How genomics and gene editing are about to turn ancient garbage into a hot commodity

For thousands of years, humans have been trying to understand the diversity of life on earth. Many of our systems of classification are based on bodies (phenotypes or morphologies). For example, as a paleoethnobotanist, I study tiny fragments of ancient plants that were left behind by people at archaeological sites – mostly in the garbage. I use the shape and size of ancient seeds to discover which species they came from, and then to describe variation in populations, especially of crop plants. The bodies of ancient organisms can tell us a lot about the origins and evolution of biodiversity. Whole fields are based on this premise: paleontologists study fossils; bioarchaeologists, paleoethnobotanists and zooarchaeologists study ancient remains of humans, plants, and animals recovered from archaeological sites. Up until recently, extinct forms were scientifically priceless, but held no economic value for agriculture or medicine. De-extinction was a topic for science fiction.

Though usually remembered for its depiction of dino de-extinction, Dr. Ellie Sattler’s reaction to this leaf (“Alan, this species has been extinct since the Cretaceous”) at the beginning of Jurassic Park’s most iconic scene implies that the thinking-machine-super-computer had also managed to ressurect lost plant species. Sadly, this never comes up again in any of the many Jurassic Park films. 

But we’re currently witnessing a revolution in the study of ancient biodiversity: genomics has cannonballed into archaeology and made an epic splash. With this methodological sea change, we have an opportunity to address fundamental questions that were unanswerable with older methods, but we also face new ethical questions. Much ink has already been spilled announcing the arrival of the genomics revolution in the study of ancient human DNA. There has been an explosion of research in the past decade, enabled by next-generation sequencing and breakthroughs in methods for separating out damaged fragments of ancient DNA from modern contaminants. Since we humans are naturally quite interested in ourselves, it is not surprising that these new techniques have been applied earlier and more widely to the study of human evolution than to that of plants and animals. Already, ancient genomic approaches have been used to better understand the sex lives of ancient humans, Neanderthals, and Denisovans (spoiler alert: they were all having sex with each other, and it was adaptive!), to test old hypotheses about the demographic effects of Neolithic Revolution, and to understand the evolution of specific human adaptations, such as the ability to digest lactase in adulthood and low hemoglobin levels in high altitude populations.

From Slatkin and Racimo, 2016, this figure shows the rapid increase in recovery of ancient human genomes between 2010 and 2016. In the past two years, this trend has continued and intensified.

These studies are revolutionizing how we understand human evolution and history, but they have also spawned very serious problems. Renowned human geneticist David Reich recently caused a snafu with an editorial in the New York Times conflating populations (in the biological sense of the word) with races and arguing that it is “no longer possible to ignore average genetic differences among “races.”” (Click here for more on this controversy). Meanwhile, white supremacists are chugging milk to demonstrate their European ancestry, in an homage to one of the studies cited above. Researchers have also raised concerns about the (sometimes) wanton destruction of ancient human remains for aDNA extraction, and how irreplaceable and rare specimens are being hoarded by a few elite institutions. These concerns are about to become major problems in the study of ancient plant DNA, too, because…

Screenshot of white supremicists chugging milk at a protest in New York City, as reported by Amy Harmon.

A revolution is also underway in biotechnology. Maybe you’ve heard about CRISPR/Cas9 and thought “That’s just too much acronym for me to process today,” but please bear with me. This discovery is already transforming our world – and especially our food. Previous methods of genetic modification involved infiltrating or blasting cell nuclei with packages of modified genes and hoping they incorporated properly (1,2). Because the new genes incorporated more or less randomly into the target organism’s genome, biotechnologists had to rely on a brute force evaluation of thousands of modified plants or animals to identify a few in which the new genes had been properly incorporated, yielding the desired new trait. Making multiple targeted changes using these methods was extremely labor and time intensive. CRISPR/Cas9, on the other hand, is a gene editing tool that allows modified or novel genes to be inserted into the target genome at precisely the right place.

Schematic of CRISPR/Cas9 system, courtesy of Wikimedia Commons.

