When female Anopheles mosquitoes take a blood meal from someone with malaria, a tiny Plasmodium parasite enters the mosquito’s digestive tract. That parasite can invade the mosquito’s salivary tissues, so when the insect takes another blood meal, the intruder can slip into the next human host and start a new malaria infection. Malaria is a life-threatening condition that infected 241 million people in 2020 and disproportionately affects vulnerable populations.

To combat the disease, researchers from the University of California, Irvine are developing genetically modified African malaria mosquitoes (Anopheles gambiae) that can’t transmit human malaria, alongside a gene-drive system that can quickly spread those genes and block the spread of the malaria parasite through the population. While this system usually operates with nearly 100 percent efficiency, a small number of mosquitoes will still wind up with mutant alleles that resist the gene drive. Could these mutant alleles sabotage the whole approach? In a paper published in GENETICS, Carballar-Lejarazú et al. looked at this phenomenon and found these mutations didn’t hamper the gene drive in their system.

Malaria-Resistant Mosquitoes

Contributing author Anthony James began exploring genetic methods for controlling vector-borne disease in the mid-1980s. Eventually he exploited mosquito genes that are only turned on in female mosquitoes after a blood meal and linked them with mouse antibodies that protect mice from human malaria parasites.

When these malaria-busting synthetic genes are inserted into mosquitoes, they can’t transmit malaria. And if it mates with a regular mosquito, the beneficial gene will be inherited like any other gene, gradually building up in the mosquito population. But what if that process could happen faster?

That’s when CRISPR gene editing technology hit the scene. “It seems like overnight when you work 10 or 15 years on something to make it work and then something new comes along and—in less than a year—you have it working,” says James.

Gene editing operates on the germline—the cells that will eventually become sperm or eggs—by snipping the normal chromosome and inserting the new sequence, in this case, the malaria fighting gene. In male mosquitoes, this works so well that each male passes on the new gene to nearly 100% of its offspring.

It’s a bit more complicated in female mosquitoes because egg cells are massive compared with sperm. When the system snips the normal chromosome and inserts the synthetic sequence, the second chromosome may be too far away to trigger the repair mechanism that sews the cut chromosome back up while including the system. That means there’s a chance the snipped chromosome will just stick itself back together—called nonhomologous end joining—possibly resulting in a mutant allele that resists the gene drive.

Exploring Gene Drive Mutations

To figure out if those mutant alleles could pose a problem for the gene drive system, the researchers linked the system to a somatic gene for eye color and marked it with a fluorescent protein. Then, the team performed various crosses to see how the genes passed on to future generations. Non-mutant progeny had black eyes (before adulthood) while those with the mutant allele had pink eyes. And all the progeny carrying the gene drive had eyes that fluoresced blue under light.

To make things a bit more complicated, some progeny were mosaics, with a mix of alleles and more complex eyes, but since the eye color gene isn’t part of the germline—it won’t pass on to the next generation—most of those mosaic mosquitoes still passed on the gene drive.

In the lab, about 25 percent of the first generation of progeny received the gene drive. By the fourth generation, the entire population had fluorescent blue eyes—meaning none of those mosquitoes could transmit malaria.

“Four generations is sufficient,” says James.  “That’s well short of one transmission season.”

James is quick to point out that this isn’t a magic bullet for malaria and there is more research to be done. There are also many thorny issues and debates for scientists and the broader community to work through before everyone is comfortable deploying gene drive mosquitoes in the wild. But James is hopeful the project to which he’s dedicated so many years may one day help ease the malaria crisis.

“There was a famous scientist who said a new idea doesn’t take hold because you change people’s minds; it takes hold because there’s a whole new generation of people that have grown up hearing about it,” says James. “I wish it was a little faster, but we’ll do our part, and hopefully people will take it up. It may not be me, but we have something to hand off.”

CITATION:

Cas9-mediated maternal effect and derived resistance alleles in a gene-drive strain of the African malaria vector mosquito, Anopheles gambiae

Rebeca Carballar-Lejarazú, Taylor Tushar, Thai Binh Pham, Anthony A James

GENETICS

2022: iyac055

https://doi.org/10.1093/genetics/iyac055

Melissa Mayer is a freelance science writer based in Portland, Oregon.

Efforts to diversify an inbred population must take into account the genetic backgrounds of the founders.

The nocturnal flightless parrot known as the kākāpō was once abundant throughout New Zealand. But after the introduction of mammalian predators, the species all but disappeared. Today, every living kākāpō is descended from a tiny handful of island survivors and a single male from the mainland. The entire population of 201 birds is closely watched over by conservationists on a few predator-free island refuges, where they hope the hefty green parrots will continue to breed.

But because their numbers dropped into the double digits, the kākāpō face a genetic bottleneck. Inbreeding can increase rates of genetic disease and contribute to poor health among the population. Geneticists studying the remaining kākāpō have quantified the amount of inbreeding among the birds as one way to understand the health of the species. Their results, reported in a new paper in G3: Genes|Genomes|Genetics, suggest that inbreeding isn’t necessarily hurting chicks’ chances of survival and that introducing additional genetic diversity may not always have the intended effect.

Saved from extinction

“Kākāpō have a really interesting natural history,” says Yasmin Foster, a graduate student at the University of Otago and the study’s lead author. “They were functionally extinct, but then a small population was found on an island in the south of New Zealand.”

