Publish or Perish

Most of my biology-focused posts have been about topics related to garden projects I've done or have been thinking about. The subject of gardening does account for much of the biology I spend my free-time thinking about, especially now in the middle of a winter-bound Minnesota. It is also far easier to examine the growth and interactions of living things in the garden than it is to go on expeditions or develop lab protocols to explore the diversity of biological topics.

I've avoided talking in detail about the biology I do at work because the work is on-going and to publicize it here might detract from the process of publishing the work in more formal contexts once the projects reach usable conclusions. Sometimes a key result can be quickly replicated once the idea behind it has been developed, so talking about results too early can be asking for some competitor to beat you to publication. I've defended my thesis and dealt with graduate school bureaucracy sufficiently to be awarded my PhD. I can now refer to myself as "Darren Abbey, PhD" in professional contexts. (I can also refer to myself as "The Doctor" (Dr. Who?) in certain social contexts.)

Along the way, I've written or contributed to several research publications. The following list is the publications I've been received name credit for my contributions, from oldest to most recent.

1. Gale CA, Leonard MD, Finley KR, Christensen L, McClellan M, Abbey D, Kurischko C, Bensen E, Tzafrir I, Kauffman S, Becker J, Berman J. (2009) SLA2 mutations cause SWE1-mediated cell cycle phenotypes in Candida albicans and Saccharomyces cerevisiae. Microbiology. 155(Pt 12):3847-59. [PMID: 19778960]
    
2. Forche A, Abbey D, Pisithkul T, Weinzierl MA, Ringstrom T, Bruck D, Petersen K, Berman J. (2011) Stress alters rates and types of loss of heterozygosity in Candida albicans. MBio. 2(4). [PMID: 21791579]
    
3. Abbey D, Hickman M, Gresham D, Berman J. (2012) High-Resolution SNP/CGH Microarrays Reveal the Accumulation of Loss of Heterozygosity in Commonly Used Candida albicans Strains. G3 (Bethesda). 1(7):523-30. Erratum in: G3 (Bethesda). 2(11):1473. [PMID: 22384363]
    
4. Hickman MA, Zeng G, Forche A, Hirakawa MP, Abbey D, Harrison BD, Wang YM, Su CH, Bennett RJ, Wang Y, Berman J. (2013) The 'obligate diploid' Candida albicans forms mating-competent haploids. Nature. 494(7435):55-9. [PMID: 23364695]
    
5. Abbey DA, Funt J, Lurie-Weinberger MN, Thompson DA, Regev A, Myers CL, Berman J. (2014) YMAP: a pipeline for visualization of copy number variation and loss of heterozygosity in eukaryotic pathogens. Genome Med. 6(11):100. [PMID: 25505934]
    
6. Ford CB, Funt JM, Abbey D, Issi L, Guiducci C, Martinez DA, Delorey T, Li BY, White TC, Cuomo C, Rao RP, Berman J, Thompson DA, Regev A. (2015) The evolution of drug resistance in clinical isolates of Candida albicans. Elife. 4. [PMID: 25646566]
    

I've also got a couple others in the pipeline. For one I'm looking for a target journal, for the other I'm still deciding exactly how to present the results. I'll let you know more details once they're further along the way to publication.

The projects a grad student works on depends on a mix of the lab they end up in and their personal style of problem solving. I ended up in a Candida albicans lab and brought to it a heavy computational approach. The mix between the two is the realm of computational biology, the topic I find myself most connected to.

The academic life can readily be described as, "Publish or Perish". With six name papers from my time in grad school, I've done alright. Now I just have to figure out the next step.

The Color of Cotton

1. www.perunaturtex.com/scientif.htm

Ever since I first heard about it, I've been interested in naturally colored cotton (also known as "color grown cotton"). The cotton plants most people are familiar with produce a pure white fiber, which can then be dyed to match any desired color. Naturally colored cotton, on the other hand, is grown with color straight from the plant.

There is archaeological evidence for the existence of cotton in various shades of yellow, brown, green, and red. The pre-Columbian Peruvian textile at left is supposedly made from colored cotton without the processing of additional dyes. There are also reports of a naturally colored blue cotton, but the internet has provided minimal evidence for this.

The majority of commercially grown cotton belongs to the species Gossypium hirsutum. Varieties of this cotton species can be found in light brown and green, but the other colors are generally nowhere to be found.