This method makes use of two defense mechanisms that bacteria use to fight off viruses. CRISPR (clustered regularly interspaced short palindromic repeats, in case you were wondering) is a piece of RNA created by bacteria to help them recognize viruses that they have previously encountered.  After recognizing a virus, bacteria deploy Cas9, an enzyme that destroys the virus by cutting apart its genome (you’ll sometimes hear Cas9 referred to as “molecular scissors.” (Read more here!) Biotechnologists can now use modified CRISPR RNA to target specific places in the genome of the organism they are modifying, and Cas9 to insert the new gene at that place. This method is a game changer because it makes genetic modification much more precise and predictable. In the US, we have already seen our agricultural system transformed by genetically modified crops created using much clunkier methods. Not only is CRISPR/Cas9 gene editing an extremely efficient method, the USDA has officially declined to regulate CRISPR/Cas9-edited crops by reframing this technology as a “plant breeding innovation.” There are already five CRISPR-edited products being developed for the market, including soybeans, camelina, lawn grass, maize, and mushrooms.

From Walz 2018, reused with permission from Springer.

What do these two scientific breakthroughs – ancient genomics and CRISPR/Cas9 gene editing – have to do with ancient garbage? That’s the million-dollar question.

This year, two reviews appeared, one in Nature: Plants, and the other in Frontiers in Plant Science, both arguing that ancient plant remains have just become a source of valuable information for the biotech and crop breeding industries. Their argument is based on two points:

  1. We are suddenly able to recover millennia worth of lost agrobiodiversity from around the world.
  2. We have simultaneously developed the tools to directly incorporate useful variation from ancient plants into modern crops.

The conventional view of the evolution of agrobiodiveristy. After Allaby et al. 2018, Fig 1.

To start with the first point, why would ag industries care about the variation contained in ancient crop genomes?  Well, first take a gander at my schematic of the “conventional view” of the relationship between genetic diversity and domestication. In this view, there is a steep drop in diversity, called the domestication bottleneck, when a small subset of a large wild population is brought under cultivation. For most crops, this happened thousands of years ago. From there, development of different varieties led to increasing biodiversity within the crop population. We call the results of this diversification landraces, which are regionally specific crop varieties, often adapted to local tastes or environmental conditions.  When crops were taken into new regions this could have led to more bottlenecks, especially if dispersal was followed by isolation. Then diversification would have begun again in multiple regions, and sometimes these distinct populations came back together through trade and migration, leading to a huge amount of agrobiodiversity worldwide.


Wheat landraces collected in Turkey. Image by Alexei Morgounov/CIMMYT.

Finally, we have a known bottleneck in agrobiodiversity that started with plantation economies and colonialism and culminated in the industrialization of agriculture during the 20th century. During this process, seed selection and crop breeding moved off of farms and most landraces fell out of cultivation, although many are still preserved in seed banks and herbaria. Today, the overwhelming majority of seed planted globally comes from crop breeders or biotech companies. They only produce a relatively limited number of “elite” varieties that have usually been bred for yield. Pant breeders and biotechnologists have always known that landraces and the wild progenitors of crops are diversity high points, and sources of useful genetic variation for crop improvement. They can use the relative diversity of these populations to breed or engineer useful traits into commercial varieties – things like drought and virus resistance, particular tastes, or different nutritional properties.

But recent insights from ancient crop genomes have called the assumptions of the conventional model of crop evolution into question. Ancient genomic data for three crops (maize, barley, and sorghum) seems to indicate that there was no domestication bottleneck. Instead of a sharp drop in diversity immediately after domestication, these crops experienced a gradual loss in genetic diversity over the course of thousands of years. One important implication of this research is that the genomes of ancient crops may hold useful traits that were lost long before colonizing botanists started to fill seed banks with landraces. In other words, the amazing variety of crops that were grown around the world when the Age of Exploration began may not actually be a high point in diversity. The high point may instead have come thousands of years ago, and there is likely a unique evolutionary history for every crop.

This would all be academic if it weren’t for simultaneous breakthroughs in gene editing. You can’t (usually) get ancient seeds to germinate, so however much valuable diversity these lost varieties hold, they would have been useless to modern plant breeders before genetic modification. But now, using gene editing, it is possible to reverse engineer living crops to replicate desirable aspects of ancient varieties.

My favorite lost crop: ~2,500 year old dessicated erect knotweed fruit from Cold Oak rockshelter, KY. Image by Natalie G. Mueller

How you feel right now is probably largely determined by your opinions about genetically modified crops in general. Personally, I find the idea of de-extincting lost crops using gene editing, frankly AWESOME. But I know enough about the history of GM crops to guess that research along these lines will not be driven by my desire to have a historically accurate garden.  Instead, ancient genomes will be mined for profitable traits, and what was once the cultural heritage of particular communities will become extremely valuable private property through the alchemy of formal breeding and genetic modification. The archaeobotanical collections that could make this kind of research possible are extremely rare, fragile, and difficult to recover. They are a limited and non-renewable resource. Curators, archaeologists, and descendent communities should get started now developing ethical and legal frameworks to guide the use of archaeobotanical specimens, before the application of ancient genomic studies expands to plant breeding and biotechnology.

