as availability of labor, availability of different [00:08:00] mechanical options for managing the. As well as storage issues. So again, you can look at the whole entire, whole entire value chain and you'll see a lot of problems out there.

Yeah. But 

Kevin Folta: so many of these at least biotic stress problems. I thought we were getting on the other side of that, because there were genes that were associated with things like, you know, like the VF gene or genes for apple scab, the genes for that were associated with resistance to Fireblade some of the disease issues rootstocks that conferred some resistance.

And so just for the audience, apples are always a clone. Or they take a cutting from a top, and then you graph that onto a rootstock and the rootstocks confer a certain degree of a resistance for certain diseases or control growth or size or whatever. So we're talking about the sign on the top part of the tree.

So are there, has there been good genetic discovery in resistant material to things like fire blight and other [00:09:00] diseases? 

Awais Khan: Yeah. So there is a lot of research which has been done to understand the genetic control of disease, resistance and apples. So as you mentioned about apple scab, there are more than 18 genetic regions in the genome of apple.

They have been, these have been identified to confer resistance to apple scab. So the level of resistance for this 18 to 20 loss is different. Some are major, some are moderate, some are minor. Perfect. When we talk about fight or flight, also a large number of QTL are legals for resistance have been identified.

To show resistance, fire blight, and so on. But I think in the man challenge is how to incorporate that resistance into breeding lines to, to use it for breeding resistant, apple cultivars. [00:10:00] And I think one of the constraint there is most of these resistances are. Identified in wild, apple germplasm apple accessions coming from different Marla species, apple belongs to genus called malice.

So those are for general audience, we can call them crab apples, and those crab apples usually have very low fruit quality, fruit size. You can also refer to. And when you make CrossFit Good quality apples, you will get first-generation, which will be very low food quality and very low far the consumer traits.

And then I mentioned previously that juvenile phase is very long for apples, so to retrieve good quality, one has to make seven crosses and. Going to be several years until you drip. Good fruit quality. So that's the big challenge. I mean, loss hive have been characterized, [00:11:00] but how to incorporate them in breeding programs.

That's a big. 

Kevin Folta: Well, that's one of the big issues. The other part of this though, is have the priorities for breeders changed over the years. I know that if you don't have disease resistance, you've got nothing. How Genesis is there on things like flavor and aroma now, maybe even flesh color for it. 

Awais Khan: So here I have, based on my own understanding of apple and breeding genetics and how apple industries, I think that more emphasis for apple breeding and genetics so far has been given to fruit quality and consumer traits rather than a disease resistance.

And there is a reason for. Because consumers. They are more into fruit, quality traders and maybe cosmetic appearance of the fruit. They don't see what is [00:12:00] going behind the screen, which is really the struggle. The apple growers to deal with disease diseases in the orchards and in the production system.

So, so far I think a lot of focus and emphasis was given to to, to food quality traits and apples in, in my opinion there have been some in past there was some programs, breeding programs like PRI Purdue, Illinois breeding program that was really focused on disease resistance. I think it takes a lot of effort and long term commitment to really breed for disease resistance.

Kevin Folta: Yeah. That's that's and you can always can control it with other types of controls, you know, you could spray or whatever. If you have a relatively small orchard, there are other ways that don't require the use of genetic controls to be able to confer resistance or, well, at least to confer tolerance, I should say to disease.

[00:13:00] So, this is all really good stuff. We're speaking with doctor of a con he's an associate professor at Cornell university, and we're talking about the genetic improvement of apples and where it is. And now after the break, we'll talk about where it's going. This is collaborative talking biotech podcast, and we'll be back in.

And now we're back on the talking biotech podcast. We're speaking with Dr. Khan. He is a, an associate professor at Cornell university working in genetic improvement of apples, primarily around the aspects of disease. And when we're leaving it off before we're talking about the problem with apple breeding, that it takes a really long time to generate a new favorite line.

