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How association genetics can find genes that help Joshua trees beat the heat

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Joshua trees in Tikaboo Valley, Nevada (jby)

This post is by JTGP collaborator Jeremy Yoder, an Assistant Professor in the Department of Biology at California State University, Northridge, who studies ecological and evolutionary genetics.

Since we first launched the Joshua Tree Genome Project, we’ve told you that one big reason we want to sequence a Joshua tree genome is to find genes that are important for adaptation to climate, which could help us makes sure that Joshua trees survive and thrive in a climate-changed future. But we haven’t discussed in detail how we’ll find those climate-adapted genes. The key to that part of the project is association genetics, a method for sifting through the genome to find the parts that contribute to traits we care about.

Figure 1. An example of a “candidate gene” experiment testing whether different diploid genotypes are associated with different trait values, or phenotypes. Points are the phenotypes of individual trees, grouped by their diploid genotypes at a candidate gene; overlaid box plots show that trees with different genotypes differ strongly in their phenotypes. (jby)

To understand how association genetics works, first consider a case in which we already know of a gene that might be important for a particular Joshua tree trait, like height or flower shape or physiological performance — or even just growing in places that are hotter or cooler. To figure out whether different variants in the sequence of our “candidate gene” are related to differences in the trait, we could measure the trait in many trees and then sequence that gene in all of those trees. We would test the hypothesis that the gene shapes the trait by comparing the trait values of trees carrying different variants of the gene sequence. Figure 1 gives an example of what this might look like in a hypothetical case, with tree phenotypes plotted against diploid genotypes at our candidate gene — “homozygous” trees carrying two copies of the G variant have higher phenotype values than homozygous trees carrying two copies of the A variant, and “heterozygous” trees with one copy of each have intermediate phenotypes. In this case, we’d probably conclude that the candidate gene has some effect on the phenotype we decided to measure, because different variants of the gene are associated with significantly different phenotype values.

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Plant physiology, the key to understanding how Joshua trees could adapt to a warming world

Karolina Heyduk is a postdoctoral researcher at the University of Georgia, and a co-PI on the Joshua Tree Genome Project. Karolina studies comparative genomics and the physiology of photosynthesis (Karolina Heyduck)

This is a post by JTGP collaborator Karolina Heyduck, a postdoctoral researcher at the University of Georgia.

Our Joshua tree proposal has been submitted to the National Science Foundation! Now we wait to hear if we’re granted to green light to study sources of adaptation in these remarkable desert species.

So what does “adaptation” mean anyway?

Adaptation refers to the process by which species over time acquire traits that allow them to succeed in a given habitat. A habitat includes both biotic elements – herbivores, pollinators, and pathogens – as well as abiotic phenomena like water availability, nutrients, light intensity, and temperature. Our research team is interested in both aspects. We suspect both pollinators and environment (temperature and water availability) are playing a role in Joshua tree speciation and adaptation, but we hope to test this with help from the NSF. Plant species that have become especially adapted to their environment didn’t arise in a single generation; instead, over many years, plants have passed down traits to their offspring that allowed these species to survive and thrive in tough conditions. However, when these abiotic environments change quickly – for example, due to climate change – plants may not be able to adapt fast enough, especially if they are long-lived with many years between generations.

For Joshua tree, which can live over 100 years, adapting to their changing climate will be critical to their survival. Joshua trees are already showing signs of trouble (check out this National Geographic article on them), and the Mojave is only expected to become warmer, forcing Joshua trees to either adapt or die. Currently Joshua trees are found across both high elevation (cooler) and lower elevation (warmer habitats). In both of those elevational levels, we also find Joshua trees in drier and wetter habitats. We might hypothesize that those Joshua trees already found in the hottest and driest habitats might survive best in the future, but we simply do not know. One big goal of our NSF grant will be to screen populations from different habitat types for traits that will help them succeed in the changing Mojave. But how do we do that?

We will measure chlorophyll fluorescence with a fluorometer to determine photosynthetic efficiency and overall plant stress. (Wikimedia Commons: Felipe Jo)

Our first step will be to collect seeds from different populations across both high/low elevations and with varying degrees of rainfall. We will grow these seedlings and plant them in gardens across the Mojave desert. Once they stabilize, we begin screening them for a long list of characteristics relating to water use efficiency and photosynthesis – two huge traits when it comes to desert survival. Water use efficiency refers to how much water a plant loses for every molecule of CO2 is gains. Plants take in CO2 through their open stomata, tiny pores on the surface of the leaf, but stomata also allow water vapor to escape from the leaves. This loss of water to the atmosphere is important for plants to pull water from the soil, forming a suction force like when you drink from a straw, but too much water loss in the desert can be deadly. Plants can minimize water loss by closing stomata, but this must be balanced by the need to take in atmospheric CO2 for sugar production. Photosynthesis is also important for plant survival, but can be impaired by extreme temperatures and a lack of water (Figure 1).

