Thursday, March 27, 2014

New Orleans

I'm in New Orleans most of the week for the 12th ASM Conference on Candida and Candidiasis. Today I presented a short talk about a genome sequence analysis tool I have been constructing. The main purpose of the tool is to provide the user with a large-scale overview about what changes have occurred in the strain, when compared to another strain (which may or may not be the reference genome for that species).

Most biologists don't have the solid grasp of mathematics or computer programming needed build such a tool, but they can recognize how the tool can help them when presented with what it does. More than one associate came to me afterwards and relayed stories of what they saw in the audience while I was talking. (The associates had seen me present on the topic, or have already been using the tool, and so didn't really need to follow my talk.) One researcher was observed taking notes on her copy of the presentation schedule, placing check-marks by each talk after it concluded. At the end of my talk, she circled my section enthusiastically. I expect she will be finding me to ask questions over the next few days.

Last night I was talking with an associate about some of the quirks involved in the data he was looking to analyze. He asked me, "How does it feel to be the only person doing this?" I was somewhat surprised by this, as I had not put serious thought to the matter.

Long before I started grad school, I had heard it was a grad student's job to become the world expert on something. Since we're investigating things that are not known, it really isn't too hard to do…  but I hadn't thought about it in a while and nobody had ever pointed it out to me.

I do wonder what I've gotten myself into with this project, as it could be something the Candida research community might want to have around for a while. As I'm the only developer on the project, I may be attached to it for some time. Once I graduate, however, I don't know how much of my time I will want to be donating. Perhaps I can get paid to assist others with their data analysis in a consultant level. This would allow me to have a day job and still contribute to the research community.

Friday, March 21, 2014

Evolution and Tomatoes

Mutations occur all the time and are random in nature. Most mutations have minimal impact, but some are dramatic. A mutation isn't intrinsically negative or positive, but becomes so in the context of how it impacts the survival and reproduction of an organism in the specific environment it finds itself.

Selection, the biased survival of certain genotypes over others, trims the random mutations to those that work. 'Artificial' selection is what we call it when we make choices about which organisms continue to reproduce. 'Natural' selection is what we call it when it is the undirected interactions with the environment and other organisms that determines which organisms continue to reproduce. There is no real difference between 'artificial' and 'natural' selection. They both push organisms around, changing how they work and what they do, in ways that are to the advantage of the organisms themselves.

The plants and animals that we have 'artificially' selected have had incredible increases to their populations. There are far more dogs than there are wolves. There are far more wheat/corn plants than there ever were of the wild species from which they were derived. You could describe the development of our domesticates as their independent evolution into a very profitable niche…  us.

I like colorful and tasty tomatoes. Given that tomatoes represent ~$2 billion in farm revenues per year in the USA, I'm not the only one. Wild tomatoes and early market varieties develop with a darker green shoulder (at right) that ripens to the final red a bit slower than the rest of the fruit. By the time the green shoulder has ripened completely, the bottom of the fruit is already beginning to get soft. For home-grown tomatoes, this isn't a problem, since you're going to be eating it soon after plucking it from the plant at its most vibrant red ripeness. For commercial tomatoes, by the time the top has fully ripened, it has become too soft at the bottom for shipping. The result is that commercial tomatoes were picked and shipped before they were ripe and would appear partially green in the stores.

At some point before the 1930s, a farmer in the USA noticed a plant producing tomatoes that didn't have green shoulders (at left) and that ripened uniformly from top-to-bottom. The tomatoes could be picked when they started to go red and would ripen evenly all over by the time they reached the grocer.

In the market those tomatoes that still had the green-shoulders didn't sell as well as the all-red neighbors. They didn't look as ripe and couldn't compete. Commercial plant breeders saw the trait as the boon it was and soon the uniform-ripening trait (u) was bred into most commercial tomatoes everywhere. In the environment of the home garden, the uniform-ripening trait didn't provide any advantage to growers and the trait remains rare in the (heirloom) tomato varieties passed down along family lines.