Sources

Allaby, R. G., Ware, R. L., & Kistler, L. A re-evaluation of the domestication bottleneck from archaeogenomic evidence. Evolutionary Applications, 0(0). doi:doi:10.1111/eva.12680

Di Donato, A., Filippone, E., Ercolano, M. R., & Frusciante, L. (2018). Genome Sequencing of Ancient Plant Remains: Findings, Uses and Potential Applications for the Study and Improvement of Modern Crops. Frontiers in plant science, 9(441). doi:10.3389/fpls.2018.00441

Estrada, O., Breen, J., Richards, S. M., & Cooper, A. (2018). Ancient plant DNA in the genomic era. Nature plants, 4(7), 394-396. doi:10.1038/s41477-018-0187-9

Ficiciyan, A., Loos, J., Sievers-Glotzbach, S., & Tscharntke, T. (2018). More than Yield: Ecosystem Services of Traditional versus Modern Crop Varieties Revisited (Vol. 10).

Haak, W., Lazaridis, I., Patterson, N., Rohland, N., Mallick, S., Llamas, B., . . . Reich, D. (2015). Massive migration from the steppe was a source for Indo-European languages in Europe. Nature, 522, 207. doi:10.1038/nature14317

https://www.nature.com/articles/nature14317#supplementary-information

Huerta-Sánchez, E., Jin, X., Asan, Bianba, Z., Peter, B. M., Vinckenbosch, N., . . . Nielsen, R. (2014). Altitude adaptation in Tibetans caused by introgression of Denisovan-like DNA. Nature, 512, 194. doi:10.1038/nature13408

https://www.nature.com/articles/nature13408#supplementary-information

Itan, Y., Powell, A., Beaumont, M. A., Burger, J., & Thomas, M. G. (2009). The origins of lactase persistence in Europe. PLoS computational biology, 5(8), e1000491.

Jaganathan, D., Ramasamy, K., Sellamuthu, G., Jayabalan, S., & Venkataraman, G. (2018). CRISPR for Crop Improvement: An Update Review. Frontiers in plant science, 9, 985-985. doi:10.3389/fpls.2018.00985

Kuhlwilm, M., Gronau, I., Hubisz, M. J., de Filippo, C., Prado-Martinez, J., Kircher, M., . . . Castellano, S. (2016). Ancient gene flow from early modern humans into Eastern Neanderthals. Nature, 530, 429. doi:10.1038/nature16544

https://www.nature.com/articles/nature16544#supplementary-information

Llamas, B., Willerslev, E., & Orlando, L. (2017). Human evolution: a tale from ancient genomes. Philosophical Transactions of the Royal Society B: Biological Sciences, 372(1713). doi:10.1098/rstb.2015.0484

Makarewicz, C., Marom, N., & Bar-Oz, G. (2017). Ensure equal access to ancient DNA. Nature, 548, 158. doi:10.1038/548158a

Prendergast, M. E., & Sawchuk, E. (2018). Boots on the ground in Africa’s ancient DNA ‘revolution’: archaeological perspectives on ethics and best practices. Antiquity, 92(363), 803-815. doi:10.15184/aqy.2018.70

Racimo, F., Sankararaman, S., Nielsen, R., & Huerta-Sánchez, E. (2015). Evidence for archaic adaptive introgression in humans. Nature Reviews Genetics, 16, 359. doi:10.1038/nrg3936

https://www.nature.com/articles/nrg3936#supplementary-information

Seguin-Orlando, A., Korneliussen, T. S., Sikora, M., Malaspinas, A.-S., Manica, A., Moltke, I., . . . Willerslev, E. (2014). Genomic structure in Europeans dating back at least 36,200 years. Science, 346(6213), 1113-1118. doi:10.1126/science.aaa0114

Slatkin, M., & Racimo, F. (2016). Ancient DNA and human history. Proceedings of the National Academy of Sciences, 113(23), 6380-6387. doi:10.1073/pnas.1524306113

Waltz, E. (2018). With a free pass, CRISPR-edited plants reach market in record time. Nature Biotechnology, 36, 6. doi:10.1038/nbt0118-6b


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Harvest time!

The summer has flown by! Yesterday we harvested our first batch of lost crops. We’ve been getting A LOT of rain this week, so my research assistant, Andrea White, and I took advantage of the beautiful sunny day yesterday to harvest three replications of our goosefoot experiment.