Measuring these traits will take us a while, and can only be done with the help of both undergraduate students and citizen scientists. Once we’re done measuring our traits of interest, we can begin to determine which Joshua tree populations are most flexible – and therefore might be the quickest to adapt – to changing environmental conditions. Understanding how Joshua tree physiology interacts with their habitat is critical for our understanding of how to help this magnificent species persist into the future

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By Invitation Only: The Next Step in Funding the Joshua Tree Genome Project

Hard at work on the full proposal. (CIS)

Back in January we wrote to you about preparing a proposal for the National Science Foundation. Now, we have some encouraging news to share.

Don’t break out the champagne yet though.

Last month we received the good news that our proposal had been ‘invited for a full proposal’.

As research funding has become more and more competitive, the National Science Foundation has turned to using ‘preliminary proposal’ system. Each January scientists from around the country put together short summaries of their latest and greatest research ideas. From the hundreds of preliminary proposals they receive, about eight dozen (approximately 25%) will be invited to submit a full proposal.

And (drum roll, please!) our proposal was one of the lucky ones invited to prepare a longer form description of our research proposal. So, while the rest of you are out enjoying the summer sun (or hiding from triple digit heat if you live in the Mojave), here at the Joshua tree genome project we’ve been hard at work trying to make the best possible case for our work. In a little less than a week we will send off our full proposal. And then we will wait …

We probably won’t hear a final funding decision until December at the earliest, and statistically, our chances are slim. But, at the moment, our thoughts are occupied with all the things that we will do if we were funded.

Here is a partial list of what we have in mind:

  • Completing, assembling, and annotating the full Joshua tree genome
  • Surveying genetic diversity across the entire range of the Joshua tree
  • Common garden experiments to identify genes involved in climate adaptation
  • An expanded citizen science program with Cal Native Plants
  • Public Lectures at the Desert Institute
  • Research internships for underrepresented minority students
  • Outreach to public school teachers in southern California

It’s a long and ambitious list. We hope that it’s enough to make our work stand out. Wish us luck!

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It’s grant proposal season

(CIS)

It was a rainy winter in the Mojave, and it was mighty quiet around the ol’ Joshua Tree Genome Project Page.

But we didn’t just huddle up around the fire.

January means grant proposal deadlines at the National Science Foundation, so while the yucca moths were underground this winter, we were hard at work trying to find funding for the next phase of this project. The generous support we’ve received from donations at Experiment.com and from The Living Desert Zoo, we’ve made huge progress towards assembling the Joshua Tree Genome. However, completing the next stage in the project – identifying the genes involved in adaptation to climate change – is going to be expensive. So, we’re looking to NSF to help us make it happen. Thanks in large part to the work we’ve been able to do so far, that proposal we wrote back in January was interesting enough to NSF for us to be invited to the next round of consideration — look for an update on that part of the process soon.

Research dollars are getting harder and harder to come by as federal spending for basic research has stagnated. So, the competition is fierce, and winning the funding game means we’ve gotta hit it out of the park. I hope what we’ve put together for tomorrow’s deadline is up to the task.

To learn more about the value of basic research check out this great story from the PBS News Hour. It’s an essay by Sheila Patek, a biologist at Duke University who studies, among other things, the biomechanics of mantis shrimp, which use their flimsy forelimbs to punch through tough snail shells. Patek’s work can seem frivolous, but it might also lead to biologically-inspired designs for stronger materials. She says

The nature of discovery is that it is impossible to anticipate what you will find. That is discovery. Discovery-based research is most fruitful when new knowledge is sought for its own sake.

If you agree, maybe phone a friend in Washington to tell her about it?

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The Living Desert funds cutting-edge DoveTail technology to assemble the Joshua Tree Genome

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The Joshua tree genome project is excited to announce a new partnership with The Living Desert Zoo and Gardens. Through a very generous gift from The Living Desert, we will use DoveTail Genomics Hi-Rise Technologies to assemble the Joshua Tree Genome.

