Thursday, February 23, 2017

Biology of the Enjoya Pepper

"Enjoya" pepper; marketing
photo from the TwitterVerse.
A few years ago a new pepper turned up in markets of Europe and then in the USA (and elsewhere). The bell peppers were a dramatic yellow splashed with red flames and were sold as "Enjoya" or "Flame" peppers.

There was no information available about the genetics of the trait, as there had been no academic literature published on the new variety. Gardeners with the habit of growing their own plants from seed took this as a challenge. People around the globe independently said, "Can I can grow seeds from that pepper and get striped fruit in my garden?" Seeds were collected by those who found the peppers in their grocers and then shared via online forums to those who had not yet found them. Soon after, there were many little green seedlings being tended to around the world.
Typical flowers and fruit.

Months later, the first reports on the plants started coming in. The plants were producing large bell peppers, but they were all ripening yellow. (I have reports of 11 plants maturing to produce yellow fruit.) As these reports were posted to the forums, interest in the plants waned. (Dreams of crossing the trait into jalapenos and other hot peppers quietly died.) If the amazing red flames weren't going to reappear, then why would anyone want to be growing these plants?



Where did these peppers come from?

The marketing site for the pepper says:
Now, 30 years later, nature has once again surprised us with a natural variation: the red/yellow striped pepper. In 2013, Wilfred van den Berg found this beautiful variety in his greenhouse in Est.
But the US patent applied for the pepper says:
[0011] `E20B3751` was discovered in a screening trial of mutants of pepper variety `Maduro` conducted at Est, Netherlands. The mutant `E20B3751` was selected based on its vertical red and yellow stripes color and propagated vegetatively (i.e., asexually).
I strongly suspect those responsible for writing the marketing site didn't want to say the variety was the result of a mutation breeding project in a high-tech lab, as such things tend to get a lot of people suspicious about their foods. This is only a slight fib, since the mutated variety is a variation of the natural pepper.

What draws my attention more is that the patent doesn't say anything at all about how the pepper plant was produced (aside from the general concept of a mutagenesis screen). The entirety of the patent starting on line [0046] is simply a rehashing of general plant biology and breeding. None of that tells us anything at all about the origin of the striped peppers. This is strongly counter to the basic idea of what patents are supposed to be. The earlier paragraphs of the patent do give a concise description of what the pepper is, as well as a listing of specific traits associated with it, so it isn't entirely a useless document.



Since there isn't any academic research published on the pepper and neither the patent or marketing information provide any biological details, we're going to have to see what we can figure out from basic principles.

Mutations in genes typically produce traits which are either dominant or recessive. (There are a few other scenarios, but we're not going to worry about them for now.) If the striped trait is recessive, then essentially all of the next generation would also have the trait.

If the striped trait was dominant, then [with perfect selfing] the next generation might all have the trait, but there are other scenarios. If the Enjoya pepper plant (remember, from the patent they are propagated assexually and so are all from the same genetic plant) was heterozygous for the dominant trait, then half of the next generation would remain heterozygous and have the trait. Another quarter would be homozygous for the no-stripes trait and the remaining plants would be homozygous for the striped trait. Dominant traits can sometimes also have recessive lethal characteristic, though it is rare. All together, at the very least 66.6% of the next generation should have stripes if the trait was due to a dominant nuclear mutation.

In either scenario, we should have the majority of the next generation with stripes. What do we see? Between my plants and those reported by other growers, we have 16 plants that have ripened fruit. All of which matured to yellow with no red stripes. This would be a very unexpected result for either model discussed above.



Meristem figure from Wikipedia.
There is another scenario that might be important. A growing meristem of a plant include multiple tissue layers which replicate independently. A mutation in one layer generally won't transfer to the other layers. As the plant grows, the mutated and non-mutated tissues will be maintained separately. As leaves or other organs develop, the different meristem layers contribute to different parts and so would result in visible variegation if the mutation had a visible impact.

Photo cropped from one at link.
After looking around a bit, I found a photo which might provide some clarity to the situation. In the cropped close-up at right, it is clear that all the seeds are attached directly to yellow tissue. There is red tissue in the core of the seed mass, but none at the surface where the eggs (and then seeds) developed.