“Functionally extinct” in this case meant no more females could be found on the New Zealand mainland; only a few males remained. In 1977, about 50 kākāpō were discovered living on Stewart Island, a large island about 19 miles south of the mainland. Predators such as feral cats roamed Stewart Island, however, so in 1982 conservationists began relocating the birds to several smaller, mammal-free outlying islands.

By then, only one male kākāpō remained on the mainland, and he was taken to a predator-free island refuge along with the Stewart Island population. The Stewart Island kākāpō had diverged from the mainland population around 10,000 years ago, giving the two groups time to develop distinct genetic profiles. Introducing the mainland bird was meant to help boost genetic diversity among a new generation of chicks.

Now, the population has grown to 201 individuals. To help inform conservation strategies, Foster and her colleagues set out to document the amount of inbreeding in the colony. Creating a large, multigenerational pedigree of the wild kākāpō wouldn’t work for a founder population of this type, so the researchers turned to DNA sequence analysis. Thanks to the availability of a high-quality kākāpō reference genome, the team could genotype the birds using genome-wide mapping of single nucleotide polymorphisms (SNPs).

Comparing measures of inbreeding

“We had this unique founding population with 50 Stewart Island birds and one mainland male,” says Foster. “From the inbreeding metrics I looked at, we found that they’re both inbred, but in different ways.” She says the study raises an interesting point about how combining two inbred populations in an attempt to increase genetic diversity can actually introduce more deleterious alleles.

Comparing multiple inbreeding metrics helped Foster get a robust view of the birds’ genome, and also to evaluate the accuracy of each one. “Some people just use one or another, but what I found was that some of them give a different story,” she says.

She started by calculating the coefficient of inbreeding, or F_H, which is the probability of an individual inheriting two copies of the same allele from the same ancestor on both sides. She compared this with a newer method of measurement, called “runs of homozygosity” or F_ROH, which looks for long sections of the genome where both copies are the same.

A third method, using pairwise analysis to construct a genomic-relatedness matrix, turned out to be the weakest of the metrics, Foster says. “Other people have found that when you have a small group of individuals that are a lot different to the larger group, they skew the outcome maybe a bit too much,” she says. “Their genetic information is more rare, and the way the maths compute this inbreeding metric, it gives more power to those rare alleles.”

‘Hybrid vigor’ – or not

In addition, they compared levels of inbreeding with survival in chicks. When the mainland male was added to the Stewart Island population, the expectation was that he would infuse genetic diversity into a new generation of chicks, boosting their health. But that turned out not to be the case: decreased inbreeding did not correspond to improved survival, partly because the less-inbred chicks had unexpectedly high levels of mortality.

“That was probably down to the mainland individuals also being reduced to a small population for a long period of time,” says Foster. The mainland male apparently brought in quite a few detrimental mutations that had been lost from the island population over the years.

The inbreeding study and others like it could help guide conservation strategies moving forward. Kākāpō have a lek mating system, which means that the males all congregate and compete to entice the females. The most popular male will produce the most offspring, and this can tighten the genetic bottleneck even more.

“One male kākāpō had fathered 22 chicks, which is really significant when there’s only 200 left,” says Foster. “Obviously his genetic material spread across the population. He was so successful, we had to translocate him to another island to give some of the other males a chance.”

CITATION

Genomic signatures of inbreeding in a critically endangered parrot, the kākāpō

Yasmin Foster, Ludovic Dutoit, Stefanie Grosser, Nicolas Dussex, Brodie J. Foster, Ken G. Dodds, Rudiger Brauning, Tracey Van Stijn, Fiona Robertson, John C. McEwan, Jeanne M. E. Jacobs, and Bruce C. Robertson

G3 Genes|Genomes|Genetics 2021; jkab307

https://doi.org/10.1093/g3journal/jkab307

Watch presentations from the conference, including talks from Katie Peichel and Jonathan Pritchard.

Now that the dust has settled from the whirlwind of the first ever standalone GSA Population, Evolutionary, and Quantitative Genetics Conference (PEQG18), we’re delighted to be able to share the audio and synched slides from the Keynote and Crow Award sessions.

We’re gratified too that attendees got so much of value from the conference. Many have approached GSA staff and the conference organizers with rave reviews of their experience, and, despite the usual growing pains of a new conference, the results from the attendee survey have also been overwhelmingly positive.

We’re excited to incorporate some of the lessons we’ve learned into planning the next PEQG. It will be held April 22–26, 2020 in the metro Washington, DC, area at The Allied Genetics Conference (TAGC20). PEQG will join the C. elegans, Drosophila, mouse, Xenopus, yeast, and zebrafish research communities for a mix of community-specific and cross-community sessions.

Stay tuned for more announcements on the upcoming conference and for several more PEQG18 blog reports in the coming weeks. Enjoy the talks below!

