The Biologist Is In: February 2017

Thursday, February 23, 2017

Biology of the Enjoya Pepper

Large bell pepper from grocery store, colored in red and yellow vertical stripes.

"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.

At left a bell pepper hanging on the plant, ripening yellow. At right a couple large white pepper flowers.

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.

A diagram illustrating three tissue layers in a plant meristem.

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.

Inside of a bell pepper, showing seeds growing from yellow tissue, with reddish tissue deeper beneath the seeds.

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).

A large bell pepper at two stages of maturity. At right, early on it has black pigment at the top of the pod. At right, later, the top of the pod is turning yellow.

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.

A large bell pepper on the plant, showing faint stripes in shades of green. At right is a pepper flower in white, with a purple spot on each petal.

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:

Marketspeak:

the-biologist-is-in.blogspot.ca/2015/11/the-color-of-peppers-2.html

Saturday, February 11, 2017

Unionids Along the Missouri

Figure illustrating the river system of Nebraska. At top right a single position is indicated by concentric red circles.

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.

Four different views of a large freshwater mussel shell. The shell is very thick, showing signs of damage and healing over its long life.

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.

Five images showing Unionid mussels with various features they use to attract fish which are hosts to the larvae of the mussel. Most show fleshy extensions that look like worms or small fish. At bottom-right is a shell edge which has sharp inward pointing teeth growing along it.

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.

References: