Genetic Assimilation.

1. [source]

Tomatoes sporadically produce fruit with horns, fleshy extensions adjacent to the calyx. Do a web search for, "Devil Tomato" and you will find several like the one in image #1. Generally, there is no evidence for these being the result of a genetic mutation. Rather, they represent the sort of thing that can happen when the normal development program of the fruit is disrupted in some way. Seeds taken from such a horned fruit will be no more likely to produce a plant that has similar fruit than seeds taken from any other fruit on the plant.

2. [source]

 There is a related species, Solanum mammosum, that has multiple such horns (image #2). (Though, there are example plants without horns.) The fruit of S. mammosum are rather toxic, so it wouldn't be a great idea to try and make a hybrid between the species and domesticated tomatoes.

3. [source]

Because there is the developmental potential for horns to be generated in tomatoes, there is the potential for a mutation to emphasize the trait. In the Tomato-TILING project, a few such mutations turned up (image #3). I'm not a professional plant developmental biologist, so I don't expect to get access to these interesting mutant seed lines any time soon.

I like the idea of looking for something that everyone else is trying to avoid. Every tomato breeder I've come across has been trying to breed away from a horned tomato, to produce a more "perfect" fruit shape, so I instead want a tomato that is all horns. I have the mental image of a tomato covered in fleshy projections featuring on a counter in some new science fiction movie.

As the previous examples have certain difficulties as a source for this trait, I've been looking for tomato lines which show a higher rate of these "deformations" to use as starting material in a project to breed a tomato that has the trait more consistently.

A rarely studied evolutionary model called "Genetic Assimilation" describes the process where an aberrant trait produced as the result of some stress is selected for and eventually becomes genetically fixed even without the presence of the stress. This mechanism sounds like Lamarckian evolution, except that it relies on the natural selection and the developmental plasticity of organisms…  rather than the personal experiences and intention of the organism that was favored by Lamark. It works because every trait is impacted by the genetic background, the combination of many subtle influences from other genes throughout the genome.

I frequent the Tomatoville forums, including the "Crosstalk: Tomatoville Research and Development™" forum. I started doing so because people there have a tendency to post lovely photos of the interestingly colored and patterned tomatoes they have been growing. Recently, a user was posted images from the results of a complex cross (["Pink Furry Boar" x "Ananas Noir"] x "Bosque Green Cherry") that they were working with. One of the diverse progeny they grew (image #4) had horns on 4 of the 20 fruit. 20% is a far higher rate than I'd otherwise come across, so I asked for a few seeds.

In a few years, I'll have a better idea of where this project is going. The good thing is that I can eat all the rejects along the way.

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References:

1. Genetic Assimilation:
1. http://jeb.biologists.org/content/209/12/2362.full
2. http://en.wikipedia.org/wiki/Genetic_assimilation
3. http://eebweb.arizona.edu/faculty/badyaev/ecol596e/assimilation.pdf
4. In tiger snakes: http://blogs.discovermagazine.com/notrocketscience/2009/10/30/big-headed-tiger-snakes-support-long-neglected-theory-of-genetic-assimilation/
5. In fruit flies: Waddington, C. H. (1942) Canalization of development and the inheritance of acquired characters. Nature 150:563-565.
2. Horned Tomatoes:
1. http://www.tomatoville.com/showthread.php?t=34162
2. https://www.flickr.com/photos/farflung/6462879911/
3. Solanum mammosum
1. https://www.flickr.com/photos/30372914@N03/3895429577/
2. https://www.flickr.com/photos/22012266@N02/7164709249/
4. Tomato Tiling project
1. http://tilling.ucdavis.edu/index.php/Tomato_Tilling
5. Tomato Varieties:
1. Pink Furry Boar
2. Ananas Noir
3. Bosque Green Cherry

A Requiem.

Jonathan Abbey, my brother.

I've never really fit in with those around me. I accept this and don't need those around me to think the way I do. All I need is for them to accept me for who I am. I have had the good fortune to find someone to share my life with who does this. Barring some unexpected misfortune, by this time next year, she and I will be married.