PEQG18 Keynotes

Jonathan Pritchard Stanford University/HHMI

Omnigenic Architecture of Human Complex Traits

Catherine Peichel University of Bern

Genetics of Adaptation in Sticklebacks

Trudy Mackay North Carolina State University

Context-Dependent Effects of Alleles Affecting Genetic Variation of Quantitative Traits COMING SOON

Finalists for the 2018 Crow Award for Early Career Researchers

Amy Goldberg UC Berkeley

A mechanistic model of assortative mating in a hybrid population

Emily Josephs UC Davis

Detecting polygenic adaptation in maize

Jeremy Berg Columbia University 

Population genetic models for highly polygenic disease

Katherine Xue University of Washington 

Evolutionary dynamics of influenza across spatiotemporal scales

Alison Feder Stanford University 

Intra-patient evolutionary dynamics of HIV drug resistance evolution in time and space

Emily Moore North Carolina State University 

Genetic variation at a conserved non-coding element contributes to microhabitat-associated behavioral differentiation in Malawi African cichlid fishes

Videos

Jonathan Pritchard 

[youtube https://youtu.be/H18k55ruCOY&w=500&rel=0]

Catherine Peichel

[youtube https://youtu.be/QRCcLixjUtc&w=500&rel=0]

Amy Goldberg 

[youtube https://youtu.be/kccUNkF7SgY&w=500&rel=0]

Emily Josephs 

[youtube https://youtu.be/CxQOrK9h6D4&w=500&rel=0]

Jeremy Berg

[youtube https://youtu.be/HqA1H24LPZc&w=500&rel=0]

Katherine Xue

[youtube https://youtu.be/fTdaAwqdt0k&w=500&rel=0]

Alison Feder

[youtube https://youtu.be/ntM0448h2lA&w=500&rel=0]

Emily Moore

[youtube https://youtu.be/aX4_HS0K1kA&w=500&rel=0]

They rattle as warning, but during the hunt their strike is silent and sudden. Any rabbit or mouse targeted by a rattlesnake is doomed—the snake’s bite carries a paralyzing venom. The toxins in this venom are proteins encoded by a large gene family that arose by gene duplication. In the July issue of GENETICS, Margres et al. tackle two models of how gene family expansion gets started by analyzing individual variation in a rattlesnake toxin gene family. They find that the most important factor in gene family growth via duplication was selection pressure to increase protein expression level.

Gene families are groups of similar genes born through duplication and are extremely common in eukaryote genomes. The different genes in a family often have sequence changes that allow them to carry out distinct functions, but additional gene copies also mean a higher overall expression level. Theoretically, both of these consequences of gene family expansion can be adaptive, but it’s unclear which is more important during the initial stages of gene family evolution. To clarify this question, Margres et al. use the rattlesnake gene family that encodes myotoxin, a component of venom.

Venom is a useful model because the connection between protein expression and phenotype is clear: the more protein is expressed, the more potent the effect. And importantly, there is still variation in the size of the myotoxin gene family among rattlesnakes. For this study, the authors collected venom and blood samples from over a hundred live-caught, wild rattlesnakes from across the Southeastern US. The number of myotoxin gene copies in these snakes ranged from zero to nearly 50, with a corresponding increase in myotoxin protein expression in venom. However, only four individuals carried gene copies with a nucleotide polymorphism within the key exons, indicating that the duplicate copies are essentially identical.

Clearly, these carbon-copy myotoxin genes aren’t carrying out different functions. The direct relationship between venom protein level and the number of myotoxin gene copies shows that the expansion of this gene family is likely driven by selection for increased expression and thus stronger venom. Animals as diverse as platypuses and cone snails use venom for hunting and defense. This study not only shows how this fascinating and frightening adaptation evolves, but it also provides a glimpse of the forces that can trigger a gene to start down the road of expansion—and ultimately—diversification.

CITATION

Selection To Increase Expression, Not Sequence Diversity, Precedes Gene Family Origin and Expansion in Rattlesnake Venom

Mark J. Margres, Alyssa T. Bigelow, Emily Moriarty Lemmon, Alan R. Lemmon and Darin R. Rokyta

GENETICS July 1, 2017 vol. 206 no. 3 1569-1580; https://doi.org/10.1534/genetics.117.202655

http://www.genetics.org/content/206/3/1569

February marks the launch of a crisp new look and improved navigation at the G3 website. Go check it out; we’re very proud of the design! We are also unveiling a new cover layout that allows the art submitted by our authors to shine. This month’s cover celebrates the first published genome assembly of the Canadian beaver.  The study is by an all-Canadian team from The Centre for Applied Genomics at the Hospital for Sick Children (SickKids), The University of Toronto, The Ontario Institute for Cancer Research, The Royal Ontario Museum, and The Toronto Zoo — just in time for Canada’s 150th birthday!

The cover shows the “3 pence Beaver,” Canada’s first postage stamp. Issued in 1851, the stamp did not feature the Queen (customary at the time) but instead honored the beaver, an iconic national symbol and the first animal ever to appear on a stamp. This unusual choice was made in recognition of the beaver trade as the economic engine that drove colonial expansion, leading to the founding of Canada. It also depicts a beaver dam, symbolizing the young country building its new towns, cities, and communities. The stamp was designed by Sir Sandford Fleming, a proponent of the worldwide standard time zone, Canada’s foremost railway engineer of the 19th century, and a distinguished scientist. The photo used for this cover was suggested by the study authors, and provided at the courtesy of an avid collector of early Canadian postal history.