It looks like some of the red core cells are able to migrate to the surface of the fruit during early development. This results in the red stripes as the fruit then expands in size.

Since the red color is carried in tissue which isn't made into eggs or seeds, it appears unlikely that the seed-grown progeny of an Enjoya pepper would produce red or striped fruit.

Sorry folks, I think the game is up. We probably won't be able to breed flame-colored jalapenos. At least we've learned something about the biology of these peppers.



That the striped trait can't be passed down through seeds tells us something about the experiments which led to the Enjoya pepper. The patent indicates it came from a mutagenesis experiment, but gives no details. One of the easiest ways to do it would have been to soak a large batch of seeds in a chemical mutagen (like EMS) and then grow them out after treatment. EMS is relatively easy to work with and it would produce point mutations all over the nuclear and cytoplasmic genomes. I bet when that first plant matured its first fruit, there were amazed expressions all around.

The classical story of pepper color genetics (described at the-biologist-is-in.blogspot.ca/2015/11/the-color-of-peppers-2.html) suggests it would take two separate mutations to produce the rich yellow color seen in the Enjoya pepper. However, there are a lot of mutations which impact pepper color that don't really seem to fit the classical story. I strongly suspect the visible difference between the red and yellow fruit tissues is down to one mutation.

However, EMS is not something that would be used to make a single point mutation. It would instead create hundreds or thousands of point mutations per seed in this sort of mutagenesis experiment. Selection of the resulting progeny, as well as backcrossing to the parent type, would normally be used to clean up any unwanted deletarious mutations... but the striped trait would not have survived this process.

This means that the genome of the Enjoya pepper is probably chock-full of other potentially interesting mutations. Many of those mutations will be recessive and so only become visible in the second generation after treatment. The plants we've been growing from saved seed represent that second generation (referred to in shorthand as M2).

Enjoya-M2 with a transient anthocyanin shoulder.
One of my seven M2 plants produced a dark shoulder of anthocyanin pigments on the unripe fruit. These anthocyanins were later broken down as the fruit matured to its [now] expected yellow. Dark shoulders are pretty common in peppers, so I'm still trying to decide if I want to save any seeds from this plant.

Enjoya-M2 with color-marked flowers.
Interesting stripes on the unripe fruit.
Another of my seven plants produced flowers with distinctive purple highlights. The fruit on this plant later showed a distinctive green striping on the shoulder while unripe. (The fruit of every other plant was solidly dark green.) I'm still expecting this one to mature to a solid yellow, but there remains the slim chance that a red cell fought its way into the seed. (The pepper has since ripened to the expected yellow.)

Two of my seven plants produced distinctively different plants. This suggests there are indeed numerous hidden recessive mutations in the Enjoya pepper. The relatively large fruit I've been getting from these plants and the potential to find other novelty mutations means I'll probably be growing quite a few of these M2 plants in the coming years.


References:

Saturday, February 11, 2017

Unionids Along the Missouri


Green is where Unionids were found in research.
Red is where I collected Unionid shells.
[Figure derived from those at link.]
Several years ago, my brother and I went on an overnight road-trip to Nebraska. Why? ...well, mostly because it was there and it was close enough to make an overnight trip. Among other memories, one of the highlights was hanging out on the banks of the Missouri River at Decatur.

As we wandered around the river edge, we found numerous large mussel shells. I collected a few, with intentions of identifying the species that made them at some later time.



Fast forward a few years and I'm digging through some boxes in the basement. I'm not sure what I was looking for, but the shells grabbed my attention. It was time to figure out what they were.

I had collected two pairs of shells. One pair was thinner and the other was thicker. One of the thinner shells broke while in storage. The remaining shell is 13.8 cm long, 8.0 cm tall, and 2.6 cm deep. After some looking around at various documents, I realized I could identify these thinner shells as a specimen of a Unionid species called the Great Floater (Pyganodon grandis). The species seems to get this name because of the penchant for their shells to float away when one has died and has begun to rot.

Thick-shelled Unionid. Lower-right
is a closeup of growth ridges.
The second pair of shells was much thicker. They're similar in size to the previous shell (12.6 cm x 7.7 cm x 3.5 cm). The shells both look scarred and aged. The best way to determine the age of Unionid shells involves destructive dissection of the shells. Instead, I used the less accurate method of counting the yearly growth ridges. I estimated the shell at ~140 years old (which is well within the age range known for these animals), but I still haven't had any luck with an ID.