The way I think about the world is very rarely linear. This has caused conflict between me and my academic advisor, as she wants me to construct lists of what I am working on and how I will set about completing then. I generally think in images, patterns, and relationships. When I am working hard on a puzzle, I tend to see my thought processes as some form of abstract math, even though I don't always have the vocabulary to convey that math to those around me. There are conceptual problems that I've thought about for a while and came to solutions that I'm absolutely certain are true, but I don't yet know how to show them to anyone else. Sometimes, I don't even have a glimpse of how to explain.

There have only ever been a few people that I looked to as role models, for inspiration. Athletes, artists, politicians, and other people who arguably have large positive (or negative) impacts on the people of the world have never felt like role models to me. The people I have ever felt this sort of connection with, that remind me of how I see and want to see the world around me, I can count (in no particular order) on one hand.

1. Albert Einstein.
2. Richard Feynman.
3. Stephen Hawking.
4. Jonathan Abbey.

None of them were biologists. Perhaps this shouldn't be a surprise, as I often don't fit the standard model of a biologist all that well. They all shared a clarity and depth of thought that I aspired to.

The first three are names you are probably familiar with. Well, you're probably familiar with them if you've had a long-running interest in science and how the universe works. Einstein and Feynman died before I became aware of them and I don't expect to ever meet Stephen Hawking. (I wouldn't know what to do or say if I did.) It was only when I started learning about how they came to the discoveries they're known for that I started looking to them as role models.

Jonathan Abbey was the older of my two older brothers, my parents' first child. A few weeks ago, he died unexpectedly. The proximate cause of his death was cardiac disease, atherosclerosis. This is what is colloquially referred to as "hardening of the arteries". The ultimate cause of his death was his inability or refusal to keep to the schedule for his medication. He had type-1 diabetes and ankylosing spondylitis, two auto-immune diseases which amplify the effect of high blood-pressure on the damage to cardiac arteries which causes atherosclerosis. He went to the emergency room in the week before with chest pain. They gave his heart a clean bill of health and sent him home.

He spent a great deal of time thinking about thinking (meta-cognition). He encouraged me to pursue a PhD and was very proud of the work I have been doing when I last visited with him. He lamented his own choice of not pursuing a higher academic degree for himself. His professional work involved designing and managing very complex systems. He liked video games, music, and poetry as hobbies. He spent time thinking very deeply about people and how the world works. He pursued knowledge and argued vehemently against "belief". He strongly felt that what was real, what was verifiable, was most important. He was a good father, but maybe not so good of a husband or boyfriend. Many people who knew him thought he was a genius. He was my brother and I'm having a hard time dealing with his passing.

I've gotten past the shock. I've gotten past the sporadic moments of denial. I've even gotten past the moments of anger. I never really went through a bargaining stage. Now, I mostly just feel old. I think this is a mix of depression and acceptance.

I don't believe in a soul or an afterlife and neither did he. Attempts to comfort me by saying, "he's in a better place", in any form or variation are misplaced. Such efforts will anger me, even if not obviously so. If I know you, they will discourage me from interacting with you in the future. If I don't know you, I'll just delete your comment and maybe ban you.

I'm in the very final stages of completing my PhD in the department of Genetics at the University of Minnesota. By the time I post this, I'll have handed off my written thesis to my committee for review. In another two weeks, I'll defend my thesis and be done with it.

I'm sad that my brother won't get to know.

What is a chicken?

We refer to them by the species name Gallus gallus domesticus, but there was a time before they had any connection to us. The wild species is Gallus gallus, also known as the Red Jungle Fowl, and it can still be found running around the wilds of south-east Asia.

There is genetic evidence that modern chickens arose from multiple independent domestication events. The diversity of alleles found in domestic chickens encompasses those found in wild populations of G. gallus spread through India (G. g. murghi), Burma (G. g. spadiceus), and Tailand (G. g. gallus). This is best explained by the early incorporation of Red Jungle Fowl from different regions into the common pool of chickens being cared for by people.

It turns out that there are three other related species of jungle fowl (grey, Ceylon, and green) roaming the area of south-east Asia. A trait found in domesticated chickens that causes yellow skin on the legs and feet is due to an allele which shows most similarity to an allele found in the Grey Jungle Fowl.