The project to sequence the Canadian beaver was conceived not only as an anniversary present for Canada, but as a test of a new method that could help diagnose genetic diseases. The goal was to assemble complex mammalian genomes directly from uncorrected and moderate-coverage long reads generated by single-molecule sequencing, an approach that could help to identify mutations in clinical samples that would otherwise escape detection. The achievement was extensively covered in the Canadian media (including the Globe and Mail and the CBC). You can read a great interview with study leaders Si Lok and Stephen Scherer here. You can also watch a video interview that includes footage of the beaver sequenced for the study, a 10-year-old resident of the Toronto Zoo named Ward. Happy birthday, Canada!

CITATION

De Novo Genome and Transcriptome Assembly of the Canadian Beaver (Castor canadensis)

Si Lok, Tara A. Paton, Zhuozhi Wang, Gaganjot Kaur, Susan Walker, Ryan K. C. Yuen, Wilson W. L. Sung, Joseph Whitney, Janet A. Buchanan, Brett Trost, Naina Singh, Beverly Apresto, Nan Chen, Matthew Coole, Travis J. Dawson, Karen Ho, Zhizhou Hu, Sanjeev Pullenayegum, Kozue Samler, Arun Shipstone, Fiona Tsoi, Ting Wang, Sergio L. Pereira, Pirooz Rostami, Carol Ann Ryan, Amy Hin Yan Tong, Karen Ng, Yogi Sundaravadanam, Jared T. Simpson, Burton K. Lim, Mark D. Engstrom, Christopher J. Dutton, Kevin C. R. Kerr, Maria Franke, William Rapley, Richard F. Wintle, and Stephen W. Scherer

G3: Genes|Genomes|Genetics February 2017 7:755-773; doi:10.1534/g3.116.038208

http://www.g3journal.org/content/7/2/755.full

When it comes to genotyping technology, poop genetics is stuck in the 1990s. While most geneticists are now awash in genome-scale data from thousands of individuals, those who depend on  fecal and other non-invasively collected samples still rely on old-school, boutique panels of a dozen or so genetic markers.

But feces — along with fur, feathers, and urine — is critically important stuff for understanding the population genetics, ecology, evolution, behavior, and conservation of wild animals. Many are too elusive or endangered to allow collection of blood samples, and even for common species it is a logistical nightmare to immobilize and draw blood from large numbers of animals in the field. In the latest issue of GENETICS, Snyder-Mackler et al. describe tools that promise to advance studies of such samples into the genomic era.

Patrick Chiyo collecting noninvasive samples from elephants in Amboseli National Park. Photo courtesy Jenny Tung.

Noninvasively collected samples have the obvious advantage of easy access. “We have freezers and freezers full of baboon poop,” says study co-leader Jenny Tung (Duke University). Tung’s group works on behavior and  genetics in a wild baboon population in Kenya. But though abundant, poop also presents serious challenges for standard genetic analysis. The DNA present in noninvasive samples is typically a fragmented mixture of host and contaminant sequence. For example, only around 1% of the DNA in a fecal sample comes from the animal that produced the poop. Most of the rest is microbial.

These limitations were first overcome in the 1980s and 1990s, and the ability to analyze DNA from noninvasive samples revolutionized the field. Using such samples not only allowed geneticists to understand the genetic diversity and viability of endangered animals, it allowed them to empirically test important theories about animal behavior and evolution.

“There are many examples. Noninvasive sampling of chimps, baboons, rhesus macaques and other primates revealed that animals really do bias their behavior towards relatives, even paternal relatives that are likely more difficult for an individual to identify as kin,” says Tung. “And in baboons, it also showed that males provide some paternal care to their offspring, which wasn’t expected for a polygamous primate.”

But the genotyping methods used in such studies have changed surprisingly little over the last twenty years. For the most part, researchers still use small groups of carefully validated markers, usually based on stretches of short tandem repeat sequences (microsatellites). This means the field has mostly missed out on the benefits of genomics that have become routine for medical researchers and those who work with laboratory organisms.

“Microsatellite approaches still work. But over the last 5 or 10 years it has become impossible to ignore the way genome-scale datasets allow you to answer entirely different questions,” says Tung.

For example, data on how a genome varies across a population can provide crucial evidence of the evolutionary and demographic forces that have shaped it. Genomic data can also trace in detail the mergers and separations of mixing populations.

Vet, a female yellow baboon, and her children in Amboseli National Park. Photo courtesy Susan Alberts.

The good news for poop genomics is that short-read next-generation sequencing methods are well suited to the fragmented DNA found in noninvasive samples. These methods have been famously adapted for analyzing a sample type that also suffers from vanishingly small amounts of target sequence: ancient DNA. The bad news is that the expensive, intensive approaches that work well for a precious sample of Neanderthal bone are not practical for a geneticist facing a freezer full of poop.

About six years ago, Tung’s friend and colleague George (PJ) Perry published a major advance that allowed large-scale resequencing from noninvasive samples. It was based on a method known as sequence capture, which enriches for host sequence using synthetic RNA “baits” to capture the target DNA. Tung was excited by the possibilities of the methods, but realized it was still too expensive for most applications. This was partly because the baits had to be custom-designed and synthesized for the species of interest. The method also had the drawback of only capturing a tiny fraction of the genome, while consuming large amounts of sample.

“Even fecal samples are exhaustible,” says Tung. “We have a lot of irreplaceable samples from dead animals, for instance. If we’re going to use them up, we want to cover all our bases and gather data on a truly genome-wide scale.”