2. Inheritance of different fiber colors in cotton.

During a recent web search, I came across an image of a very dark brown naturally colored cotton. The image also shows a nice orange cotton, in addition to the more typically seen tan and light brown colors. Seed for these varieties aren't generally available, however, but they can be accessed from different seed collections (such as GRIN) if you can show you have some worthwhile research, education, or other public good rationale for having them.

These more interesting colors are seen in relatively wild varieties of a second cotton species, G. barbadense. G. hirsutem and G. barbadense don't naturally cross in the wild due to different timing of pollen maturation and other mechanisms. These are all easily circumvented by directed hybridization efforts. The resulting F1 hybrids grow well and are fertile, though fertility issues do arise in some F2 generation plants. These issues wouldn't interfere greatly with the intentional recombination of pigment alleles from both species, which I think has interesting potential to create new interesting colored varieties.

After I've done some further experiments with growing the cotton lines I already have in Minnesota, I might request some of the interesting colored forms from the collections.

3. Cotton and I.

...wait, growing cotton in Minnesota (red star)? ...a thousand miles north of where cotton is grown in the USA (green regions)?

A few years ago, on a whim, I planted some cotton seeds I had come across in south Texas. Cotton is typically described as needing a long and hot growing season to mature. I don't know how the production of my plants compared to similar plants in the South, but their production was dramatically higher than I expected up here in the North. After the first hard freeze of winter, I broke the plants off at their base and hung them to dry in the garage. A few weeks later, the cotton fibers had completed drying and were easy to collect. This process might not be something that can be scaled up to a proper crop, but it might. Further research is needed.

References:

1. www.perunaturtex.com/scientif.htm
2. Dark brown cotton: www.scielo.br/scielo.php?pid=S1984-70332014000400008&script=sci_arttext
3. GRIN: www.ars-grin.gov/npgs/orders.html
4. Crossing: www.ogtr.gov.au/internet/ogtr/publishing.nsf/content/cotton-3/$FILE/biologycotton08.pdf

The Case of the Marvelous Meiosis

1. Meiosis in male (A) and female (B).

The typical story of meiosis is described in detail in many a biology textbook. It all starts with a diploid somatic cell. The chromosomes duplicate and homologs align along the cell's mid-line. The aligned chromosome doublets crossover and then the doublets are divided into two new cells (Meiosis I). The chromosome doublets align along the mid-lines and the new cells divide again (Meiosis II) to produce the final gametes. This model well matches the process of spermatogenesis (Fig 1A), which was used as a model to study and understand the process. Oogenesis follows basically the same process, except that each division is asymmetric. The result is one large gamete (Fig 1B) and two or three non-viable cell fragments with the extra chromosomes.

2. Meiosis in male (A) and female (B) Caninae group roses.

Roses in the group Caninae (Dog Roses) do a peculiar version of the process. The basic example is tetraploid, but produces haploid male (Fig 2A) and triploid female (Fig 2B) gametes. During meiosis I, two homologs for each chromosome align to form a bivalent and the rest remain as monovalents. The monovalents are lost during spermatogenesis, but retained during oogenesis. This system is referred to as "permanent odd polyploidy".  It even works with extra uneven chromosome copies. The extras form monovalents and are discarded or retained just like the red and blue chromosomes in Figure 2. This system allows fertility to be maintained even with odd chromosome counts that would normally make a plant sterile.

I came across this peculiar variant of meiosis while researching what complications might develop in a cross between Rosa pomifera (4n, group Caninae) and R. rugosa (2n). Both parent species have relatively large fruit. My initial plan was to hybridize the two species, generating at least one F1 plant, then allow them to self and screen many F2 progeny plants for increased fruit size.

That the two roses are not very closely related and both species have numerous smaller-fruited relatives suggests their large fruit was evolved separately. This is useful for a breeding project as it means that different mutations accumulated as each plant developed large fruit. Those separate mutations can possibly be recombined via hybridization to generate a progeny plant with even larger fruit.

I expect the project of domesticating roses as [more of] a fruit will take decades. It should be an amusing hobby and I expect to have enough time left to see some nice results.

References:

1. Meiosis: en.wikipedia.org/wiki/Meiosis
2. Rose groups: en.wikipedia.org/wiki/List_of_Rosa_species
3. Dog rose meiosis:
1. www.nature.com/hdy/journal/v101/n4/abs/hdy200863a.html
2. www.ncbi.nlm.nih.gov/pubmed/24685720

Mathematical Recreations : Homeomorphically Irreducible Trees of size n=10

A homeomorphically irreducible tree is an acyclic graph where there are more than two branches from each internal vertex. The size n=10 means there are ten vertices, internal or edge, in the tree.