A. Green stars indicate putative domestications.
B. Domesticated chicken.
C. Red Jungle Fowl. (Range in red in A.)
D. Grey Jungle Fowl. (Range in grey in A.)

At least four different populations across two (of what we consider) separate species contributed to modern domesticated chickens.

How could the process of domestication start in multiple places at the same time? Well... it can't, but it can happen close enough in time to be indistinguishable to modern researchers.

It is a common pattern in domestication for the idea of domesticating an animal or plant to spread faster than the newly domesticated organism can spread. This results in multiple independent domestication of a single species, or of similar species, found across a wide area.

Cattle appear to have been domesticated two or three times (from Bos tauros, B. indicus, and possibly B. africanus). Sheep and goats appear quite distinct to us now, but when they were domesticated, they were very similar creatures.

Chile peppers have been domesticated at least five times (Capsicum annum, C. chinense, C. frutsecens, C. bacatum, C. pubescens). Squash were domesticated at least five times (Curcurbita pepo, C. moschata, C. maxima, C. mixta, C. ficifolia). Carrots (Daucus carota), parsnips (Pastinaca sativa), celery (Apium graveolens), parsley (Petroselinum crispum), Dill (Anethum graveolens), and chervil (Anthriscus cerefolium) all belong to the family Apiaceae and look very similar in their wild state.

So.  What is a chicken?

It is an example of how the rapid spread of ideas through human culture impacts the process of wild things becoming integral to our civilization.

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References

1. http://en.wikipedia.org/wiki/Red_junglefowl
2. Multiple domestication : http://www.biomedcentral.com/1471-2148/8/174
3. Hybrid between red and grey jungle fowl : http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1000010 
4. Cattle : http://archaeology.about.com/od/domestications/qt/cattle.htm
5. Chile peppers : http://archaeology.about.com/od/cbthroughch/qt/Chili-Peppers.htm
6. Squash : http://en.wikipedia.org/wiki/List_of_gourds_and_squashes
7. Apiaceae : http://science.jrank.org/pages/1240/Carrot-Family-Apiaceae-Edible-species-in-carrot-family.html

Evolution

While thinking about the evolvability of different artificial life simulations, as discussed some in my last posting, I realized that it would be helpful to talk about what is required for a system to evolve. It comes down to four basic traits.

1. Reproduction: Some unit in the system has to reproduce. This unit could be bacterial cells in your gut, or it could be numerical representations in a computer. (Even fire can be described as reproducing when it spreads through a house or forest.)

2. Inheritance: During reproduction, each new unit in the system has to gain traits from its parent(s). The traits could be hidden, as in recessive alleles, or it could be obvious, as in dominant alleles. The number of parents can be one or more than one. (We have two, but maybe some aliens have three or more.)

3. Mutation: At some point in the reproductive cycle, there has to be the potential for changes in the traits (mutations) that are inherited.

4. Death: Death is generally required to remove individuals from a population, thus freeing up room for the next generation. However, there are scenarios where death isn't required. If the population is continuously expanding into new territory, the front-line sub-population can evolve over time without individual death. In this case, the older organisms being left behind fills the same role of actual death.

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It is relatively easy to prove mathematically that a system with these four traits will experience evolution.

Lets give it a go in a simulation that has a maximum population of four organisms represented by letters and driven by the following rules.

1. Reproduction with inheritance: A -> AA; B -> BB
• A or B can duplicate.
2. Mutation: A -> B.
• A can mutate into B.
3. Death: A -> A 
• Only A can die.

We start the simulation with "A" .

"A" -> "AA" -> "AAAA" -> "AAAB" -> "AAAB" -> "AB" -> "AABB" -> "AABB" -> "ABB" -> "ABBB" -> "ABBB" -> "BBBB"

This may not look like the sort of math you are familiar with, but it is math nonetheless. Math is the manipulation of abstract symbols that represent precise concepts with the extremely rigid rules of logic. 2+2 always equals 4. A system with the described traits will always experience evolution.

Now, this little toy system I've described has an extremely low evolvability. The starting state of the system ("A") does meet the four requirements and thus evolves. However, once the system has reached the final state ("BBBB"), it no longer meets the four requirements and thus cannot evolve further.