And that the new lines not only have to have all the resistance to disease, but also have to have size shipping and be reasonably flavored and colored. I mean, all the things consumers want. And so what you're trying to [00:14:00] find is this very tiny little overlap in the Venn diagram. That happens only once in a very long time.

Like once in thousands of trees that you may get a favorable combination of genes that is commercially viable. So are there other ways to do this? And so I'd like to focus on biotechnology and what's being happened. What's happening right now in breeding programs with respect to like say genomic selection and marker assisted breeding.

Awais Khan: So Kevin, you have seen that there a lot of developments On genomics and genome sequencing site and especially for fruit crops. And so if you remember, the apple genome sequence was published first in 2010, and now we have many genome sequences of apples available and the very high quality of genome sequences.

And then. Other hand, there are a lot of [00:15:00] developments on biotechnology side. We have seen new opportunities with transformation, genome editing, and at the same time on genetic analysis side, new tools and analytical platforms have been developed and pipelines have been assembled. For example. And flawed genomic selection and also marker our genome assisters selection.

So all this developments with the within apple genomic sequencing and also a broader community of biotechnology, biotechnologists and geneticists we are in a very good spot at this time that we can make big differences in accelerating the improvement. Of of prenatal trees, specifically apples and develop new varieties, which are really re really good for the production systems environment.

And as well as for the. 

Kevin Folta: Yeah. Well, let me backfill that just a little bit, just to keep the [00:16:00] consumers engaged, who are trying to figure out what is this genomic selection thing, and when you have genomic selection, it means that you're able to look at members of a population or members of a, of, well, any plants, genome, and identify specific regions that may be associated with a trade that you're interested in.

Say a certain flavor or color of the. Or disease resistance, and then be able to look for these little signatures in seedlings. So you make a cross look to see if those seedlings carry those little signatures in that way. Instead of having to grow the tree for five years to find out if it makes a fruit that is reasonable and that resistant to disease, you can have a certain statistical likelihood that that seedling will.

Have a favorable presentation of the traits of interest. And so this kind of genomic selection really is accelerated breeding and challenging fruit crops, especially in tree fruits. So let's talk about the idea of [00:17:00] transgenics that happen. And we, we think of these as the genes, where are these plants where we can move a gene from one to another.

We know that you know, there's this Arctic apple that was, was available. What is that? And is it something that's really. 

Awais Khan: Yeah, of course. So first I go a little bit to the genomic selection. I think genomic selection, because we can develop now a lot of large number of DNA markers across the whole genome of apples or any, any crop.

And we can develop this prediction models to to predict what trait will be in the progeny without phenotyping them in the future. So depending on the trades, if they're highly heritable or if they have no heritability, we can apply genomics selection too, to a certain extent, but market messages, selection it can be a way to move forward for highly heritable traits.

We have diagnostic markers, for example, disease resistance. Now to your second part of the question, Arctic apples. That is a big big development and encouraging [00:18:00] for all of us in who are working with biotechnology and crop improvement, because this are our NAI interference based technology to silence this PPO gene, which if you don't sign as it, it can create Browning of fruit was a huge success.

And it has been approved from by the us by the, by FDA. And those Arctic apples are now available. You can also go to Amazon and fresh market and in certain places you can get. You can purchase those Arctic apples. They have been developing Arctic apples in the background of golden delicious, as well as gala Ganesh mate.

And I think they will be exploring in different backgrounds as well. So it's really encouraging and a huge opportunity for all of us. We're working with biotechnology. 

Kevin Folta: That's exciting that that went through because it was really the first fruit other than the papaya. To, to get approved, but also has a consumer [00:19:00] facing trait, right?

All the other traits in genetic engineering work to make things better for the consumer. I'm sorry for the farmer. This one was making a product that was better for the consumer. And so that made things pretty exciting, but aren't there varieties of apples that have very low PPO oxidase activity already so that you wouldn't have to use a transgenic to get to the same place.