A. Homeomorphic irreducible trees of n=10.

In the movie "Good Will Hunting", the puzzle of calculating all of such trees with a total of ten vertices was presented to a class as a difficult challenge that took a couple years to calculate.  The puzzle came to my attention after watching a Numberphile video on youtube, where the guest mathematician rightly pointed out that it really isn't that difficult of a puzzle as he drew out all ten possibilities.

There are lots of blog posts pointing out how simple the problem actually is, but neither the Numberphile mathematician or any of the various bloggers point out how the ten possibilities are connected by a set of very simple transformations.

B. Transformation rules.

The first transformation (fig B1) takes three leaves (edge vertices) from an internal vertex and generates a new internal vertex with two leaves.

The second transformation (fig B2) moves a leaf from one internal vertex to another.

You can't apply a transformation such that it will generate a tree with only two branches from an internal vertex, as the resulting tree will no longer be homeomorphically irreducible.

C. Transformation relationships.

With these transformations, it is a simple task to generate all the possible homeomorphically irreducible trees for higher numbers of vertices. (At least for every case that I've examined.) You start with the simplest case of the pinwheel and apply as many of the transformations as you can at each step until you run out of steps. Proving that this process will generate all possibilities for any number of vertices is a more difficult challenge.

I suspect there might be a set of such trees where individuals are isolated from others, where another transformation would be required.

D. Transformation graph.

The map of trees connected by transformations generates a new graph (such as fig D). My limited scribbling of the trees for a set of 11 and 12 nodes shows that there is more than one valid transformation graph for some sets of trees. I'll have to explore the properties of these transformation graphs further at some point.

The Trouble with Seeds (3/3)

For someone interested in breeding plants, that some plants often don't produce seeds can be a major barrier. A breeder would be limited to looking for selectable mutations, rather than using the more general mixing and segregation of genetics to increase crop diversity.

Many of our common vegetables, fruits, and landscape plants are traditionally propagated by clonal divisions. In some cases (apples, pears, etc.) the complex genetic diversity of the crop means that every seedling would produce a distinct plant. The market demands for consistent production then encourage growers to clone the plants. In other cases, the clonal tradition is enforced by the plants themselves due to their inability to produce seeds. There's also a third category of plants which have such long life-cycles that breeding projects become prohibitive.

Researchers have figured out tricks to get seed and allow breeding to be done with plants that might otherwise prefer not to.

The Trouble with Seeds (1/3): Garlic, Horseradish, Potato Onion, Walking Onion, and Banana.
The Trouble with Seeds (2/3): Pineapple, Lily of the Valley, Potato, and Sweet Potato.
The Trouble with Seeds (3/3): Babington's Leek, Crosnes, and Bur Oak.

Most of this post is an accumulation of information from other sources (in colored quotes below) about how to get seeds from these crops. I've also included my thoughts and experiences where I felt something needed to be clarified or extended.

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How to get Babington's leeks (Allium ampeloprasum var. babingtonii) to set seed.

Many alliums have forms which produce clonal bulbils in the flower head along with some flowers, but generally don't produce any viable seed. Research in garlic (A. sativum) has shown that you can often get seeds to mature if you extract the bulbils from the flower head at an early stage. The bulbils seem to draw nutrients away from the flowers and cause them to age too quickly for seeds to mature. When the bulbils are removed, the few flowers then get sufficient energy to grow and mature seeds. This process will probably work for Babington's leaks, or any other allium which produces bulbils and no seeds.

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How to get Crosnes (Stachys affinis) to set seed.

[1] "Stachys palustris performed much better here than S. affinis this year - larger tubers, greater yields, no difference in flavor that I can detect. I've never grown either one before. Both set some seed, which is apparently common for S. palustris but not S. affinis. I wonder if I got some crossing."

If S. palustris shows self-incompatibility, the clonal Crosnes will generally fail to set seed. The plants would then be desperate for usable pollen, even from related species, in order to set seed. If they're crossed, the plants that grow from any of those seeds should be highly homogeneous, but distinct from either parent.

1. alanbishop.proboards.com/thread/4973/stachys-affinis?page=2#ixzz3PznHBzAA

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How to get Bur Oak (Quercus macrocarpus) to set seed, when very young.

Bur Oak trees can live for hundreds of years and generally don't start fruiting until they're 35+ years old. The wild trees produce very large acorns that are relatively sweet, requiring minimal processing to use as a food item. Domestication efforts could readily convert the species into a useful nut crop, but the long life cycle means it will generally have to be considered an intergenerational project.

