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The genomics of peppermint are not as fresh as their flavor but scientists from the University of California, Davis, have found a way to breathe new genetic variation into the species.

Similar to strawberries, potatoes and many fruit trees, peppermint plants (Mentha × piperita) are reproduced asexually by a process called clonal propagation. In the case of peppermint, this means that their genomes have remained unaltered for more than 200 years. This lack of genetic variation leaves them susceptible to disease and means that properties such as yield and flavor have remained stagnant.

�鶹��ý plant biologists used radiation to induce mutations in the leading peppermint clone grown in the United States, resulting in over 250 new and genetically distinct variants. Altogether, they introduced 1,406 large genetic mutations, which can now be used to identify key genes for breeding or selecting new and superior peppermint varieties. 

The findings, published May 8 in , could help the mint industry develop new varieties of peppermint and provides a roadmap for improving clonal crops more generally.

“Our results provide a powerful resource for studying mint genomics and a low-cost, non-GMO method for inducing genetic variation and improving sterile crops,” said first author Nestor Kippes, a former fellow in the �鶹��ý Department of Plant Biology and Genome Center.

Can peppermint plants get even mintier?

The project began when Mars Inc., the global candy maker that acquired Wrigley in 2008, asked the team, led by Project Scientist Isabelle Henry and Distinguished Professor at the �鶹��ý Genome Center, to inject long-needed genetic variation into peppermint. In particular, mint growers and industry are interested in identifying genes that confer resistance to Verticillium wilt, a soil-borne fungal disease that is threatening peppermint farming worldwide.

The researchers focused on Black Mitcham, a peppermint clone that was discovered in England in the 1880s. Since all Black Mitcham plants are sterile clones, they can’t shuffle their genomes through sexual reproduction, which is how genetic variation usually arises in sexually reproducing species.

“Black Mitcham peppermint oil is used by companies from all over the world for candy, chewing gum and toothpaste,” said Comai, who is corresponding co-author on the study. “This clone is getting jeopardized by disease, so the mint industry needs to make sure that they can continue to grow it, or a similar variety with equivalent or better properties.” 

Instant genetic and aromatic variation

To introduce genetic variation, the researchers exposed peppermint cuttings to gamma radiation and then grew the cuttings into 261 full-sized plants. When they sequenced the new plants’ genomes and compared them to the Black Mitcham genome, they found that each new variant had acquired 5.83 large-scale mutations on average.

“This method of mutagenesis is almost 100 years old, but it still provides a powerful tool for probing gene functions and plant development, especially when we combine it with today’s cutting-edge genomics,” Comai said.

To understand how these mutations affected the plants’ traits, the researchers grew the new variants at the University of California Intermountain Research and Extension Center field station in Tulelake and distilled oil from the plants’ leaves to characterize their oil profiles.

Surprisingly, they found that two of the clones produced drastically lower amounts of menthol, the molecule that gives peppermint its cooling properties, compared to Black Mitcham. Whereas menthol accounts for 42% of the scent molecules in Black Mitcham’s oil, it accounted for only around 4% in these clones.

“Mint obviously likes to be variable in its oil composition,” Comai said. “These oils are not there for us to make chewing gum — they are defense compounds — so having variation in oil properties enables plants to adapt to the arrival of new herbivores and pathogens.”

One plant, multiple genomes

The vast majority (250/261) of the new variants were chimeras, meaning they carried multiple distinct genomes in different cell layers. In line with , another clonal crop, the team showed that mutations occurred more than twice as frequently in the stem cells that produce a plant’s skin or epidermis (L1 stem cells), compared to the stem cells that produce a plant’s sperm and eggs (L2 stem cells).

“This supports our hypothesis that L1 cells might accumulate mutations faster in order to provide useful variation with little long-term genetic consequences, whereas L2 stem cells might have evolved to be more resistant to mutations in order to protect the plant’s sex cells,” said Henry, the study’s senior author.

Being able to alter a plant’s genome in a single stem cell layer could enable breeders to create tissue- or organ-specific changes without altering the plant’s other properties. 

“This means we could introduce disease-resistance in the roots without affecting the plant’s leaves or architecture,” said Kippes. “Our method provides a non-transgenic and cost-effective roadmap for doing that.”

Additional authors on the study are Meric Lieberman, Isabelle DeMarco, Helen Tsai and Kanae Masuda, �鶹��ý; Darrin Culp and Robert Wilson, University of California Cooperative Extension; and Jordan Lopez, Mars Inc. 

The work was supported by Mars Inc. and the National Science Foundation.

This project utilized several �鶹��ý research core facilities, including the , and the , and the DNA Technologies and Expression Analysis Core at the .