Through the process of trying to identify these shells, I accidentally identified a shell I had found in central Texas when I was in highschool. This shell is from another Unionid that is called the Threeridge (Amblema plicata). I had long ago given up on finding the name of this shell, so this was a cool bonus.



Unionids have all sorts of interesting biology. Like most bivalves, they make their living filter feeding water as they hide buried in the sediment. The live in freshwater river systems worldwide, with the most diversity present in North America. Adult Unionids can only travel very slowly by shifting their foot, so you would think they'd have a difficult time traveling up rivers. The Unionids have developed a very special trick to get around this limitation. They use fish to transport their babies.

Unionid larvae (called glochidia) spend some time as a parasite in the gills of fish. The fish can travel upstream or downstream, much further than the adult could ever crawl. After some interval, the glochidia drop from their fishy host and start living the traditional life of a c.

Images from unionid.missouristate.edu
How the glochidia get into a fish is kinda awesome. The general strategy is to convince a fish (of an appropriate species) to inhale a bunch of the babies. How they do this varies all over the place. The females of some Unionid species develop large flanges/flaps of tissue that are shaped and colored to mimic small fish or other aquatic creatures. These organs have musculature and so can even move in a realistic manner, which all aids to draw the interest of fish. When the fish come close to try and get a meal, they instead get a mouthful of larval bivalves. Other Unionid species release their larvae in sacs (ovisacs) that attach to rocks or the parent shell and are buffeted around by the current. These sacs look like little fish, again drawing the interest of fish looking for a meal. One group of Unionids (the Epioblasma) even bites onto the heads of fish (using tooth-lined shell edges), so they can shove their babies directly into the fishes' mouths.



These species are long-lived, but sensitive to environmental disruption. They can't survive in a river that dries up and are they're unable to get out of the way when water quality is impaired by human activities. Because of this sensitivity, there are legal restrictions on their harvest (in every state I've checked).

All the shells I have were collected on dry land, which sidesteps the legal restrictions designed to protect the live animals from harm. Frankly, I couldn't imagine collecting living animals to get their shells. You have to respect your elders, even if they happen to be living on the bottom of a river.


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Tuesday, January 24, 2017

Snail Locomotion

During a recent conversation with a biologist I'd just met, the topic of a jumping nematode came up. (It is a type of tiny nematode that parasitizes insects, so you don't have to worry about these critters jumping out of the woods at you.) I then mentioned some of the unexpected ways that snails can get around. This inspired me to write up this short post on the topic of snails, with some helpful links to illustrative videos.



Your typical, every day garden snail gets around by gliding on mucus it secretes. The gliding motion is driven by continuous waves of muscle contraction along the bottom of its foot.

Some aquatic snails (such as the charismatic Turbo snail) have evolved a division down the length of their foot, separating their foot into two "feet" which can move independently. This allows the snails to walk around by alternately moving each foot forwards, while holding securely to their substrate. These same snails can also glide around when they're not in such a hurry. (I've witnessed this behavior in a saltwater aquarium setting, but I don't have a nice video link illustrating it.)

The next advancement in snail locomotion comes in the form of a shoreline living creature called the Plough Snail. It eats fish and jellies that have washed ashore, but has to be quick about it because so many other things are looking to eat this rich source of protein. In conversation, I had described these snails as "running", but really what they're doing is more of a breast-stroke style movement that propels them quickly along the surface of wet sand. By altering how far they move either side forward, they can change direction equally quickly. (This is the fastest snail I've ever seen, so I'll probably keep referring to them as "running" when they come up in casual conversation.)

There are also various kinds of snails that get around by swimming. One group are referred to as Sea Butterflies because the motion of their "wings" looks like that of a butterfly.


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Tuesday, January 17, 2017

A Pretty Little Weed

One of my favorite roadside wild-flowers is the invasive Eurasian weed Lotus corniculatus (Bird's Foot Trefoil).  It is almost always found in a bright yellow, but I've been keeping a look out for forms with other colors. I occasionally find plants with enhanced red/orange streaking, but never as intense as the one in the photo below. I did once find plants with distinctly [pale-]orange flowers in a southern California ocean-side park. (Unfortunately there were no seed pods to be found.)

