Tuesday, October 28, 2014

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.



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.



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.

Wednesday, October 15, 2014

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.



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.



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.



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.


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 
2tt2tt3TT3TT2tt2tt3TT3r2tt2R3TT3TT2tt2R3TT3r
2tt3r
2tt2tt3TT3r2tt2tt3r3r2tt2R3TT3r2tt2R3r3r
2R3TT
2tt2R3TT3TT2tt2R3TT3r2R2R3TT3TT2R2R3TT3r
2R3r
2tt2R3TT3r2tt2R3r3r2R2R3TT3r2R2R3r3r

… 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.



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.



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

  5. 7. Derived from S. peruvianum.
    • [unnamed] gene: pigment on fruit shoulder, similar to "Aft". Seen in variety "Purple Smudge". (image #7)
  6. S. pimpinellifolium
    • gene #1: pigment on fruit shoulder, similar to "Aft". Described here.
    • gene #2: pigment throughout plant, similar to "atv". Described here.
  7. Conventional tomatoes
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!



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

Tuesday, October 14, 2014

Something to grow.

From reference #5.
Parochetus communis

There aren't all that many true-blue flowers around. P. communis ("Shamrock Pea", "Blue Oxalis") is one that originally came from the mountains of Africa and Asia and has been grown in Europe since the 19th century. It is low-growing and has leaves much like the oxalis which lends it one of its common names. There are occasional photos of versions with pink or white in the flower and many with different patterns of red and white on the leaves.

I haven't grown this plant yet, but I will sometime. 

References:
  1. http://en.wikipedia.org/wiki/Parochetus
  2. http://gardenbreizh.org/photos/AberBenniget/photo-151458.html
  3. http://www.plantillustrations.org/illustration.php?id_illustration=147323
  4. http://onlinelibrary.wiley.com/doi/10.1111/j.1467-8748.1991.tb00353.x/abstract;jsessionid=54B76FD485128C6BED70A4F8E4BB99E1.f01t04
  5. http://discardedlies.com/entry/?11568

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. annuusH. 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.