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If you argue that life doesn't evolve, then you are logically arguing that life does not meet one of the four requirements discussed above. Unequivocally, life meets the four requirements.

Life evolves. The math doesn't provide any other possibility.

Artificial Life

Way back in 1989, I read an article in Scientific American ("Mathematical Recreations") which described a simulation which showed evolution of a very simple "bug".

The genome of the bugs consisted of six numbers that weighted a random selection of which direction each bug would go in the next time step (forward, left, hard-left, backward, hard-right, and right). The bugs would eat "bacteria" which rained down around them, reproducing if they ate enough, but starving to death if they didn't.

Along the way, bugs would evolve different strategies depending on the environment they found themselves in. If they found themselves in a highly food-rich environment, it was more advantageous for them to stay in the same place. If they found themselves in food-poor environments, it was more advantageous for them to keep moving whatever direction they were going.

I thought this was a really cool idea and set about writing my own version of the program. My bugs had a genome of eight numbers, but otherwise they were identical to the original. I worked on the project periodically over several years, eventually producing the version seen at left in 2006. Thousands of bugs (green) cruising around a screen full of bacteria/food (blue) that rains evenly over the screen (at a higher rate in gardens) can be seen at left.

In the later versions of the program, I took some effort at visualizing how the bugs were evolving. At first, I simply plotted the population size over time and compared the resulting curves across different runs of the program. I figured out that the system would consistently support a higher number of bugs if they were allowed to mutate instead of just replicate. The bugs reached population densities about 10% higher than in the no-mutation control.

I then realized I could convert the genome into a 2D coordinate, describing the propensity of each bug to go in each direction. This would let me observe the behavior of the population at a glance.


The video at right starts with the population of genomes clustered around the center. They have no initial tendency to go any where and walk around randomly.

The distribution soon widens to the right and left, as many of the bugs replicating in the gardens are turning in tight circles.

By about 0:02, the population has split into three groups. The left and right have moved downwards as the garden-bugs are increasingly turning around during each time step, going nowhere at all. The center region has started to move upward, showing the specialization of the wide-open-bugs for moving forward at increased rates.

 The three populations continue moving through the remainder of the video. The wide-open-bugs experience large periodic population cycles as they deplete their food source and die back, while the garden-bugs with their much more rich food source show a more constant population over time.

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The video above shows a version where mutations are introduced every time a bug divides. If mutations are introduced only when a bug is starving, which potentially allows a single bug to mutate into a better strategy and so keep on living, a much tighter population distribution results. In the figure at left, three separate runs have been overlaid (in red, green, and blue, respectively). In these simulations, six gardens were available, but only a limited number were colonized. Each colony is represented by a single cluster of genomes in the lower half of the image.

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Being able to experiment with evolutionary concepts on my computer helped me learn about biology and represented a stepping stone on my way to my current approaches to understanding biology. This system is limited, it can only evolve to a few end points.

Other systems can evolve in a much more complicated way. In Tierra, the evolution of short computer programs develops into a rich ecosystem of interacting organisms. Parasites evolve, followed by hosts that are resistant to those parasites.

The term "evolvability" is used to describe these differences in how different systems can evolve. Our biology has a very high evolvability, while my little simulation has a very low evolvability. What allows a system to have a higher evolvability seems to be related to how complicated the interaction of an "organism" is to its environment, as well as how complicated its genome is.

My bugs can only interact with the density of food and their genome is eight numbers. The bacteria living in my gut can interact with me, my food, other bacteria, radiation from the sun, etc. and their genome can grow or shrink as needed. Simulations with higher evolvability invariably show more of the features that we see in living things and so are more useful/interesting for studying real living things

Systems that show any evolvability at all are interesting and included in the subject of "artificial life". Hypothetically, you could be a "biologist" and never look at a messy living thing. I like studying the messy living things too.  ;-)

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Links:

1. http://martin-gardner.org/MGSAindex.html
2. http://lifesciassoc.home.pipeline.com/instruct/evolution/
3. Tierra :
• http://life.ou.edu/tierra/ 
• https://www.youtube.com/watch?v=Wl5rRGVD0QI
4. Critters :
• https://www.youtube.com/watch?v=GuWJ1ZiwO2g
• http://www.scottschaferenterprises.com/alife.htm
5. https://www.youtube.com/watch?v=ZpW_ojpmTWk
6. Polyworld : https://www.youtube.com/watch?v=_m97_kL4ox0

Sunflower crosses.