I found the following representative images from various sources online.

L. corniculatus in yellow.
L. corniculatus in orange.
L. corniculatus with red streaks.

I'm really quite surprised I haven't found any evidence for a white flowered variation. All it would take is a single mutation to inactivate one of many genes involved in the early steps of the pigment pathway, which should make it a pretty easy variation to arise. Maybe white flowers don't attract whatever pollinates this species and so the trait would rapidly die out after being formed.

The plant family containing this species (Fabaceae) has flowers covering the whole spectrum of flower colors. Reds, blues, yellows, and whites are all common. These colorful relatives suggests there may be the genetic potential for more color diversity within this species, even though they are not yet apparent.



Additional interesting flower color genes can be found in close relatives. If we're very lucky, maybe these related species could cross to L. corniculatus. Improving the floral characteristics of a common weed isn't the sort of project that is going to get much research funding, so there might not be much information available about the possibility of inter-species crosses within the genus. For now, lets just make pretend that we can do these crosses and have a look at what genetics might be available in the genus Lotus.

L. pinnatus
The closely related L. pinnatus (Bog Bird's Foot Trefoil) is an uncommon species found in western Canada and the USA. Its lower two petals are splayed open and a bright white color. These flower traits suggest this species would be interesting to cross to the more common weedy species. L. pinnatus grows as a sprawling plant in boggy wetlands.

This dramatically different lifestyle of this species means there would be many non-floral traits that would need to be cleaned up via back-crossing to L. corniculatus if we wanted to maintain the growth form of the original weed. I like the weed's growth habit, so this would be a goal for me.

L. formosissimus
A similar species to L. pinnatus is L. formosissimus (Seaside Bird's Foot Trefoil). This one has the same fancy flower shape, but comes in a greater diversity of color forms.

L. tetragonolobus
Another close relative is L. tetragonolobus (Asparagus Pea). This species produces edible (and tasty) winged seed pods. The flowers are a brilliant scarlet-red, which would be a lovely trait to bring into the mix. The plant grows as a low mound, much like L. corniculatus, so I wouldn't need to worry about cleaning up the genetics of a cross so much. They also seem to come in versions with yellow flowers. The traits that result in this species having tasty pods might also be a cool thing to select for in any hybridization projects. (Maybe I'll just grow this species in the garden anyhow.)



Some more research into this group reveals there is abundant information about hybridization between species. The reason is that the various species are common forage plants in pasture. Any species impacting agriculture will have a lot of research done on it.

Some of the hybrids I can find information for:
  • L. corniculatus x L. tenuis
  • L. uliginosus x L. tenuis
  • L. corniculatus x L. stepposus
Unfortunately, none of these species have much of anything going on for interesting flower colors. That these crosses work, however, suggests other crosses in the genus might also work. Knowing something about how many chromosomes are found in species of the genus might help sort out what crosses have a better chance of working, but it really comes down to simply trying the crosses and seeing what happens.


References:

Sunday, January 8, 2017

Black Nightshade 2

The potential toxicity of Solanum nigrum fruit has been much debated. I had previously written about this species and some thoughts about why people might think it was poisonous. I generally consider it's ripe fruit to be edible and I routinely eat small numbers of them with no negative effects. However, there are still reliable reports of toxic reactions to the fruit (Joseph Lofthouse's reporting) that should be cause for concern to those unfamiliar with the plant.

I think the species had great potential as a future domesticated plant for the vegetable garden. The plants don't seem to have any real disease issues, especially when compared to the related tomatoes. Improvements in fruit size, sweetness, or total production would be worthwhile traits to develop. To that end, I've been on the lookout for wild plants with improved traits to help start a breeding program.

Last summer (2016) I grew several plants from saved seed. In our fenced in garden area, several additional wild weedy plants grew. I was pleased to find one of the wild plants had large berries, on average maybe twice the volume of the berries I'd planted. Rabbits were getting into the garden, so I kept a close watch on the plant while waiting for the fruit to mature.