So Tung’s group and their collaborators worked to modify and scale up Perry’s protocol. They also constructed the baits in a considerably cheaper way, using in vitro transcription of RNA from baboon DNA templates, sidestepping the need for custom synthesis. The new protocol had more modest input DNA requirements and could enrich the target DNA by 40-fold.

But getting enough sequence per sample was just the beginning. Xiang Zhou (University of Michigan) led the group’s efforts to develop tools to analyze data from the new method. Zhou says one of the reasons microsatellites became so popular was the availability of standard and easy-to-use software for assigning paternity from the data. “If people are going to transition to a new method, we thought it would be incredibly important that we package our models into software that will make it as easy as possible,” says Zhou.

But to develop something comparable for low-coverage sequence, the team faced two major challenges: the data is simultaneously much richer (more sequence) and much lower quality (more uncertainty). To deal with the large quantity of data they needed much more computationally efficient algorithms. They also had to factor in the lower data quality, which makes it  impossible to use the simpler approaches that work when the genotype at each site is known with certainty. Instead, they incorporated the error rate across all the sites in the genome, generating a sophisticated statistical model.

One of (several) freezers in the Tung lab containing boxes of fecal samples. Photo courtesy Jenny Tung.

Using the new capture method and the paternity assignment software (called WHODAD), the team were able to construct pedigrees from baboon fecal samples that almost perfectly  matched those created using traditional analysis of high-quality DNA from blood. In short, despite the low coverage of the genome (typically less than 1x), and the resulting very high uncertainty of the genotype at any one site, the trends in the data were more than enough to reconstruct family relationships.

But what about cost? Lead author Noah Snyder-Mackler gave the project the pet name “fecal alchemy” because it aims to transform poop into a data goldmine. But not every researcher can afford gold — most labs must use the cheapest tool that will get the job done. Tung says they included a cost analysis in the paper because they are regularly asked about the price of making the switch.

“Right now it costs about twice as much to produce 1x coverage of the entire baboon genome as it does to type 14 microsatellites. But the amount of information you get is much greater! So if you’re thinking in terms of cost per genotype, our method is way more cost effective. But in terms of absolute amounts it’s more expensive. In the end the cost-benefit decision depends on what questions you’re trying to answer,” says Tung. “Of course we’d like to get it even cheaper and more efficient and more robust. We’re working on it!”

FUNDING

This work was partly funded by the National Science Foundation DEB through an EAGER grant, with co-funding from NSF Biological Anthropology.

CITATION

Noah Snyder-Mackler, William H. Majoros, Michael L. Yuan, Amanda O. Shaver, Jacob B. Gordon, Gisela H. Kopp, Stephen A. Schlebusch, Jeffrey D. Wall,Susan C. Alberts, Sayan Mukherjee, Xiang Zhou, Jenny Tung (2016). Efficient Genome-Wide Sequencing and Low-Coverage Pedigree Analysis from Noninvasively Collected Samples. Genetics, 203(2), 699-714.

http://www.genetics.org/content/203/2/699

DOI: 10.1534/genetics.116.187492

The sex of many reptile species is set by temperature. New research reported in the journal GENETICS identifies the first gene associated with temperature-dependent sex determination in any reptile. Variation at this gene in snapping turtles contributes to geographic differences in the way sex ratio is influenced by temperature. Understanding the genetics of sex determination could help predict how reptiles will evolve in response to climate change.

In crocodiles, alligators, and certain lizard and turtle species, an embryo can become either a male or a female depending on the temperatures it experiences while in the egg. Rapid climate change may threaten the future of some of these species by skewing the sex ratio. For example, by some estimates temperature rises over the next century will cause painted turtles to produce only females. Such species may also evolve in response to climate change. Biologists are trying to understand how these animals will be affected by and adapt to rising global temperatures.

But little is known about how this temperature-dependent switch between ovaries and testes is regulated. To look for clues to the molecular mechanisms behind this process, study leader Turk Rhen (University of North Dakota) and his colleagues investigate how genes influence sex determination in common snapping turtles. The advantage of focusing on this rugged-looking North American native is that sex is determined in a brief five-day window during the 65-day egg incubation: the temperature-sensitive period. If the incubation temperature during the temperature-sensitive period is changed from a “male-producing temperature” (26.5°C or 79.7°F) to a “female-producing temperature” (31°C or 87.8°F), all the eggs will hatch into females.

Snapping turtle adult. Photo: Turk Rhen

In previous work, the team identified a gene—CIRBP—that is activated within 24 hours of such a temperature shift. Two days later, several genes known to be involved in ovary or testes development are either activated or repressed. The new study confirmed that CIRBP is expressed at the right time (very early in the temperature-sensitive period) and the right place (the gonads) to be involved in specifying sex.

To test whether this hypothesis is correct, the researchers investigated DNA sequence variation at the CIRBP gene, and whether it influenced the chances of an individual turtle becoming male or female.

They found that some of the turtles carried a slightly different version of CIRBP: at a specific position in the sequence, an “A” in the four-base DNA code was substituted with a “C”.

This change rendered the gene unresponsive to temperature: instead of being induced by the female-producing temperature, the “C” version of the gene remained at steady levels.

Individuals carrying this unresponsive “C” version were more likely than average to be male. This single-letter DNA difference between turtles could explain around a quarter of the genetic variability in sex determination temperatures, which suggests that the activation of CIRBP is a crucial event in specifying sex.