Last year I crossed the perennial (tuber-forming) sunflower Helianthus tuberosus (image #1) to an annual sunflower H. annuus "Russian Mammoth". I used the much larger, 1ft wide, flower of "Russian Mammoth" (image #2) to pollinate as many of the tiny H. tuberosus flowers as I could.

2. H. annuus "Russian Mammoth".

1. H. tuberosus & seeds.

At the end of the season, I collected ~70 seeds from the H. tuberosus seed heads. Many seed heads had already been destroyed by the local birds which resulted in some scattered seed.

Squirrels got to the seed heads of the "Russian Mammoth" (image #2), because I later put them out in the sun to dry. As a result, I don't have the many more seeds of this variety I was expecting to have. Fortunately, the variety has been available since roughly 1870 and should be easy to find more seeds for.

3. Giant hybrid.

I didn't plant any of the seeds I collected from the H. tuberosus plant, since I would be moving before the plants had matured. However, three of the seeds that the birds had scattered managed to grow up out of the weed patch. Of these, two were obviously hybrids (they had traits found separately in both parents). One of the hybrid plants has a thin stem and flopped over (yellow flowers at lower-left in image #3), even with my efforts to keep it upright. The second hybrid has a robust stem that has let it withstand all the wind and rain of this season. So far, this plant is pretty much exactly what I was hoping the F1 hybrid plant would be.

I finally got a picture of me (6'4") standing next to the hybrids yesterday. Both hybrid plants are still green and thriving, even though the H. tuberosus plants have all shut down for winter. Once we get a killing freeze, I'll cut down the plants and dig up any tubers they've produced.

With luck I'll be able to collect some F2 seed off these plants, but since I no longer live where the plants are, I'm expecting the birds to get to them before I do. As the F1s are supposed to produce tubers generally, I should be able to regrow these plants next year from the tubers they are now likely to be producing.

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4. en.wikipedia.org/wiki/Perennial_sunflower

The sunflower genus (Helianthus) contains a wide range of species. Some species are difficult to cross, while others will cross readily. Image #4 illustrates the use of hexaploid species to break down reproductive barriers between annual and perennial diploid species (at left and right). Crossing the tetraploid hybrids to either parent type results in uneven chromosome sets and high rates of infertility due to aneuploidy. The tetraploids can easily cross, however, allowing genes from diverse sources to be recombined in their progeny.

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5. www.edenbrothers.com

The common sunflower (H. annuus) has been bred to produce a range of colors in addition to the yellow of wild sunflowers (such as those in image #5). The genes for these color changes could be added to a perennial sunflower using the same method I'm using to add traits for giant growth. (Someone else has this project under way.)

Because of the differences in ploidy between the annual sunflowers from the commonly available perennial (H. tuberosus), it would likely be in the F3 generation or later before such rich colors could be regained. This is discussed in the link below.

the-biologist-is-in.blogspot.com/2014/10/genetics-in-sunflowers.html

Making a new "Blue" tomato

1. Tomato "Indigo Rose".

"Blue" is the color label applied to the new breed of anthocyanin rich tomatoes. "Indigo Rose" (image #1 at left) is the first officially available variety with the trait. The variety was bred at OSU, using two genes from wild relatives of tomatoes. The atroviolaceum ('atv') gene was introgressed from Solanum cheesemanii. The anthocyanin fruit ('Aft') gene is a transcription factor introgressed from S. chilense. The two genes combine to result in a tomato with dark purple anthocyanin pigment production when exposed to sunlight.

The high-anthocyanin traits managed to escape from the OSU breeding program before the official release, under the names "OSU Blue" or "P20". This variety was not yet stable and didn't taste very good to most people, but it did successfully introduce tomato breeders to the interesting traits a few years early. Breeders quickly took to trying to incorporate anthocyanin expression into better tasting types of tomatoes.

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2. F2 tomatoes, showing pigment on fruit and calyx.