Top: Extra-large and extra-toxic.
Bottom: Typical and edible.
Once most of the berries had ripened, I collected a sample from the different plants. I took the berries inside to photograph and save seeds. The smaller berries tasted just as I had expected. The larger berries...  I immediately spit them out. The larger fruit had a significantly higher level of solanine than I had ever experienced in this species. I knew exactly what this agent tasted like from tasting the closely related S. dulcamara, which is consistently toxic to humans.

This plant exemplifies some of issues leading to the ongoing debate about this species. I'll eat berries from most plants of this species, but there is no way I would ever eat a handful of berries (or even a single berry) from this plant. I know how to recognize the plant's poison, but you may not.

It took me several years of sampling fruit from every plant I found of this species before I found one that was poisonous. You might find a poisonous one on your first try. Your risk of tasting a couple berries will be minimal, but you should always take great care when eating a plant that you're not very familiar with.



Even though the larger berries were toxic, I'm still going to plant the seeds I collected from them. I'm hoping some of the plants in the next generation will have the larger fruit and yet not be toxic. Even if the two traits are closely linked, eventually I should be able to find a plant that separates them. Wish me luck.


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Wednesday, January 4, 2017

Early Chile Domestication

I first discussed Capsicum annuum var. glabriusculum in an earlier post. I've collected wild seeds for this species from a few different sources throughout the desert southwest.

The two views at right are the same plant, grown from seed collected in Phoenix-Az. The comparison of above and side views shows how well the down-hanging fruit of this plant are hidden from above, where birds would be more likely to see them while flying overhead. Since birds are the primary distributors of wild chile seeds, this trait would not encourage dispersal of the seeds and so the trait would not spread.

Early human farmers would have found this trait useful, as it would help reduce crop loss to birds. The majority of non-ornamental chile varieties grown today share this trait.

The lack of any other traits associated with domestication make me think this plant either represents the impact of selection pressure by early farmers, or a much more recent introgression event where all the other modern traits were heavily selected against. The seeds were wild-collected in Arizona (where humans have been living and using chiles for thousands of years) and modern chiles are commonly grown in the area, so either scenario is likely.



An approach which might help clarify the plant's ancestry is to compare its genome to more domesticated types. If it has this trait due to a modern introgression, there will be regions of its genome that match the relatively low diversity found in domesticated chiles. If it has had this trait for far longer, it's genome will be covered in variations not found in domesticated chiles.

Processing out some genomic DNA (gDNA) is a pretty easy thing. The difficulty will come in getting the material sequenced. I do have reasonably simple access to a nanopore DNA sequencer, but the data that comes from that technology has been problematic for some whole genome analyses in my experience (and in the experience of others). Ideally I'd have the gDNA sequenced using Illumina technology. This technology also introduces errors into the resulting data, but at lower levels and in ways that I have already written software to compensate for.

As I continue to build out my lab, this is probably one of the projects I'll be investigating further. Clarifying the ancestry questions for this chile would be personally rewarding, but also might fill in a tiny little detail about how people of the desert southwest lived. This might be very interesting to a large number of people.



I expect the other seed lines I have for C. annuum var. glabriusculum will grow to have upright fruit, but otherwise match the characteristics of these plants. I like the small size and pungency of the fruit, so I'll probably keep growing them even if I can't think of a breeding project to use them in.


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Wednesday, December 28, 2016

Relict Ecosystems

Part of what I like about biology is that it includes topics ranging in scale from the analysis of how a single protein functions all the way to how species and whole ecosystems change over geological time. At every level, there is dramatic complexity to be explored.


As the glaciers from the last Ice Age retreated northwards, a whole landscape of ecosystems followed them. Along the way, isolated fragments of those moving ecosystems were sometimes left behind as a species found a microclimate to their liking. The first such relict ecosystems I learned of are the Lost Pines and Lost Maples areas of central Texas. Each of these locales are known for the large numbers of a type of tree not seen over a wide area around them. More recently I learned about Texas Wild Rice, a tiny relict of the ecosystem that northern Minnesota is now known for.

If these relics persevere long enough, they may rejoin the parental populations when the next ice age happens. The relict populations may then blend back into the larger population, or (if they have fully speciated) they may retain a distinct biological identify. American Sycamore trees and their old-world cousins can hybridize, so it isn't certain the extended time apart would result in speciation.


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