Remarkably, this CIRBP variant may partly explain a curious fact about snapping turtles: the sex ratio in populations from different latitudes responds differently to temperature. For example, if you collect eggs from snapping turtles in Minnesota and Louisiana and incubate them all at 27°C (80.6°F) in the lab, the eggs collected in the North will produce nearly all males, while those from the South will produce mostly females. This variation suggests subpopulations of the species have evolved and adapted to their local climate.

The team found that the “C” version of CIRBP was more common in turtles from northern Minnesota than those from 250 miles away in southern Minnesota, and it was not detected in a population from even further south, in Texas. Though this is only a small sample of locations, the trend is consistent with the sex determination pattern in each population: the “A” version (which makes turtles more likely to be female) was more common in populations that produce females at a lower temperature.

The protein encoded by the CIRBP gene (cold-inducible RNA-binding protein) is likely involved in sensing temperature and converting it into a developmental signal to trigger the formation of either testes or ovaries, says Rhen. Studies from other organisms suggest that this protein can regulate temperature-dependent processes by binding to the RNA “messages” produced by specific genes.

“CIRBP seems to play a crucial role in sex determination,” says Rhen. “The striking part is that we see a consistent association across multiple levels of biology: the variation at the DNA level influences the gene’s activation (expression into RNA messages), which is in turn correlated with whether an individual turtle becomes male or female. That association with sex holds whether we look at individuals or families, and we even see differences at the population level.”

But CIRBP is not the only gene important for specifying sex in snapping turtles, the data show. “There is a common misconception that there must be a single “magic bullet” gene that determines sex in response to temperature,” says Rhen. “Our data suggests that multiple temperature sensors control sex by acting together. We’re trying to identify the other components of this system and to determine how they interact to influence sex. Better understanding variation at these genes may one day allow us to predict how reptile species will evolve under a new climate regime.”

CITATION

A Novel Candidate Gene for Temperature-Dependent Sex Determination in the Common Snapping Turtle

Anthony L. Schroeder, Kelsey J. Metzger, Alexandra Miller, Turk Rhen

GENETICS May 1, 2016 vol. 203 no. 1 557-571; DOI:10.1534/genetics.115.182840

http://www.genetics.org/content/203/1/557

In 1996, when I started researching the conservation genetics of New Zealand’s critically endangered parrot, the kākāpō (Strigops habroptilus), little was known of the species’ genome.  On many occasions after a long day in the molecular lab on the hunt for an elusive gene, I found myself imagining that I had the complete genome of kākāpō at my fingertips. Fast forward 20 years, and that dream has been realised with the sequencing of the kākāpō genome by researchers at Duke University and PacBio, as part of the Bird 10,000 genomes project. It was like Christmas the day the whole genome landed in my inbox (22 January 2016). Elusive kākāpō genes could no longer hide from me and the possibilities for kākāpō conservation and species recovery seemed endless.

Many people will be familiar with the species thanks to a kākāpō called Sirocco (the species’ “spokesbird,” who tweets @Spokesbird) and his unusual mating habits. Stephen Fry’s narration of Sirocco mating with Mark Carwardine’s head on YouTube has had over 6.7 million views. For many others like me, Douglas Adams introduced kākāpō to them in the moving account of the species’ plight in the book Last Chance to See. Historically a widespread species, the kākāpō reached a low of only 51 birds in 1995, and the world population now stands at a hard-won 125 individuals. To maintain genetic diversity and ensure species recovery, all birds are now intensively managed by a team of dedicated conservationists.

Photo courtesy Andrew Digby / DOC

Conservation genomics – the application of genomic analysis to the management of threatened species – is a fast-emerging field propelled forward by the dramatic advances in genome sequencing.  These technological developments have also led to considerable cost savings, and we are to the point where we can once again dream big for kākāpō. In a world first, the Kākāpō 125 Genomes Project is sequencing the genome of every surviving individual of a species.  

This ambitious project is a collaboration among researchers from the New Zealand Department of Conservation’s Kākāpō Recovery Group, the University of Otago (New Zealand), Duke University (USA), the Genetic Rescue Foundation and NZ Genomics Ltd.

For the Kākāpō 125 Genomes Project, we’re using the high-quality reference kakapo genome developed by Duke University and PacBio as a scaffolding to assemble all other 124 kakapo genomes against. This project is being funded by the Genetic Rescue Foundation, a not-for-profit organization dedicated to advancing the scientific techniques required to prevent species extinction through genetic intervention. The Genetic Rescue Foundation makes use of the latest crowdfunding and online scientific services such as Science Exchange and Experiment.com to fund and conduct research. The Genetic Rescue Foundation’s latest crowdfunding campaign is currently in progress and in just 20 days has raised over $32,000 toward a target of $45,000.

A genomics approach to kakapo conservation will be a great boost to the recovery program. A genome-wide understanding of genetic variation will help to develop breeding strategies to retain variation at genes important for species persistence, such as the immunity genes and their role in kākāpō diseases. We will also be able to explore the genetic basis of infertility in kakapo; only 60% of eggs hatch (normally this should be about 90% in birds) and sperm abnormalities contribute to infertility. Solving the issue of infertility will greatly aid species recovery by maximising reproductive effort.