Each replication is divided into four quadrants so that we can study the effects of different plant densities on yield and biomass. One quadrant of each replication has all three fall maturing lost crops growing in it (that is, sumpweed, erect knotweed, and goosefoot — click here to get caught up on the lost crops). We think it is unlikely that these plants were grown as monocrops by ancient people, since descendant communities are renowned for their innovative polycultures. We’ll soon have data about how these species do as a community vs. on their own, which Andrea will be analyzing for her independent study this semester.

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Sumpweed and goosefoot growing together and getting along just fine.

Andrea and I harvested the senesced goosefoot plants by uprooting them, then hand stripping their seeds. We’re using big funnels, paper bags, and a tarp, but if ancient people used this technique they probably used tightly woven baskets, bags, and mats like the ones that have been found in rockshelters in Arkansas. I decided to harvest the plants this way to minimize seed loss, and so that I could also measure the weight of the stripped plants as a compliment to our plant density data. We can now say how density effects plants size, and how both of these measurements interact with yield. Even though these are small plots, I am hoping this will help us reconstruct what ancient fields might have looked like.

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An ancient bag full of domesticated goosefoot seeds from Eden’s Bluff, Arkansas. Image from Fritz and Smith 1988.

We also timed ourselves to see how labor intensive the harvest would have been. This is one way that archaeologists have characterized the efficiency if growing lost crops in the past. Although the harvest is certainly an important job, Andrea and I agreed that it was much easier and more pleasant than the hours (cumulatively, days) of weeding we did in the spring and summer. Harvesting was actually relaxing.  We enjoyed the perfect weather, bopped to some tunes, and felt a keen satisfaction from the pitter-patter of seeds raining into our bags. Looking forward to another break in the rain to get back out there!

 

Plasticity and domestication

Yesterday I had a chance to share my research at the Cornell School of Plant Science Horticulture Seminar. This is the first time that I’ve spoken about how I became interested in plant plasticity —  the way and degree to which plants react to their environment. In the context of domestication, you can think of plasticity as the way plants behave when they meet humans. Some animals are easier to tame and bring into the family than others, and I think some plants were predisposed to join human society, too. My current project at Cornell is investigating how developmental plasticity may have played a role in the process of domestication.

Several years ago, when I was working on describing the lost crop erect knotweed, I found its changeability frustrating. Documenting what it looked like was like grasping at sand — there was no typical form. Gradually, I came to see this plant’s responsiveness to its environment as the subject of my research, rather than a barrier.

If you’ve never heard of lost crops, this talk is also a good way to introduce yourself!

Growing lost crops: It has begun!

The only reason I started writing this blog post 2 days ago, instead of  continuing to furiously tend my lost crops, is because it was pouring rain. When it started raining, I was where I have been nearly every daylight hour for the past three weeks: in my field (or “patch,” as my father calls it). I was weeding an experimental plot of maygrass. Undeterred, I kept weeding for another 2 hours, until the mud was sticking to my fingers and I could no longer pull weeds with the precision necessary to avoid pulling lost crops, which are also weeds. I said a silent but sincere Thank you to the Earth for finally watering my babies, then packed it in for the day.

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My Dad, Tim Mueller, weeding one of the goosefoot plots after some much needed rain. It’s surprisingly difficult to weed when you’re cultivating…weeds.

Average precipitation for June in Ithaca, New York, is 3.85 inches –we have received only 2 inches this month. It shouldn’t be necessary to irrigate weeds, especially in a place as wet as central New York, but I needed to keep the seed beds damp to encourage as much germination as possible. When I got home from a conference on June 14th and found the parched, clayey soil actually cracking, I made an emergency trip to Tractor Supply (one of many) for hoses and a sprinkler. Not the most high-tech irrigation solution, but pretty much all the tap near my field can handle– and works in a pinch. The best time to be out in the field moving a sprinkler around is just before sunset on these long summer days. I can bring my dog and book, or puzzle over small oddities in my plants in peace.

The saga of getting these plants to germinate began back in December, when I started experiments to see what kind treatment the seeds of each species needed make them sprout. Domesticated plants have been selected and bred by people for thousands of years to germinate as soon as they are planted. The thing about wild seeds is that they have never been able to count on humans to collect and store them in safe places, then plant them somewhere that they will like the next year. Instead, they have evolved diverse means of protecting themselves from freezing, flooding, drought, getting chewed on and digested by animals– and then, when conditions are just right, “knowing” that it is time to germinate.