The genome is the complete set of DNA letters that spell out the ‘instructions’ for how to build an organism. By sequencing the genome of the Joshua tree we hope to be able to understand its evolutionary history, how it’s relationship with yucca moth pollinators originated and evolved over time, and how Joshua trees might adapt to ongoing global climate change.

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Ewww-eww! That Smell! Why do Joshua Trees Smell Like that?

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If you’ve ever sat under a flowering Joshua tree on a spring afternoon, you’ve probably noticed a peculiar odor. “What … is … that?”

A little investigation reveals that the odor is coming from the flowers. The smell isn’t bad, exactly. Just odd. And strangely familiar. What does it remind you of? Wild mushrooms? Blue cheese? Windex? Overripe cantaloupe?

Figure 1: The source of the odor: A Joshua tree flower. (William Godsoe)

Figure 1: The source of the odor: A Joshua tree flower. (William Godsoe)

The early American botanist William Trelease described the scent of Joshua Tree Flowers as, “Oppressive” and “intolerable in a room”, but also commented a more positive note that previous descriptions of the odor as “fetid” was “not strictly accurate.”

A paper just published in the American Journal of Botany uses cutting-edge chemistry to unravel the mystery of why Joshua tree flowers smell the way they do. Glenn Svensson, a chemical ecologist at The University of Lund in Sweden, led an international team of scientists, including members Joshua Tree Genome Project, in collecting samples of Joshua tree scent. Using a hacked aquarium pump the team sucked up samples of air around Joshua tree flowers, and collected the odor molecules using some custom-made filters containing a special absorbent.

Figure 2: Sampling scent from a Joshua tree in the Spring Mountains, Nevada. The black pump is connected to hoses that draw air through carbon filters. One filter is placed inside a plastic oven bag containing a Joshua tree inflorescence. The second filter draws in air from the outside, providing an environmental control. (Chris Smith)

Figure 2: Sampling scent from a Joshua tree in the Spring Mountains, Nevada. The black pump is connected to hoses that draw air through carbon filters. One filter is placed inside a plastic oven bag containing a Joshua tree inflorescence. The second filter draws in air from the outside, providing an environmental control. (Chris Smith)

The filters were then taken back to the lab, and analyzed using process called Gas Chromatography Mass Spectroscopy (or GCMS). Gas Chromatography separates the different odor molecules in a long heated column, so that different compounds are retained the column for different lengths of time. Mass spectroscopy ionizes each molecule and produces a “fingerprint” or “mass spectrum” based on its mass and charge. The combination of retention time and mass spectrum data can be used to identify the different molecules contained in the odor mixture.

When Svennson and his team looked at the data from the Joshua trees, they found that up to 80% of the molecules found in Joshua tree’s scent was a complex 8-carbon compound called mushroom alcohol. The technical, less beautiful, name is (R)-1-Octen-3-ol, or pentyl vinyl carbinol.

Figure 3: The chemical structure of Mushroom Alcohol ((R)-1-Octen-3-ol), the primary compound found in Joshua tree scent. (Wikimedia Commons: Ju

Figure 3: The chemical structure of Mushroom Alcohol ((R)-1-Octen-3-ol), the primary compound found in Joshua tree scent. (Wikimedia Commons: Ju)

Mushroom alcohol occurs naturally in many plants and mushrooms, as well as in many foods, including artichokes, wheat bread, and soybeans. At least one other flower is known to emit odors containing mushroom alcohol: the orchid Dracula lefleurii, which mimics mushrooms to attract fly pollinators. Mushroom alcohol is also used commercially as an artificial flavor. The chemical manufacturer Sigma Aldrich describes the flavor as “cheesy, creamy, fishy, green, meaty, mushroomy, earthy, and herbaceous”

So why would a Joshua tree want to smell like a mushroom? The most likely explanation is that odor attracts the yucca moths that pollinate Joshua trees. Many flowers use odor as a way to attract pollinators, and it seems likely that the peculiar odor of the Joshua tree is somehow related to their peculiar pollination biology.

Svensson and his team compared the odor profiles of different species of Joshua trees that are pollinated by different species of moths, and found that they are indeed significantly different from one another. Joshua trees from the eastern Mojave produce less mushroom alcohol and more of another chemical, poetically called (E)-4,8-dimethyl-1,3,7-nonatriene, which is also found in cardamom.