Tomatoes still went soft on the way to stores, costing the industry a fortune in lost revenue. In the 1960s, a tomato was found by Henry Munger (a professor of plant breeding at Cornell) that showed strongly delayed ripening. This ripening-inhibition (rin) trait, when bred into commercial tomatoes, resulted in plants which produced fruit that remained hard for a longer time. Because they didn't go soft during ripening, they could be shipped with fewer losses. This made it cheaper to get a tomato to market and soon the trait had been bred into most commercial varieties. The ripening of these tomatoes is sped up by exposure of ethylene gas (a common plant development hormone), so they can be perfectly ripe upon arrival at the store.

In 1974, researchers realized that the ripening-inhibition (rin) trait resulted in changes in the chlorophyll and carotenoid composition of the fruit, in addition to the obvious inhibition of ripening. Several years later in 2002, the rin trait was identified as being caused by a mutation which impacted two adjacent transcription factors central to the development of fruit and flowers in tomatoes.

In 2012, other researchers realized that the uniform-ripening (u) trait was also caused by a mutation impacting a transcription factor. The broken transcription factor resulted in reduced production of carbohydrates and carotenoids in the fruit, in addition to the apparent change in ripening.

That transcription factor changes were found in both important-market traits is interesting and highlights their potential importance in other traits of interest.

At some point during this process, people began to notice that the tomatoes bought from the store didn't compare to those they or their friends grew at home. This realization has contributed to the recent resurgence of interest in heirloom tomatoes (and other crops) that have been maintained within families who saved seeds over the years. In the case of tomatoes, many of the varieties originated well before the 1930s when the story I tell above began.

The combination of mutation and selection is a very creative and powerful process, but our interests can only direct this process if we're involved in it. If we let others do it for us, it is their interests that will direct the process, as is now evident in the case of tomatoes.

An important aspect of my gardening philosophy is that anyone who grows vegetables should also save seeds. This keeps the interests of the gardener involved in the evolution of the crops they grow and, over the longer term, ensures the vegetables remain worthwhile (by diverse definitions) to grow.

Now that we know how the market-oriented selection of tomatoes has led to their decline, there is the potential to breed varieties which have the better flavor we associate with the heirloom varieties and the traits that make a tomato a market-sucess. However, even if market tomatoes are returned to the glory they should have always been, it will remain important for home growers to keep saving seeds if their interests are to remain involved in the plant's future.

  1. uniform-ripening (u)
  2. ripening-inhibition (rin)

Monday, March 10, 2014

The Genetics of Backcrossing

"Tatume" squash.
"Lemon" squash.
A user over at the Tomatoville forums recently asked for thoughts on a squash breeding project they're thinking of starting. Tomatoville is generally focussed on all-things-tomato, but it has a large range of people interested in breeding of other garden vegetables (including squash).

The goal of the proposed project is to combine the color, flavor, and production of "Lemon" with the vining habit, vigor, and insect resistance of "Tatume". They were seeking input on what strategy to pursue with the F1 plants; backcrossing to "Tatume" or selfing.

Backcrossing is generally used to transfer a limited number of traits from one genetic background to another, without dragging along unrelated genetic baggage. This technique was used to transfer anthocyanin pigment production from wild tomato relatives (Solanum cheesmanii & S. chilense) into the garden tomato (S. lysopersicum var. "Indigo Rose") [1]. It was also used to transfer the red factor from the Pine Siskin (Carduellis cucullata) into Canary birds (Serinus canaria domestica) [2]. In both cases, there were many other traits of the donor species which were not desired in the final product and that were successfully filtered out via backcrossing.


The genetics of squash color is relatively well worked out: two loci interact to produce green (wwgg), yellow (wwG_), or white (W_G_) immature fruit [3]. The genetics of vining habit is also known: one locus produces bush (Bu_) or trailing (bubu) vine habit [4]. The genetics of flavor, yield, and many other traits are less well understood.

If you don't know the genetics of the traits you're interested in, you should first grow out a reasonable number of F2 progeny in order to characterize the inheritance patterns. Growing 20 or so F2s from a selfed-F1 might not give you every expected combination of traits, but it will allow you to examine the ratios of each of the traits you're interested in. The recessive version of each trait will be in the minority.


Backcrossing has specific consequences to a breeding program depending on the genetics of the trait(s) you're looking to transfer.