Over the course of the winter, my research assistants and I developed some pretty good guidelines for treating the seeds of each species. Although we weren’t getting the 80% germination that you can expect from most store-bought seeds, we were getting at least 25% and sometimes as high as 70% with our most successful treatments, and we applied those treatments to the seed stock for the main experiments I had planned for this summer. Between May 2nd and May 18th, we planted everything in experimental plots and started the vigil for sprouts.

Even after months of preparation, I was far from confident that all five species would grow. Those germination experiments were conducted in the greenhouse. The greenhouse might as well be a different planet when it comes to experimental results. On Planet Greenhouse, every day is 12 hours long. It’s always 80 degrees during the day and 65 degrees at night. Planet Greenhouse is made of homogenous, rich, loose, potting soil, which is always perfectly moist, but not too wet. There are almost no bugs, and no larger animals to chomp on tender seedlings. It’s a wonderful planet for baby plants, but not at all like Planet Earth.

After 8 weeks of weeding and watering and watching, we have now counted over 5,000 seedlings representing all five lost crops in three different experiments. Some are behaving a little strangely, but that’s to be expected given that I’ve taken them out of their habitats and life cycles.

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Some of the freshly weeded and watered plots, full of thousands of seedlings at last — I had a lot of help and moral support from my Dad (background) this week. 

For example, consider the two lost crops that are spring maturing grasses, maygrass and little barley. In the wild, they germinate either in late fall or in very early spring, and by June they have already produced all their seed for the year and died. I couldn’t plant them in early spring here at Cornell, because there were several feet of snow on my field in March. That’s normal western New York stuff, but by mid-April things were getting a little upsetting. I had to push back the preparation of my field for weeks, until after the last snow, which occurred as I was laying out experimental plots in mittens and boots on April 30.

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Laying out the field on May 1. There was snow the day before.

That’s one of the many downsides of leaving Planet Greenhouse for Planet Earth: weather. I decided to make lemonade from crappy spring lemons and look into something that my colleagues and I have been wondering about: Would it have been possible for ancient people to get two crops per year out of these species? If you plant seeds after the first harvest in May, could you get another crop in late summer?

I planted maygrass and little barley in May, and they are just starting to flower now. The jury is still out, but its looking good for two-crop lost crops! The maygrass is doing something odd. Instead of growing to ~40 cm before flowering, some of my seedlings are producing flowers when they are only 4 cm tall. If they can muster the energy to produce seeds with so few leaves, this is not necessarily a bad trait from a farmer’s perspective: more seeds in less space and time. It is also interesting to see how the maygrass and little barley interact. We’ve gotten the best germination and the earliest flowers in the plots where the two species are grown together.

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Maygrass and little barley germinating together at the beginning of June.

Other experiments have not been as successful. Out of thousands of seeds sown, I’ve counted only 12 sumpweed seedlings. I spent four entire work days weeding empty sumpweed plots, hoping they would germinate if I kept them clear and moist, but so far no luck. The dry weather this June is probably worse for sumpweed than for the other species, since this plant grows in marshy locations (its other common name is marsh elder). We are still hoping for some late comers, but even if they never sprout we’ve learned something valuable about sumpweed – how not to grow it.

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My research assistants, Peter and Andrea, transplanting knotweed last week. This was the last experiment we set up, and the only one that relied on plants started in the greenhouse.

 

As of yesterday, everything is finally laid out, built, planted, and weeded (for now…), and this week we can concentrate on collecting data for the first time. It’s been the busiest two months of my entire life, but it’s wonderful to see all of these plants growing together and I am looking forward to learning from them everyday for months to come.

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This final experiment we set up is comparing knotweed responses to sun and shade — but more on that next time!

Lost crops and living knowledge profiled by CBC’s Quirks and Quarks

I spoke with CBC’s Quirks and Quarks on Sunday about my research on eastern North America’s lost crops. Listen here!

I was pleased that Quirks and Quarks chose to pair my interview with the fascinating and vital work of Kim Recalma-Clutesi (Ogwiloqwa) to preserve and apply traditional ecological knowledge in her community. “Lost things found!” are always fascinating (they’re the bread and butter of archaeology), but they are only a tiny part of the story of Indigenous North American ethnobotany.  I can’t emphasize enough how important Native American crops still are to food security and cuisines all over the world. Even more importantly, there are Indigenous communities all across North America who are advocating for the right to tend and care for the plants and animals in their homelands. Community knowledge based on thousands of years of experience is more important now than ever, as we face population growth and climate change. I wish every story about lost crops could appear beside a story about ecological knowledge that is preserved, passed on, and applied.