Awais Khan: There is variation in the expression of PPO genes within apple, but I think still to get to that reasonable decrease in the expression and to ha to keep it non Browning for that long time. This is a really the way to go forward. So there are close to 10, 11 PPO related genes in apples, and this RNI based silencing reduces, it reduces the expression up to 10% of the [00:20:00] original level of this enzyme.

So that's really really big advantage of this technology. Whereas if you use natural variations, there will be still still variation in there. And we cannot achieve you to that level of what about 

Kevin Folta: other transgenic, apple modifications that have been particularly exciting or particularly useful?

Awais Khan: Also again, my research in Fireblade that this is a resistance, we have seen that. People have used. Trust genes of, I will say, assist genes, genes from the other Mala species and then transform gala apples, which are highly susceptible to fire blight. And. They reduced significant level of fire blast susceptibility in gala against fireplace.

So there are several diseases is genes which have major a major impact on. Susceptibility and resistance people have [00:21:00] functionally proved that you can get very high level of resistance if you transform apples with those genes. But those genes are present in wild crab apples. And if you use breeding approach, even if it's marker assisted breeding still is a long way to achieve that level of.

But resistance to brooding of the Sam gene. 

Kevin Folta: Well, I think they demonstrated that very well with the VF genes. So the genes associated with apple scab, right? That's apple scab. That they began breeding for this in what the 1940s with, as a Malus floribunda that had the resistance or Sylvestri or wherever it was one of the wild apple species had a resistance gene that they bred in and it took some something like 50 or 60 years.

Whereas a group in the Netherlands took the gene out of that wild apple and plugged it into domestic apple and was done in five. 

Awais Khan: Absolutely. And you're referring to VFD in which is coming [00:22:00] from mother's floribunda 81, which is Japanese cap apple. And if you look at that apple I have that apple growing in my apple collection here.

These are very small crab apples, as you don't want to eat them, but you will never try them again. But the breeding, as you said, this PRI program was a breeding for resistance, a scab, and they use that to, to bring it to commercial background, but it took very long time. That's right. And with genome.

Based biotechnology based approaches. It really takes a very less time compared to conventional approaches. Absolutely. 

Kevin Folta: Well, what about the trans genes that control flowering and how can those be helpful inside a breeding program? 

Awais Khan: Yeah, this really brings a very close to my own specific interest.

And I will say that my program is [00:23:00] really interested in. To develop pre-breeding lions it lines. Which are available to the breeders to use for developing final cultivar, a final product, I will say. And I have been thinking for a long time. Okay. What is the best way to achieve that in a reasonable time to bring the diverse diversity present in plants?

So that can be used in breeding programs and consumers and producers can use. And enjoy it. So as we said, apple breeding takes long time. So one approach I see is pre-breeding programs can use different approaches to develop disease pre-breeding lions, and I'm using one of this approach. And that approach is I'm using early flowering, transgenic, nines of apples.

Which were developed by a [00:24:00] research program in Germany. And I have those lions in my greenhouse and uses lines to make cross with wild apples. So basically this lines, which make early flowering If you plant seeds from these lines, they will start flowering in one year instead of five to six years.

Okay. So now we use these lines to CrossFit wild apples. And because we know with gene, we have in this lines, And we also know which gene is present in the viral apples for disease resistant. We can use DNA markers to do selection of first-generation. Gary, both early flowering gene, as well as disease resistant gene.

And then first-generation fiddling, which has both of these traits. We cross it [00:25:00] back to your favorite apple, for example, Honeycrisp and then when we crossed with Honeycrisp. The next generation, which we get we against select for early flowering and that disease resistant gene, which we were intending to incorporate in Honeycrisp background.

And, you know, Vivian trying to increase the proportion of good quality, good quality traits of apples in together with the disease resistance. Bye. Excellent. Rating it through early flowering gene. And in this process, we can also incorporate another disease that is a gene from another background as well.

In this process, we can also speed up and make cross of this second generation early flowering and diseases and dying with another good quality apple so that we don't have the issue of. [00:26:00] Self-fund compatibility in a crossing with the same background again again. So in that way, I foresee that in four, five years, you will have pre-breeding lines which have two, three diseases, instant.