I've been growing a miniature tomato variety called "Tiny Tim" for the last several years. I saved a batch of open-pollinated seeds two years ago, as my previous batch was running out. Last year, one of the seedlings turned out to grow much faster and larger than all the others. It was the result of a cross to one of the other tomatoes growing the previous year. I grew several F2s this year, allowing me to identify the other parent as a "Roma" tomato.

Among the F2s, I noted a range of anthocyanin phenotypes in the fruit and leaves/stems. The anthocyanin pigment produced on the fruit when sun-exposed came in three levels (none, middle, and high in image #2.)

3. F2s (top two) & "Indigo Rose" (bottom).

The anthocyanin pigment produced in the calyxes also came in three levels (high, none, and middle in image #2), but independent from the fruit pigment. The pigment produced in the rest of the plant wasn't as obvious. The no-pigment plants were entirely green. the medium-pigment plants had the anthocyanin highlights on the calyxes and leaf edges. The high-pigment plants showed increased pigment over the entire plant where sunlight hit, at a level about half of that seen in "Indigo Rose" tomato plants (image #3).

4. Original; color-enhanced; postureized.

The high-pigment plants also appeared more of a red/brown color rather than the purple of "Indigo Rose" plants. Image #4 shows a section of the image #3 after using the color-enhance filter in GIMP (center) and then the posterize filter in GIMP (right). The enhanced images more clearly convey the difference in color which is visually seen on examining the plants. This either indicates a different mix of anthocyanin pigments, or is a visual artifact caused by the blending of green chlorophyll with the anthocyanin purple. I would need to do some chromatography or micro-dissection experiments to discriminate between these possibilities.

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5. Anthocyanins on unripe "Tiny Tim".

The level of pigment in the F2s was a surprise as I hadn't noted any anthocyanin expression in the parent varieties. After seeing the F2s, I re-examined some "Tiny Tim" plants grown this year and found they did have anthocyanin expression. The fruit show a level of anthocyanin production comparable to the high-fruit-pigment F2s (image #5), but the small size of the fruit made it hard to notice. The F1 showed a pigment level like the medium-fruit-pigment F2s, suggesting it is a single trait with partial dominance. The color of the calyx and leaf/stem was comparable to the F2 plants with the middle level of pigment for each feature. Looking into the lineage of "Tiny Tim" suggests the middle-pigment trait was contributed from S. pimpinellifolium, used in the breeding to contribute small fruit size to "Tiny Tim". As anthocyanin pigments on the shoulder is common in many wild tomato relatives, I suspect the fruit trait also came from S. pimpinellifolium.

"Tiny Tim" is an open-pollenated variety, so it should be homozygous for any alleles impacting pigment production. The increased calyx/leaf/stem pigment intensity in the F2s over what is seen in "Tiny Tim" suggests the involvement of a second gene from the "Roma" parent that enhances the expression of the first gene. This second gene would have been hidden in "Roma" because that variety doesn't have any anthocyanin pigment production.

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What are the expected genetics for this cross?

The fruit pigment appears driven by one gene. Under the model of partial dominance, the cross ...

1tt1tt x 1R1R

… produces an F1 …

1tt1R

… that shows a low level of anthocyanins in the fruit. Low amounts of anthocyanin pigment was noted in the fruit of the real F1. Selfing the F1 produces F2s …

	1tt	1R
1tt	1tt1tt	1tt1R
1R	1tt1R	1R1R

… where 1/4 have high-pigment on the fruit (1tt1tt) and another 1/2 have low pigment on the fruit (1tt1R). I only grew 10 F2s this year, so it is hard to estimate real ratios, but all three color classes were observed.

