// Twitter Cards // Prexisting Head The Biologist Is In: ploidy
Showing posts with label ploidy. Show all posts
Showing posts with label ploidy. Show all posts

Monday, February 12, 2018

Chromosome Painting

Microscope image of chromosomes, false-colored to help visually distinguish individual chromosomes. The figure at left shows the metaphase chromosomes of a pepper root-tip, in all their squiggly false-color glory. In it you can count the number of chromosomes and (with some little background research) determine the overall ploidy of the source plant. (It has 24 chromosomes, so is a diploid.)

The original image had all the same information, but it was much harder to look at and learn from. This is a fundamental lesson of, and reason for, data visualization.


Microscope image of a cell with condensed chromosomes visible.
Step 0.
The original image comes from Twitter user @ChaoticGenetics. They're studying chile genetics and routinely post cool photos derived from their work. The question paired with this image was, "How many chromosomes does everyone see?" I figured I'd take a stab at it.

Lets dive into the details of how I made my figure. I use GIMP for essentially all my image editing needs. With each step figure I'll include the menu options for each command I use in brackets, so others can repeat the procedure.

0) Load the image with GIMP. Open "Tool Options" [Control-B] and "Layers" [Control-L] windows.

initial image cropped to just show the condensed chromosomes.
Step 1.
White color of the image has been converted to transparency.
Step 3.
1) Select a rectangular region around the interesting looking chromosomes, then crop [Image > Crop to Selection] the image.

Background of the condensed chromosomes has been erased.
Step 2.
2) Select the "Eraser Tool" and erase all the background color and spots that don't appear as chromosomes.

3) Right-click on the image in the layer window. Select, "Add Alpha Channel". Discard the color information in the image [Colors > Desaturate]. Remove the background color [Colors > Color to Alpha (Set "From:" color to white.)].

White background is restored to the image.
Step 4.
4) From the layer window, make a new image layer filled in white. Move this layer beneath the image layer. Select the image layer.

A single condensed chromosome has been false-colored with purple.
Step 5.
5) Using the "Free Select Tool", draw around a visually distinct chromosome. Invert the color of the selection [Colors > Invert]. Change the color of the selection [Colors > Components > Channel Mixer... (red=50,0,0; green=0,0,0; blue=0,0,50)].

A second condensed chromosome is false-colored, this time in green.
Step 6.
6) Many of the chromosomes in this example are adjacent or overlapping with another. For these, we have to use some knowledge about chromosomes and some artistry. Lets have a look at the cluster here highlighted in green.

Green false-colored chromosome broken up into three parts, each colored part colored differently (blue, red, and green).
Step 7.
7) At this scale, chromosomes are essentially linear structures. They don't branch and they don't loop. From this we can tell the green feature in step 6 is actually three chromosomes. I cut each chromosome out of the image and pasted into a new layer. From there I could clean up their shape a little before changing the colors and recombining them.
Several chromosomes have been highlighted in various colors. Large aggregate of condensed chromosomes that can't be visually separated is colored in pink.
Step 8.

8) Going progressively through the image, isolating and coloring the most apparent chromosomes at each stage, we come to 16 chromosomes that we can be confident about. (So, our cell isn't a haploid with 12 chromosomes.)

We're left with the region at left I've highlighted in pink. This region would need to account for a further 8 chromosomes to reach the expected diploid count of 24 in total. Though there are probably a few chromosomes in this region that we can confidently separate, much of it is down to guesswork.

It is possible for this specific pepper plant to have fewer chromosomes. Though it is unlikely for a chromosome pair to be lost, since each has been conserved over a long time period and likely contains critical genes, it is common enough evolutionarily for chromosomes to fuse. That pink mess could hypothetically be 6 or 4 chromosomes, though this one image isn't sufficient evidence to make me think it is likely. If the same pattern is shown in a few more images from the same plant, especially if the chromosomes are better spread, then I'd start to consider that as increasingly likely.


For now, the balance of the evidence leads me to think there are 24 chromosomes and they're just not perfectly isolated. So, I divided the uncertain pile of chromosomes into the number that I expect are remaining. Any figure you make will invariably include your assumptions. The key is to try and make those assumptions reasonable or at least apparent to the reader (though this may require some nice caption-writing).

Interestingly, there's a protocol which can experimentally produce the sorts of painted chromosomes we're simulating here. Fluorescent In-Situ Hybridization (FISH) relies on making DNA probes which are stained a unique color for each chromosome. When the probes are applied to a chromosome spread, the result helps visualize chromosome crossovers, deletions, and other large scale alterations that can be important in diagnosing cancer and other disorders. The setup work for this is pretty intense, so it's probably not going to be used for the simple task of seeing how many chromosomes a plant has.


While I was in grad school, I routinely modified figures from papers I was reviewing for in-class (or in-lab) presentations. Usually highlighting different components of the figure in different colors (like here), to make them stand out more when displayed. I was doing the hard work of figuring out the important parts of the figures so students watching my presentation didn't have to. My goal was for them to focus on what I was saying about the figures and see what wanted them to see at a glance.