Dominant : color genetics.

"Tatume" x "Lemon"
P1 : (wwgg) green x (wwGG) yellow
F1 : (wwGg) yellow

"Tatume" x F1
P2 :  (wwgg) green x (wwGg) yellow
BC1 : (wwGg) yellow + (wwgg) green
Recessive : hypothetical flavor genetics.

"Tatume" x "Lemon"
P1 : (BiBi) bitter x (aa) sweet
F1 : (Bibi) bitter
F2 : 3 (1 BiBi; 2 Bibi) bitter + 1 (bibi) sweet

"Tatume" x F1
P2 : (BiBi) bitter x (Bibi) bitter
BC1F1 : (1 BiBi; 1 Bibi) bitter

"Tatume" x F2
P2 : (BiBi) bitter x (bibi) bitter
BC1F1 : (Bibi) bitter

In the dominant case, it is apparent which of the progeny carry the trait of interest. You can immediately choose which plants to use in the next stage.

In the recessive case, it is not apparent which of the backcross progeny carry the trait of interest.  You will have a 50% chance of losing the recessive allele you're interested in during every backcross generation. The solution to this is to screen a set of F2s in every cycle to find progeny carrying two copies of the recessive allele.

Dominant : color genetics.

"Tatume" x BC1
P3 :  (wwgg) green x (wwGg) yellow
BC2 : (wwGg) yellow + (wwgg) green
Recessive : hypothetical flavor genetics.
"Tatume" x BC1F2
P3 : (BiBi) bitter x (bibi) sweet
BC2F1 : (Bibi) bitter
BC2F2 : 3 (1 BiBi; 2 Bibi) bitter + 1 (bibi) sweet

After several cycles of recurrent backcrossing with a dominant trait, the final progeny will be very much like the backcross parent ("Tatume"), but with the exception that they will have both dominant and recessive alleles for the selected locus. A round of selfing to produce F2s, then another round of selfing to produce F2 families, then selecting those F2 families which did not produce any green squash will get rid of the undesired recessive allele from the "Tatume" parent line.

After several cycles of recurrent backcrossing with a recessive trait, the final progeny will be very much like the backcross parent ("Tatume"), with the exception that they will have two recessive alleles at the selected locus. No further selection will be required to stabilize the line, but it will have taken twice as many years to get here compared to the case of transferring a dominant trait.

If you instead screen large numbers of F2s from the original "Tatume" x "Lemon" cross, you might find the combination of dominant and recessive traits you're looking for in the second year. The recessive traits will already be stabilized, while the dominant traits will require more work. Self each F2 plant and save the seeds separately to make F2 families. Growing out the F2 families will let you see which F2 plants had a recessive allele hiding under the desired dominant trait. In addition, this method will let you examine a large number of combinations of genes you weren't expecting in advance.

If one parent was poisonous or had many other negative traits, you would get very few edible progeny and your time would probably be better spent using a backcrossing strategy to filter out everything except the few traits you're interested in transferring.

Both parents in the proposed project are highly edible and only differ in a few charismatic traits, so the vast majority of F2 progeny will be highly edible and have some combination of those traits. In this case, the potential to uncover interesting combinations of hidden alleles out-ranks the limited potential to produce inedible progeny.

Tuesday, March 4, 2014

Domesticating Garlic Mustard

Garlic Mustard (Aliara petiolate) is considered a noxious invasive weed throughout North America. Very few animals find the garlicky taste palatable (deer will preferentially browse on native plants), so it spreads unchecked and can take over in areas where it gets a foothold. It was brought to this continent by European immigrants, who planted it in their gardens for use as a potherb (imagine boiled spinach).

The plant is entirely edible, from root to flower. Analysis has shown it to be high in vitamin C, fiber, and other nutrients. Garlic Mustard does contain cyanogenic compounds and so should be cooked to break down these compounds if it is consumed routinely. This trait puts it in the good company of lima beans, cassava, and flaxseed.

As long as you don't routinely eat it raw in large quantities, you shouldn't have a problem with the cyanide levels you would be exposed to. Remember, the dose makes the poison.