In the same background together with early flowering gene. And now at that stage, depending on the goal I can select against early flowering I can keep early flowering and make future crosses to incorporate more disease resistant are other traits in that background. But if I selected against it, then I can make.

Pre-breeding line without early flowery gene to the breeder to make next cross tests for final cultivar development. So that's my approach at this point. That's 

Kevin Folta: really cool. I [00:27:00] think it's using these kinds of tools to accelerate because the end product is not genetically engineered, right? You're just using the rootstock.

That's genetically engineered to induce the on the flower. Is that. 

Awais Khan: So in this case the science is a transgenic. The gene, which we have in this line is from silver Birch is from another plants, a little Birch. And the sign is transgendered because we are collecting Polands are we are using the, that transgenic line as a matter of.

But as I said that we have, we know which gene we have put in this this transgenic line and we have DNA markers. We can select it precisely selected out precisely when we really want to check it out and to further confirm it. Now we can also do deep [00:28:00] genome sequence. And make sure that this newly developed early newly developed disease does this 10 lines.

They don't have any trace of a trans gene or even early flowering gene from Silverberg. And that can be done very precisely with the high depth genome sequencing. 

Kevin Folta: Yeah, I totally remember. Now I've forgot. I read that paper about the silver Birch gene. That's kind of how we connected. I think because I was interested in how do you make something flower faster?

I'm intrigued with ancient apple germplasm that is present in the south. I live in the deep south and. In the 18 hundreds, every yard had a tree throughout Mississippi, Mississippi, Alabama, Georgia, South Carolina. And in north Florida, you didn't really have it, but there are some trees here that are spectacular and you find some varieties that are old varieties from the ninth or the [00:29:00] 1830s, 1860s, 1880s, where these trees are, are alive and well on their own roots are on maybe just generic, apple roots.

So putting them on modern. Rootstocks may really be an exciting opportunity, but these trees have resistance to disease. Just, you know, they were all selected before you had pests and pathogen controls like fungicides. So you pick these things that are super Hardy that maybe don't have the best fruits in the world and maybe not the most storable fruits, but this is really what I'm interested in.

How much work is there being done right now to kind of curate that old germplasm and maybe look at it for genes associated with diseases. 

Awais Khan: Yeah, that's a very good question. And I really think that we should go a little back to in history to, to really talk about this collection and digital plans.

So if, if we really think that [00:30:00] when we got this domesticated apples in the in north America, in this continent and it's 1600. The Europeans came to America and they brought apples with them. And we also know that the host and pathogen deco evolve in the same environment when they are there for a long time.

So now we can really think that and we really know that apples domesticated apples never had chance to co-evolve with the pathogens. Present in this continent until before the camp here in 16 hundreds. So most of this apples are susceptible to domesticated apples are susceptible to a wide variety of pathogens and insects, but luckily we all have access to.

Lugs, one of the [00:31:00] largest collection of Mullah species here in Geneva, New York, which is which is which is in responsibility of USDA and Cornell as well. And we have more than 6,000 different apple accessions in this collection is world's largest collection and it has more than maybe 30 different species of malice in this collection.

And you can see apples from Kazakhstan, apples, from Turkey, apples, from From Asia from everywhere around the world. And this diversity of apples has a lot of traits, which are very important for breeding for biotech, nearby distress tolerance, as well as production traits. Many researchers in the us and around the globe, they have been exploring and screening for and characterizing this diversity for many [00:32:00] traits.

So my program has also used this collection for a long time to look at scabbard resistance, to look at firebrand resistance, to look at powdery, mildew resistance and so on. So a lot of Djarum plasma novel sources of resistance have been identified in this journal Plaza. Now we have to move to another lab a stage, and that stage is really how to make use of this diversity and put it in the production system so that we can actually experience and enjoy that diversity for flavor, for fruit quality trays, for disease resistance, for the production traits.