Grafting methods are routinely used to increase fruit production or control the growth of fruit trees. Such grafting methods might also be applied to the Bur Oaks. When a host oak tree goes into bloom, the hormonal signals flowing through its sap will encourage the grafted Bur Oak to also go into bloom. There are numerous shrub oak species (Q. cornelius-mulleri, Q. dumosa, Q. durata, Q. palmeri, Q. sadleriana, Q. turbinella, Q. vaccinifolia, or Q. wislizeni) that mature much younger than the Bur Oak. One of these might be ideal as a host for grafted seedlings. The shorter life of the host oak would mean that you could plant as many as you need to host whatever number of Bur Oak seedlings you're working with. This process should shorten the 35+ year breeding cycle of the Bur Oak down to 2-3 years, making it a much more reasonable project to take on.

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Most of the lovely plant diagrams in this post were derived from public-domain images hosted at botanicalillustrations.org. Some other diagrams are public-domain images from the same era that I found via google. I chose the original images which depicted the plants under discussion in the way I appreciated and then did some coloring or color correction using my favorite image editor (GIMP)

Hybrid Sterility and Speciation

1. Burbank's Fragaria x Rubus.

Luther Burbank (1849-1926) was a widely renown botanist and scientist. He bred numerous interesting plants. He liked to attempt wide crosses; crosses between distantly related species. One of the more unexpected crosses he attempted was the cross between a strawberry (Fragaria spp.) and a raspberry (Rubus spp.) (fig. 1). To the then (and now) commonly held Victorian ideal of plant species, this cross shouldn't have had a chance at all of working. However, the cross appeared to succeeded and fruit developed. The plants that developed from the seeds grew with a combination of characters from the parents, thus showing their hybrid nature. The hybrids flowered abundantly in the second year, but no fruit was ever produced. Burbank found that at most a few seedless drupelets (fruit segments like in a raspberry) would form and so he abandoned the project.

There is a lot that Luther Burbank didn't know about plants. His exuberance for performing crosses and doing selections let him produce some wondrous results, but his lack of knowledge was a limitation.

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Chromosomes were discovered in the 1880s, but the process of meiosis wasn't made clear until 1905-1911. The dates suggest it is possible that Burbank was aware of meiosis, even if he wasn't aware of the consequences for his work. Fortunately, such knowledge is now widespread and biologists are well aware of the consequences.

2. Meiosis and failures of meiosis.

Strawberries and raspberries show a diversity of genome sizes, but they all have a basic chromosome count of 7. They species range from diploid with 14 chromosomes to decaploid with 70 chromosomes. An even number of chromosomes is found in all cases, as this is required for the formation of gametes (fig. 2A).

If two species with different chromosome counts are crossed, the resulting hybrid can have an uneven number of chromosomes and will be generally unable to generate gametes (fig. 2B). (Example: 2n x 4n => 3n; this is how seedless watermelons are made.)

If two species with the same number of chromosomes are crossed, but the chromosomes are too unrelated, the resulting hybrid will also fail to generate gametes (fig. 2C). In this case the hybrid will have an even number of chromosomes, but they won't line up during meiosis and the result will be a haploid with an increased basic chromosome count. This can be caused by a high level of structural rearrangements in the chromosomes of strawberries vs. raspberries, even if the genes are otherwise compatible.

Because Burbank performed the cross with whatever strawberry and raspberry plants were convenient and the cytogenetics of the parent plants wasn't examined, either of the above scenarios could be responsible for the hybrid infertility that he saw.

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I have one raspberry (Rubus occidentalis, isolated in my yard in Minnesota) and one strawberry (Fragaria vesca, isolated in central Wisconsin) in my collection and I think I will set about crossing them during this year. Both species have been examined in detail and happen to be diploid with 14 chromosomes, so the first incompatibility mechanism isn't a concern.

3. Meiosis after allotetraploidy.

The second incompatibility mechanism can be overcome by inducing tetraploidy in the hybrid. This would be done using Colchicine or Oryzalin, herbicideal compounds that interfere with cell division and result in a doubling of the number of chromosomes in treated tissue. Induction of tetraploidy generally produces one branch that has larger fruit, thicker stems, and other visible features to distinguish it from the original diploid parts of the plant. Because this tetraploid would contain two full copies of genomes from different species, it would be referred to as an allotetraploid.