The Lettuce (Lactuca scariola/serriola) and Endive (Cichorium intybus) we enjoy as delectable salad greens were once spiny, bitter weeds. These plants have been shaped by our desires and have hitched their evolutionary destiny to our own.  This process is referred to as 'domestication' and can happen with or without the conscious action of the people growing the plants.

The idea of domesticating random plants amuses me. Garlic Mustard already makes a decent vegetable and I suspect that with some work, it could be made into an excellent vegetable.

A key requirement for breeding a new variety of a plant is having some genetic diversity to work with. To get this starting diversity in Garlic Mustard, I could wait a few hundred years in cryostasis while my henchmen scour the planet looking for some interesting variations of the plant… or I could induce a bunch of mutations and be done with this step in a few years.

I've decided to go with the quick option.
'Kinnow' -> 'KinnowLS'

Agricultural research labs routinely use various chemicals, particle radiation, or x-rays to induce mutations in plants. They then screen the mutated results for improvements in some characteristic to make the plant more useful/tasty/nutritious/etc. The process is known generally as 'mutation breeding' and has been widely used since the 1970s. The seedless orange in the figure at right was generated by mutating the seeded orange, then screening through the mutated progeny for the desired improvements.

I don't feel like dealing with aggressively mutagenic chemicals and I don't have ready access to a particle accelerator or an X-ray generating machine, but I do have ready access to another form of mutagenic radiation. Ultraviolet (UV) light is the component of sunlight which is responsible for giving us a sun-burn. The thymine bases in our DNA absorbs the UV light and becomes chemically altered.   The alterations can result in base mismatches or strand breaks as the excess energy from the UV dissipates. Shortwave UV, or UV-C, is particularly good at damaging DNA and can be produced by commercially available bactericidal-UV fluorescent light bulbs.

Garlic Mustard seeds are small enough that sufficient UV-C light should get to the embryo and cause the mutations I'm looking for. There's no risk to me, as long as I don't get exposed to the UV-C bulbs, so it is a major plus over X-rays or mutagenic chemicals.

After acquiring some UV-C lamps and setting up a light-tight enclosure (so I don't sunburn my eyes/etc.), I will have to determine the radiation dosage needed to successfully mutate the seeds. A reasonable method is to irradiate batches of seeds for different times, then compare their germination to an un-irradiated control. The dosage corresponding to a 50% reduction in germination will give me the most bang for my buck…  producing lots of mutations, but still giving me lots of viable seeds to work with.

Damaged DNA by itself is not a mutation. The damaged DNA has to be repaired with some new change to count. A method to encourage the repair of the damaged DNA is to wake up the seeds by soaking them in water a few days before mutating them, allowing them to restart their paused metabolism. This should result in a higher survival rate with more actual mutations, rather than dead seeds with shredded DNA.

Any dominant-effect mutations will be apparent the first generation after mutation (M1), but many mutations are likely to be recessive and will only become apparent in later generations (M2, etc.).

The first thing to consider relates to the current problems caused by the plant. It is highly invasive, spreading seeds everywhere and taking over wild-lands. An ideal garden vegetable would be a bit more polite and stay where we want it. How would we select for a more polite weed?

A primary difference between wild and domesticated plants is that wild plants drop their seed (referred to as 'shattering') and domesticated plants retain them until we intervene. This single trait would go a long way to convert the noxious, invasive weed into a polite garden inhabitant.

Fortunately, all I need to do to get this trait is to break the existing system the plant uses to drop its seeds. Since I'm going with a mutagenic approach, which essentially is breaking things randomly, I can expect to break this system in some plants. They key detail is to actively screen the M1 and M2 plants for modifications to the shattering trait and then to only work with their descendants. This will keep my potentially interesting garden plants from spreading and taking over wild lands like the wild Garlic Mustard plant.

At this stage of the project, I will have to develop some idea of what my breeding goals are.

Food quality related traits are likely to be easy to examine and select for. Larger roots/leaves/flowers? More delicate, sweeter leaves? Making the plant better for something that people already use it for will produce it something that someone will want to use.   People already use Garlic Mustard for a wide range of recipes, so there are many potential directions.