The differences in scent does suggest that odor is important for attracting pollinators but, counter-intuitively, the two different species of yucca moth that pollinate Joshua trees seem to be unable to tell the difference between the different trees; where the two Joshua tree species grow together, the moths get confused and visit both trees equally. Why the flowers differ in their scent, even though the moths can’t seem to tell the difference, remains an evolutionary mystery for the moment.

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Getting to the essence of a Joshua tree: DNA extraction

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As we’ve talked about before on this site, the genome is the complete set of DNA letters that spell out the ‘instructions’ for how to build an organism. By sequencing the genome of the Joshua tree we hope to be able to understand its evolutionary history, how it’s relationship with yucca moth pollinators originated and evolved over time, and how Joshua trees might adapt to ongoing global climate change. This summer we started the process of decoding the genome.

Figure 1: Samples of Joshua tree leaves for DNA extraction. (Ramona Flatz)

Figure 1: Samples of Joshua tree leaves for DNA extraction. (Ramona Flatz)

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Thanks!

(Photo: Chris Smith)

(Photo: Chris Smith)

Our crowdfunding campaign at Experiment.com concluded last night at midnight, with $10,643 raised — 124% of our original funding goal. That means we’ll have funds for the DNA sequencing we’d wanted to assemble a Joshua tree genome sequence, and some additional funding towards our stretch goal, to develop a gene expression atlas based on that genome sequence. Thanks to every single one of the 325 backers who pledged support, and to everyone who helped spread the word on social media, and to the partner organizations who supported the campaign! We couldn’t have done this without you all.

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Let’s stretch!

Can we go higher yet? (Photo and stunt-man: Chris Smith)

Can we go even higher? (Photo and stunt-man: Chris Smith)

We’re unbelievably gratified by the support for our crowdfunding campaign — we’ve won the Experiment.com challenge to recruit the most backers for a project at a liberal arts college, and the bonus from that blew us past our funding goal. But we’ve still got a few days in the campaign, and assembling a genome is a big project. If we had a little more money, there’s more cool work we could do.

That’s where “stretch goals” come in — Experiment allows projects that meet their goals ahead of schedule to propose additional research, and set a new funding goal to support it. We’ve currently raised $10,523 — with about $3,000 more, we’d be able to go beyond assembling a Joshua tree genome sequence, taking the first steps to understand that sequence. We’d do that by building a gene expression atlas.

An assembled genome sequence is really just a long string of DNA nucleotides. What that code actually means — the proteins it codes for, their responses to different environments — is not simple to understand. We can make some headway in understanding a new Joshua tree genome sequence by using what we know about the general structure of protein-coding genes, and comparing genes found that way to other sequenced plant genomes about which more is known, like maize or Arabidopsis thaliana. But that will only get us so far. To really decode the Joshua tree genome, we need to understand what genes are expressed, or turned on, to form different parts of the plant, or to respond to different environmental conditions.

Every cell in a Joshua tree contains the tree’s complete genomic code, but not every gene in that code is expressed in every cell — genes that are important in a leaf cell are not necessarily the same ones that are important in a flower cell, or a root cell. We can take samples of different types of Joshua tree tissue like leaves, flowers, and roots, and specifically sequence the regions of the genome that are active within the cells in those different samples. Doing this will help us identify what parts of the genome actually are protein-coding genes, but it will also tell us something about those genes’ functions — a gene that is strongly expressed in a leaf, but not in flowers or root tissue, is probably important for the specific functions of leaves. Similarly, sequencing expressed genes in leaves from trees experiencing drought stress and trees that aren’t stressed can identify genes that are important for coping with that stress.

So that’s our stretch goal: funding to do the additional sequencing we’d need to target those expressed genes in an array of tissues and maybe more than one environment, too. In total, it’ll bring our project budget to $13,582 — but we’ve already raised enough that all we still need is $3,059. We’ve got five days left in the campaign. Can we do it? If you haven’t pledged your support yet, now’s the time! And if you have, keep spreading the word on Twitter and Facebook.

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We won!

(Photo by Jeremy Yoder)

(Photo by Jeremy Yoder)

We’re delighted to announced that we’ve just gotten word that we won the Experiment.com challenge for projects at liberal arts colleges — of all the projects in the competition, ours received the support of the most individual backers. The prize is $2,000 in bonus funding, which we can put towards more of the expenses of sequencing and analysis that go into assembling a reference genome sequence.

We literally could not have done this without the support of over 300 backers, and all the folks who’ve taken an interest in this project and spread the word on social media and by good old word-of-mouth. Many, many thanks. The collaborators are all excited to get underway.

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