Beyond being of considerable significance to kākāpō conservation, the Kākāpō 125 Project will make an important contribution to the emerging field of Conservation Genomics. Our project will provide a genome dataset for every member of an entire species, serving as a much needed case study to further develop analytical pipelines and methodologies. Only with studies such as ours will Conservation Genomics’ true potential be realised.

Bruce Robertson is an Associate Professor studying the conservation genomics, molecular ecology and wildlife management of New Zealand native species in the Department of Zoology at the University of Otago, Dunedin, New Zealand.

The views expressed in guest posts are those of the author and are not necessarily endorsed by the Genetics Society of America.

When an eastern diamondback rattlesnake bites its prey, it injects a cocktail of toxic proteins and peptides that attack on multiple fronts. These toxins destroy blood vessels, block the blood clotting cascade, cause necrosis, and inflict crippling pain.

But the precise recipe for this noxious mix is generally thought to depend on where the snake was born. Cycles of co-evolution between predator and prey are expected to drive extensive genetic divergence between locations; the data so far have mostly fit this model, and venom composition seems to vary with geography.

In a recent issue of GENETICS, Darin Rokyta and colleagues reported the results of a large survey of venom diversity across two snake species sharing nearly identical ranges and similar habitats in the southeastern United States. As expected, the mix in one species—the eastern diamondback rattlesnake—varied considerably from place to place. But the eastern coral snakes told a completely different story. In contrast to its rattlesnake neighbors, no matter where a coral snake came from, its venom was always the same.

Rokyta says the team was shocked by this lack of variation. “This is the first time anyone has looked at venom variation at this scale, and everybody has assumed that the co-evolutionary arms race would cause local populations to diverge quickly.”

The results not only challenge this assumption, they provide crucial information for rattlesnake conservation and coral snake antivenom development.

When an eastern diamondback rattlesnake bites its prey, it injects a cocktail of toxic proteins and peptides that attack on multiple fronts. These toxins destroy blood vessels, block the blood clotting cascade, cause necrosis, and inflict crippling pain. But the precise recipe for this noxious mix is generally thought to depend on where the snake was born. Cycles of co-evolution between predator and prey are expected to drive extensive genetic divergence between locations; the data so far have mostly fit this model and venom composition seems to vary with geography. In a recent issue of GENETICS, Darin Rokyta and colleagues reported the results of a large survey of venom diversity across two snake species sharing nearly identical ranges and similar habitats in the southeastern United States. As expected, the mix in one species—the eastern diamondback rattlesnake—varied considerably from place to place. But the eastern coral snakes told a completely different story. In contrast to its rattlesnake neighbors, no matter where a coral snake came from, its venom was always the same. Rokyta says the team was shocked by this lack of variation. “This is the first time anyone has looked at venom variation at this scale, and everybody has assumed that the co-evolutionary arms race would cause local populations to diverge quickly.” The results not only challenge this assumption, they provide crucial information for rattlesnake conservation, and coral snake antivenom development. Image credit: Joseph Pfaller

Rokyta (Florida State University) and his colleagues study venom to generate powerful datasets for testing quantitative models of adaptation. These theoretical models of adaptation need to be evaluated and guided by data from the field, but the types of real-world traits evolutionary biologists typically study can’t be used in this way because they’re hard to link to specific genetic variants. That’s because these complex traits—like coat color, jaw shape, stress tolerance—are affected by many networks of molecular and developmental pathways, and many candidate genes are likely to contribute to their variation.

For venom, in contrast, the link between genotype and adaptive phenotype is relatively direct. Simple changes in expression of a particular toxin can boost or dampen venom potency, or alter its specificity for certain types of prey. And because venom is produced by a specialized gland, the genes encoding the toxins can be rapidly identified by analyzing the venom gland transcriptome. These data could help in the identification of relevant gene variants and the signatures of the corresponding proteins in proteomic analyses of venom samples. The bottom line? Rokyta sees analysis of snake venom as a shortcut to a detailed genotype-phenotype map of an ecologically critical trait and an ideal source of hard data to test and shape quantitative models.

Creating this genotype-phenotype map is a long term project, but as an important first step, Rokyta and his team have assembled the venom gland transcriptomes of the two snake species and collected venom and blood samples from individuals collected at many locations.

Venom samples were collected from 65 adult eastern diamondback rattlesnakes (circles) and 49 adult eastern coral snakes (triangles). Different colors represent different putative populations. AR, Apalachicola River; SMR, Saint Mary’s River; SR, Suwannee River. From Margres et al.

Using sensitive proteomic analyses, Rokyta et al. were able to divide the eastern diamondback samples into five different geographic groups that each brews its own brand of local venom. For example, in venom from snakes on Caladesi Island, near Tampa, Florida, two major toxins normally produced in this species are undetectable.

But for the coral snakes, there was no significant variation. Whether the snake had been caught in the Everglades or several hundred miles north in Florida’s panhandle, the venom was indistinguishable.

Rokyta says there are several possible explanations for this surprising result. One hypothesis is that differences in diet between the species might result in distinctive patterns of co-evolution. “Coral snakes tend to eat long, slender reptiles, while rattlesnakes tend to eat mammals,” he says. “So there may be big differences in the dynamics and genetics of their prey.”

Another idea is that the coral snake population could be less genetically diverse than the eastern diamondbacks, from a recent range expansion, for example, or a selective sweep. To figure out what’s really going on, the group is now examining the population genetics of the two species. “We have an intriguing pattern, but now we need to understand which driving selective forces—if any—caused this difference between the species.”

