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International Team Sequences Truffle Genome

By Andrea Anderson

NEW YORK (GenomeWeb News) – An international research team reported in the early, online version of Nature yesterday, that they have sequenced the largest fungal genome so far: that of the Périgord black truffle, Tuber melanosporum.

When the researchers — from France, Italy, and Germany — compared the genome with those of other sequenced fungi, including another fungal symbiont, they found that the black truffle genome has a distinct genome structure, gene repertoire, and transcriptome.

"It's very strikingly different," lead author Francis Martin, a plant biology researcher with the French National Institute for Agricultural Research (INRA), told GenomeWeb Daily News, noting that these differences may reflect the fact that T. melanosporum belongs to one of the oldest known fungal lineages.

Those involved say the results not only hint at multiple adaptations for symbiosis in fungi, but also provide the foundation for using information in the truffle genome to improve truffle cultivation and certification.

Truffles, which form symbiotic interactions with trees such as oak and hazelnut, are sought after for their edible fruit bodies. At the moment, the researchers explained, the most popular and pricey truffles are the Périgord black truffle and Piedmont white truffle.

The Périgord black truffle grows in basic, limestone soil in southern Europe, Martin explained, as the fruiting body, which matures in December, cannot withstand frost.

While it's possible to inoculate host trees with the truffles, the approach has variable and unpredictable success. Consequently, researchers and producers are interested in the genetics behind the truffle's fruit production, reproduction, and symbiosis with its host plants.

"We would like to get better knowledge of the interaction," Martin said. "To do that, we need more information. We need the genome."

To get this genomic information, collaborators at the French sequencing center Genoscope used the Sanger approach to sequence the125 million base pair genome of a Périgord black truffle haploid strain called Mel28.

Going into the study, the researchers predicted that the truffle genome would contain many genes, large gene families, and an extensive gene repertoire — features that the team believed to be trademarks of symbiosis based on their sequencing of the fungal symbiont Laccaria bicolor in 2008.

In contrast, though, the black truffle genome contained relatively few protein-coding sequences: the team identified just 7,496 protein-coding genes.

Using custom microarrays, expressed sequence tag sequencing and RNA-sequencing with the Illumina platform, they were able to detect expression of most of the genes in various truffle tissues.

Just a few dozen transcripts were detected in only the ectomycorrhiza form of the truffle, the fruiting body, or the mycelium. For instance, the team pinpointed 64 potential membrane transporters that are more highly expressed in the ectomycorrhiza and may help the fungus swap nutrients with its host.

The researchers also found evidence that the truffle genome contains all the enzymes needed to produce so-called truffle volatiles — flavorful compounds that contribute to the truffle's characteristic aroma and flavor. The expression of these genes seems to get ramped up during fruiting body maturation.

"All of the enzymes needed for the main, the major volatiles … all the pathways are there," Martin said. "The mushroom itself is producing most of its aroma."

And in contrast to previous theories that the black truffle might reproduce clonally, the researchers found evidence for two black truffle mating types, consistent with sexual reproduction in this fungus.

Meanwhile, the team's comparisons with other fungal genomes suggest 3,970 predicted truffle proteins are similar to those in Saccharomyces cerevisiae, while around 5,600 each overlapped with proteins predicted in the fungal species Neurospora crassa and Aspergillus niger.

Despite its overall size, Martin explained, the researchers found that the black truffle has a small and streamlined genome when it comes to its actual gene content.

Instead, the size and complexity of the truffle genome stems, in large part, from a preponderance of transposable elements in the genome. Some 58 percent of the genome is comprised of these transposable elements, which are particularly common in parts of the genome where genes are scarce.

The overall organization of the truffle genome also appears to be distinct from other fungi. For instance, Martin noted, it shares almost no synteny with other fungi, even in conserved regions of the genome.

Such differences are consistent with the notion that the black truffle belongs to one of the oldest fungal lineages. The team estimates that the lineage leading to T. melanosporum diverged from other fungal lineages more than 450 million years ago.

Moreover, distinct features in the black truffle and L. bicolor genomes suggest symbiosis may have evolved several times using different molecular toolkits in the fungal tree of life, Martin said.

With the black truffle reference genome in hand, the team is currently taking a crack at sequencing the white truffle genome using Roche 454 Titanium sequencing and Illumina sequencing to fill in the gaps.

They also plan to genotype about 200 black truffle populations in Italy, France, and Spain to try to find DNA fingerprints that can be incorporated into a database to help truffle producers from different regions certify their products, Martin said.

Other follow-up studies in the works involve using a combination of transcription analyses and mass spectrometry to try to fish out more information about the genes involved in the production of truffle volatiles, he added. The team is also thinking of developing kits to help truffle growers identify truffle mating type.