Using colors to present different partitions of a larger dataset ended up being central to my last large graduate project (YMAP) as well as an important part of my current [non-academic] job. While using colors for data presentation, it is important to keep in mind that not everyone has the same ability to see color. The most common forms of color-blindness are often called Red-Green-colorblindness. From this, it is a good idea to try and avoid the commonly used Red-Green color scheme seen so often in biology research figures. (Blue-Yellow is a good alternative, but there are subtleties I'll have to go into later.) Being conscious of the issues means they will inform your decisions, even if you're not fully aware of the topic.


This post was inspired by a conversation over on Twitter. (You can follow me there as @thebiologistisn.)

The original picture of the chromosome spread was made by @ChaoticGenetics, who gave permission for me to use it in this post.


References:
https://twitter.com/ChaoticGenetics/status/9602424397463060
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Tuesday, May 31, 2016

Night of the Hopping Amphibians!

I recently went out to my car after dark to retrieve a bag of pine-cones (for a later posting). On my way down the stairs, I heard a "churp" and saw a grey, patterned frog a few steps down. I politely stepped aside, then scooped it up. I set it down in a shallow puddle on one of the steps and continued to my car. When I opened the door, the light cast downwards onto a toad who was hiding flat-like against the gravel. I scooped it up too, then set it on a nearby wall where I would be able to find it in a minute or two. I retrieved the items from my car and quickly returned inside to grab my camera and a light source.

Grey treefrog on purple surface.
Hopper #1
On the way back down the stairs, I picked up the frog again and placed it on a vertical part of the railing. It took a few pictures before I got one I liked. What I thought was a very bright LED flashlight turned out to be just barely bright enough to get decent exposures with the lens I was using.

American toad tucked down into dirt.
Hopper #2
The toad was hunkered down into a shallow spot on the wall. This was perfectly fine for taking its portrait, as it let me brace the camera against the very stable concrete. I picked the toad back up and placed it on the ground near the base of the wall.

Two green-colored treefrogs.
Hopper #3
After finding the two critters, I took a walk around the house to see if I could find any more. I didn't immediately notice any, but I did notice I had left a pepper plant where deer would be able to get to it. I started to move the pepper to a more protected location when a little green frog came popping out.

Single grey spotted treefrog.
Hopper #4
After another quick search, I headed back inside. As I came up the steps, I found the first frog was where I had left it. I also found a smaller grey frog about a foot further up the railing.

We've just had an intensely rainy day and the warm season is far enough along that there are plenty of bugs around. This was apparently the perfect night to go looking for amphibians.


The three frogs likely belong to the same species. They could be either the "Cope's Grey Treefrog" (Hyla chrysoscelis) or the "Grey Treefrog" (H. versiclor). Both species change color from a pale grey, to near black, to bright green depending on their emotional state and the surroundings they're trying to hide in. It can be difficult to be sure which species is in hand. They have slightly different patterns of coloration and different pitches to their calls. Biologically, the biggest difference between them is in how many chromosomes they have. H. chrysoscelis is diploid, while H. versicolor is tetraploid. I suspect the frogs in my yard are H. versicolor, based on the example photos I've found, but I'm really not certain.

The identification of the toad is much simpler because the "American Toad" (Anaxyrus americanus) is a distinct member of the few toad species known in Minnesota.


References:
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Thursday, January 22, 2015

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.


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.


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:
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Tuesday, October 7, 2014

Genetics in Sunflowers.

Plant #1 (H. tuberosus).
Plant #1 leaf.
Last year I started a genetics experiment with sunflowers. I grew a perennial sunflower (Helianthus tuberosus) and an annual sunflower (H. annuus cv. "Russian Mammoth") and tried to cross them. I shook pollen from the very large H. annuus flower all over and around as many of the very small H. tuberosus flowers as I could. This cross has been done before ([1], [2], & [3]), but not necessarily with a giant variety like "Russian Mammoth" as the father variety. I was working under the assumption that I'd be able to recognize any hybrids.

At the end of the season, I dissected 12 seed heads and recovered approximately 70 seeds. I ended up not planting any seeds this year, as I knew I was moving to a new house and wouldn't be able to maintain the experimental plants.

As I was doing a final cleanup of my flat, I noticed a sunflower blooming out in the backyard. On closer examination, there were three sunflower plants poking up through the weed patch. The only sunflowers growing in the area last year were my experimental parents. The H. tuberosus mother was growing in a planter and the H. annuus father wasn't allowed to mature seeds in the garden, so I was pretty sure these sunflowers had grown from seeds dropped by the mother plant.

The first plant to bloom appears to be completely H. tuberosus, though it shows much more red in its stems than the mother.  The other two plants came into bloom later and appear to be hybrids.