In the meantime, the results have practical significance. For example, they could aid efforts to conserve eastern diamondback populations. The species is currently in decline and under consideration for listing as threatened under the Endangered Species Act. These declines are thought to have been caused by habitat loss, compounded by persecution from humans. “We find a surprisingly large number of rattlesnakes dead on the road where people have obviously stopped, chopped the head and tail off, and left them for dead,” says Rokyta. He suggests conservation managers could use the new data to ensure the full range of local venom brews is conserved.

“Usually when you’re deciding which populations to focus your efforts on, you look at genetic differentiation,” says Rokyta. “But this type of functional differentiation in an adaptive trait might be even more useful; if you lose one of these five populations, that entire venom phenotype is gone.”

The lack of variation in eastern coral snake venom could be a boon for those developing a sorely-needed antivenom. Co-author Jack Facente (Agritoxins) runs a venom production lab to supply companies working on antivenom, and many of the coral snakes sampled in the study were originally collected by Facente for his lab. But if snakes born in different locations delivered significantly different venom, the resulting antivenom could also vary in effectiveness, depending on the range of subtypes sampled. Luckily for Facente, it doesn’t seem to matter where he finds his coral snakes—they always provide the same product.

Hopefully—after we learn more about the genetics of these beautiful snakes—we’ll one day know why.

CITATION:

Margres M.J., M. Seavy, K. P. Wray, J. Facente & D. R. Rokyta (2014). Contrasting Modes and Tempos of Venom Expression Evolution in Two Snake Species, Genetics, 199 (1) 165-176. DOI: http://dx.doi.org/10.1534/genetics.114.172437 http://www.genetics.org/content/199/1/165.full

How does evolution rewire an animal’s sensory system? In time for both National Bat Week and Halloween, new research in G3 investigates this question by comparing the genomes of bat species that “see” the world in different ways.

The black flying fox Pteropus alecto forages for fruit mainly by smell and sight. In contrast, the insectivorous Myotis davidii has vision adapted for dim light and hunts using the animal version of sonar—echolocation. Though it is not clear whether M. davidii evolved echolocation before or after the split from flying foxes, Hudson et al. reasoned that comparing the genomes of species with such divergent sensory preferences could shed light on the evolutionary mechanisms involved.

The researchers started by looking for evidence of functional decay in the visual system, examining bat orthologs of visual perception genes that are either inactivated or missing in the naked mole rat, a mammal with poor vision that lives most of its life in darkness. Of the 19 orthologs, five were inactivated by premature stop codons in echolocating M. davidii but not in the flying fox P. alecto. This evidence hints at loss of visual function associated with M. davidii’s poor day vision and small eyes.

The black flying fox Pteropus alecto uses smell and vision to forage and does not echolocate. Photo credit: Susanne Wilson/CSIRO

But there is more to sensory rewiring than loss of function. To identify potential signals of gain-of-function changes, Hudson et al. explored genome-wide patterns of codon usage bias between the two bat species. Codon usage bias refers to the nonrandom frequency of different synonymous codons. Extreme codon usage bias is thought to result partly from selection operating on the efficiency of translation.

The authors have previously found that extreme codon usage bias tends to occur in genes that are particularly characteristic of and important to the organism under study, such as genes involved in hair formation genes in mammals, mitochondrial function in birds, and chloroplast physiology in plants. They argue that extreme codon usage bias and selection on translation efficiency at the whole-pathway level may play an underappreciated role driving change during the evolution of new or enhanced functions (gain-of-function changes).

Of genes with extreme codon usage bias in the echolocator M. davidii, the functional class most overrepresented was retina development. Other statistically enriched functions included axon development and light and sound perception. In contrast, none of these classes were enriched for codon usage bias in P. alecto.

To learn whether this association between echolocation and codon bias in sensory genes holds up for other species, the authors performed a similar analysis for M. lucifugus (an echolocating bat) and P. vampyrus (non-echolocating). In this case, the authors did not find a statistically-significant enrichment of sensory perception genes among those with extreme codon bias. But when they examined the sensory genes that were enriched in the original analysis of M. davidii, all of these genes were more biased in echolocating M. lucifugus than in P. vampyrus.

Myotis davidii or David’s myotis echolocates and has small eyes adapted for low light. Photo credit: Susanne Wilson/CSIRO

This suggests that during the evolution of echolocation a set of genes involved in vision and hearing was under selection for new or enhanced functions—perhaps reflecting development of enhanced sensitivity to dim light and ultrasonic sound. Remarkably, genes involved in both hearing and vision also showed up in a 2013 study of convergent evolution in bats and dolphins, two groups that independently evolved the ability to echolocate. These latest results add to our developing understanding of sensory rewiring and paint a picture that combines both functional decay and novelty.

Hudson N.J., N. S. Hart, J. W. Wynne, Q. Gu, Z. Huang, G. Zhang, A. B. Ingham, L. Wang & A. Reverter (2014). Sensory Rewiring in an Echolocator: Genome-Wide Modification of Retinogenic and Auditory Genes in the Bat Myotis davidii, G3, 4 (10) 1825-1835. DOI: 10.1534/g3.114.011262 http://g3journal.org/content/4/10/1825.full