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The calyx/leaf/stem pigment appears to involve two genes. If we assume both involved alleles are recessive, the cross …

2tt2tt3TT3TT x 2R2R3r3r

… produces an F1 …

2tt2R3TT3r

… that shows no anthocyanins in the calyx/leaf/stem. No anthocyanin pigments were observed in the calyx/leaf/stem of the real F1. Selfing the F1 produces F2s …

	2tt3TT	2tt3r	2R3TT	2R3r
2tt3TT	2tt2tt3TT3TT	2tt2tt3TT3r	2tt2R3TT3TT	2tt2R3TT3r
2tt3r	2tt2tt3TT3r	2tt2tt3r3r	2tt2R3TT3r	2tt2R3r3r
2R3TT	2tt2R3TT3TT	2tt2R3TT3r	2R2R3TT3TT	2R2R3TT3r
2R3r	2tt2R3TT3r	2tt2R3r3r	2R2R3TT3r	2R2R3r3r

… where 1/16 are expected to express the recessive alleles from both parents and thus show the high-pigment trait. Another 3/16 are expected to express the recessive allele from "Tiny Tim" and show the medium-pigment trait. The remaining 12/16 should only have green chlorophyll evident in the unripe fruit. This year I grew 10 F2s and only one shows the high-pigment trait. 1/10 approximates 1/16 reasonably well for the numbers I grew. Some, but not all showed the middle-pigment trait. I didn't note exactly how many F2s showed the middle-pigment trait and they've begun dying back from the cold, so I will have to screen more F2s next year at an earlier stage to better estimate the true ratios of the different color classes.

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The dark pigment of "Indigo Rose" fruit is due to the interaction of two traits, the anthocyanin fruit ('Aft') trait combined with the atroviolaceum ('atv') trait. The 'Aft' trait by itself only produces a small amount of pigment on the fruit shoulder. The 'atv' trait by itself only produces dark pigment on the calyx/leaf/stem of the plant.

If the fruit pigment in the F2s is driven by a single gene, as it appears, and two genes are responsible for the calyx/leaf/stem pigment, then 1/64 of the F2s will contain both high-anthocyanin traits.

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6. Derived from S. hirsutum.

There are several anthocyanin traits floating around that have been introgressed from different wild tomato relatives.

1. S. cheesemanii
• "atv" gene: pigment throughout plant. Seen in variety "Indigo Rose" (image #1).
2. S. chilense
• "Aft" gene: pigment on fruit shoulder. Seen in variety "Indigo Rose". (image #1)
3. S. hirsutum
• [unnamed] gene: pigment on fruit shoulder, similar to "Aft". Described at maprc.blogspot.com. (image #6)
4. S. peruvianum




7. Derived from S. peruvianum.

• [unnamed] gene: pigment on fruit shoulder, similar to "Aft". Seen in variety "Purple Smudge". (image #7)
5. S. pimpinellifolium
• gene #1: pigment on fruit shoulder, similar to "Aft". Described here.
• gene #2: pigment throughout plant, similar to "atv". Described here.
6. Conventional tomatoes
• gene #3: modifier of pigment. Described here. (A similar modifier impacting "Aft" and "atv" is described by Fusion_power in a discussion at the Tomatoville forums).

The four fruit pigment traits and the two plant pigment traits seem to behave similar to the others in each category. Because the species are so closely related, the traits may represent different alleles of the same genes. If so, combinations of a trait from each category (like the "atv" and "Aft" in "Indigo Rose") should result in a strong increase in the total pigment produced relative to either trait alone, especially when the modifier trait (gene #3) is also present.

I've isolated a line that appears homozygous for gene #1 and one that appears homozygous for both genes #2 and #3. Unfortunately, crossing these two lines would simply recreate the F1 (heterozygous for all three traits) rather than help me generate a triple-homozyous line.

Comparing the lightly-pigmented fruit in images #6 and #7 to my pigmented F2s suggests they are showing a different mix of anthocyanins from the other "blue" lineages. I look forward to finding one of the rare segregants which contains all three genes, so I can find out!

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References:

1. "Indigo Rose" tomato: http://extension.oregonstate.edu/gardening/purple-tomato-debuts-indigo-rose
2. 'Aft' gene: http://www.esalq.usp.br/tomato/Aft.pdf
3. 'atv' gene: http://www.esalq.usp.br/tomato/atv.pdf
4. Escape of "P20" tomato : http://www.tomatoville.com/showthread.php?t=16989
5. "Tiny Tim" tomato: http://tatianastomatobase.com/wiki/Tiny_Tim
6. "Roma" tomato: http://tatianastomatobase.com/wiki/Roma
7. "Orange Smudge" tomato: http://tatianastomatobase.com/wiki/Purple_Smudge