There are several traits which distinguish H. tuberosus from H. annuus cv."Russian Mammoth":

Plant #3.
  • H. tuberosus leaf margins extend down the entire petiole, while in H. annuus the leaf margin ends at the top of the petiole. All three plants showed the H. tuberosus version of this trait.
  • H. tuberosus has an elongated leaf, while H. annuus has a wide leaf. The blooming plant showed the H. tuberosus version of this trait. The other two plants had leaves of an intermediate form.
  • H. tuberosus flowers are very small, while H. annuus cv."Russian Mammoth" flowers are very large. The first plant to bloom has flowers the size of those on H. tuberosus. The other two plants now have flowers which are about twice as wide as those on H. tuberosus.
  • H. tuberosus plants are highly branched with many flowers, while H. annuus cv."Russian Mammoth" has a single stem with a single terminal flower. The first plant to bloom has lots of side-branches and lots of flowers. The second plant to bloom has a few small side-branches and a few flowers. The third plant has no side-branches and a few additional flowers growing from leaf-axils.
  • H. tuberosus plants have thin stems, while H. annuus cv."Russian Mammoth" plants have a very thick stems. The first two plants to bloom have thin stems and need support to stay upright. The third plant to bloom has a thick stem, capable of keeping it upright without support.
  • H. tuberosus plants grow to near 6 feet tall, while H. annuus cv."Russian Mammoth" plants grow to near 11 feet tall. The first plants to bloom topped out below 5 feet tall. The second reached about 7 ft and the third reached over 8 feet tall before blooming. I'll get a better measurement of their height when they have died for the season.
  • H. tuberosus produces underground tubers, while H. annuus does not. I'm waiting until frost has killed the plants before digging them up, so they have the best chance to produce tubers.

Plant #3, showing winged petiole.
What predictions can we make about a cross between H. tuberosus and H. annuus? H. tuberosus is hexaploid and H. annuus is diploid, so the resulting hybrid will be tetraploid.

H. tuberosus is usually propagated by tubers, so it can contain lots of hidden genomic diversity across it's six sets of chromosomes. The progeny plant which appears to be completely H. tuberosus has much higher levels of red pigment in its stems, indicating heterozygosity is present in the H. tuberosus parent. H. annuus cv. "Russian Mammoth" is an annual variety that has been stable since 1880, which means it is highly inbred and therefore highly homozygous.

With this information, the cross would look something like:

A1A2A3A4A5A6 x BB

The resulting F1 progeny would be:

AaAbAcB

There are 20 potential combinations of three alleles of of the possible six homologs at a each locus of the 17-chromosome sets found in the H. tuberosus parent, so it isn't surprising that the two observed hybrids aren't entirely alike.


Selfing the F1s...
AaAbAcB x AaAbAcB

…has all kinds of possible outcomes. If we simplify the calculation by assuming the H. tuberosus parent is homozygous, we get a cross...

AAAB x AAAB

…where half of the gametes are AA and the other half are AB. The Punnett square for this cross...
AA
AB
AA
 AAAA AAAB
AB AAAB AABB

…shows there's no way to get an F2 which is homozygous for the alleles from the H. annuus parent. A goal of this project is to breed up a sunflower which has the tuber-generating trait of H. tuberosus and the super-sized growth of H. annuus cv. "Russian Mammoth". If I can isolate some F2s that appear AABB for some of the interesting traits, perhaps by isolating those that have the best tubers and the most overall growth, then I might later be able to select from F3 families which cover the whole range of allelic combinations at the loci important for these two traits. The Punnett square for selfing the AABB F2s...

AA
AB
AB
BB
AA AAAA AAAB AAAB AABB
AB AAAB AABB AABB ABBB
AB AAAB AABB AABB ABBB
BB AABB ABBB ABBB BBBB

Plant #3 flower buds.
…shows the very diverse combinations expected at each locus. From such an F3 family, I would then have the best odds of selecting out a plant with several alleles driving tuber formation and driving extreme plant growth. The combination of which, I hope, would then result in extreme tuber production.

In a typical diploid cross, the F2 generation is where the most combinations of alleles appear and where selection is most important. In this example, it wasn't obvious before calculating through the probabilities that the F3 generation would be where the most combinations of alleles would appear.

Hopefully, the deer at my new place will leave my sunflowers alone for the years it will take to complete this project.


References:
  1. Encheva, J., M. Christov, and P. Ivanov (2003). Characterization of Interspecific Hybrids Between Cultivated Sunflower H. annuus L. (cv. Albena) and Wild Species Helianthus tuberosus. Helia 26: 43-50.   (http://www.doiserbia.nb.rs/img/doi/1018-1806/2003/1018-18060339043E.pdf)
  2. http://bulbnrose.x10.mx/Heredity/sunflowerXchoke/sunflowerXchoke.html
  3. Kantar, M. B., K. Betts, J. Michno, J. J. Luby, P. L. Morrell, B. S. Hulke, R. M. Stupar, and D. L. Wyse (2014). Evaluating an interspecific Helianthus annuss x Helianthus tuberosus population  for use in a perennial sunflower breeding program. Field Crops Research 155: 254-264 (http://experts.umn.edu/pubDetail.asp?id=84888291084&o_id=199&t=pm)
Information about traits from references and observations.
  • Reference #2 indicates tuber formation is dominant in the F1s.
  • Winged petioles are dominant in F1.
  • Secondary branches are mostly dominant in F1. There seems to be allelic variation for this trait in the H. tuberosus parent.
  • Large flower size is mostly recessive in F1.
  • Tall growth is partly dominant in F1. There seems to be allelic variation for this trait in the H. tuberosus parent.
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