4. Tragopogon spp. hybrids.

This process has been observed to happen naturally. Three species of Tragopogon (T. dubius, T. porrifolius, and T. pratensis) were introduced into the Pacific northwest region of the USA in the early 1900s from Europe. By the 1950s, scientists realized there were two new species of Tragopogon to be found in the region (T. mirus and T. miscellus). The new species were fertile allotetraploid hybrids between pairs of the introduced species. The hybrid species have even been recreated in the lab. In this case, it appears the allotetraploids came about because the parent species occasionally produce aberrantly diploid gametes which merged to form the fertile allotetraploid. The precise pathway is different than what I expect would be going on with the strawberry/raspberry cross, but it is a wonderful case-study for hybrid speciation.

If everything works out, it will be a few years before I have a fertile strawberry/raspberry cross. I wonder what the fruit would taste like? I'll keep you informed as it goes.

References:

• Burbank plants: mentalfloss.com/article/57818/10-crazy-creations-plant-wizard-luther-burbank
• Luther Burbank cross: bulbnrose.x10.mx/Heredity/Burbank/Burbank_raspXstraw.html
• Strawberry chromosome counts : strawberryplants.org/2011/02/genetics-of-strawberry-plants/
• Raspberry chromosome counts : hortsci.ashspublications.org/content/30/7/1447.full.pdf
• Rubus occidentalis: en.wikipedia.org/wiki/Rubus_occidentalis
• Fragaria vesca: en.wikipedia.org/wiki/Fragaria_vesca
• Colchicine: archive.org/stream/useofcolchicinei3424derm/useofcolchicinei3424derm_djvu.txt
• Colchicine and Oryzalin: hortsci.ashspublications.org/content/43/7/2248.full 
• Tragopogon 1: www.massey.ac.nz/~jtate/index_files/Page573.htm
• Tragopogon 2: nzprn.otago.ac.nz/wiki/NZPRN/PeopleSymonds
• Tragopogon 3: scienceblogs.com/observations/2010/10/20/evolution-watching-speciation-1/

Astrobiology : Mars & Isotopic Analysis

Two stable isotopes of carbon are found, carbon-12 (¹²C) and carbon-13 (¹³C). Carbon-12 is the vast majority, at 98.89% abundance. Individual samples of carbon from different sources can vary strongly from the general abundance ratio, depending on where they came from.

The reactions of biology tend to prefer the lighter form of carbon, so over time biological sources of carbon tend to become relatively enriched in carbon-12 vs. carbon-13. A consequence of this is that carbon samples from a living source are distinct from carbon samples from a geological source.

We've examined the carbon ratios from many sources here on Earth. We've now also examined the carbon ratios from a few sources on Mars. A study was published in the December 2014 edition of Meteoritics & Planetary Science that discussed the examination of carbon ratios in a recently recovered Martian meteorite called Tissint.

There are significant differences in the carbon pattern between Earth and Mars. I put together the above figure from one in references #1 and #2, to better compare the known carbon ratio differences by sample type.

The carbon ratios found in carbonates on Mars suggest they are of inorganic origin. Those from freshwater or marine sources on Earth have ratios consistent with their known biological origin.

The red data-points are from organic material found in Tissint. They compare very well to the blue data-points from coal here on earth. On both planets, atmospheric carbon is relatively reduced in carbon-12 compared to the organic material. This is pretty good evidence that the organics in the meteorite came from some living thing, just as the organics in coal did.

The carbon could still have come from contamination here on Earth, after the meteorite landed. To test for this, the researchers examined the isotopic ratios of the hydrogen in the sample. Mars is enriched in deuterium relative to Earth and this same enrichment was found in the examined organic material.

This research happens to be the best direct evidence so far for the existence of life on Mars (at some point in the past). This still isn't entirely conclusive physical evidence. For that we will have to go to Mars and either find an unambiguous macro-fossil, or culture some micro-organism from one of the brine-water seeps we've been looking at from space.

I've been convinced that there was in the past (and is most likely today) living organisms on Mars, from theoretical considerations about how life works, and how it must have spread in the early solar system. However, I wasn't really expecting to see any convincing physical evidence any time soon.

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References:

1. Tissent Carbon ratios: onlinelibrary.wiley.com/doi/10.1111/maps.12389/full
2. Earth Carbon ratio: www4.nau.edu/meteorite/meteorite/book-glossaryc.html 
3. Mars Carbon ratio: www.astrowatch.net/2014/12/scientists-find-new-evidence-for-life.html
4. Organics on Mars: www.sciencedaily.com/releases/2014/12/141216144137.htm