And as I was saying earlier, that now we have so many tools available and oppurtunity. Around around genomics genetics, as well as biotechnology and accelerated breeding. And we are, and many programs are really now trying to make use of this diversity to the. 

Kevin Folta: Oh, that's really cool. I think [00:33:00] maybe it's kind of a goal in my lifetime.

I only got maybe 50 years left to come up one for Florida. Well, but I think that we can do it. I mean, there's some really interesting things that came out of Israel came out of university of Florida that were low chill apples, but they're also have seen, and you can tell me if I'm crazy that we've planted things like Honeycrisp here in Florida and they flower.

With 400 chill hours and other a friend of mine who was going to be my student down in Orlando. He has apples growing on trees with probably a hundred chill hours. So it seems like there are other factors at play with, with males. That maybe we haven't, we just haven't tried it with the right rootstocks and the right cultural conditions and that things like the foliation, which they do in the tropics, other ways may allow farmers in warmer areas to be able to start to grow this crop, if you can manage the [00:34:00] disease.

So it may just be that I'm kind of thinking that apple may be finding an interesting new Renaissance between the efforts with molecular breeds. Things that are going on in your program. Things that are going on in Minnesota and in Washington, but then in all around the world for that matter.

But also, you know, trying to expand its range a little bit and looking at some of the old germplasm. So, you know, what do you think is the future for apple? Where are we going next? 

Awais Khan: You're right. I have Ana apple from Israel and this is also, you know, one of the earliest earliest maturing apple here in upstate new.

So there are a lot of apples, which are grown in Southern regions, for example, in Israel, as you were saying, but also South Africa. And they have very low chill hours and I think it should be explored in more depth. Another thing, interesting thing, I will say that we were talking about malice.

PCs and did afford [00:35:00] species, which are native to north America Melissa Inc's, Corona area angustifolia and also malice Frisco. And a very interesting thing is Montessori angustifolia. Madison goes to folia, which is the crab apple. It's really, the native range is close to Georgia and Florida up Northern side.

So I think some programs now I know that they have started to explore the adaptation of those wild multi-species in this area and look at what genes are controlling that. And I think that research, the result of that research will be very interesting to. To, to see what genetic potential Marlo species hold for this Southern environment.

Kevin Folta: Well, I was excited to try to find angustifolia just to use it as a rootstock. 

Awais Khan: Yeah, exactly. And as, as you said, the rootstocks modify a lot of traders have signed over a gifted sign. So I think. Is another area of exploration that [00:36:00] how rootstock modifies sine traits is it through, you know, the movement of transcripts from the sign to the, the from the rootstock to sign to the graft union and what level of control that could be.

And then again, I think this chilling requires. The how it will be drafting on different rootstocks. So I think it will be very exciting research really, to look at, but you're, you're I think the question about future of apple breeding and future of apple production, I think I will say that there are so many opportunities now for apple breeding.

We can even think of designer, apple cultivars. We have so many. Niches which need, you know, specific traits, for example the environment in Northeast is very different from the environment in west coast. Washington state and New York so far, we were really [00:37:00] going for, you know, kind of brush approach, a broad approach, trying to fit one apple in all these different ranges.

But now we can really think about the the regional needs be more More aware of what pathogens are in this region. What pathogens are in that region? What are the consumer demands in in this region, in what are the consumer demand in other regions also, depending on your shelf, life and storage facilities?

The availability. So there are so many things Are really creating this opportunity for breeding specialist cultivars specific to those environments and those , trips. Well, 

Kevin Folta: very cool. Well, if you ever develop anything, you want to try it and Florida, which is the hellscape for disease for apples you know, give me a call.

So. Dr. con thank you so much for your time today and talking about the future of all kind of the past present and [00:38:00] future of apple breeding. And when something exciting happens, give me a call and let's let's do this again. So thank you very much. Thank you very much. And as always, thank you for listening to the talking biotech podcast, ranked among the top podcasts in the life sciences on iTunes, and that's because of your loyal listenership.

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