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Science, Vol. 320, 25 april 2008

Uncorking the Grape Genome

Among wine connoisseurs, opinions differ about whether 2007 will prove a good year for Pinot Noir. But among plant geneticists, it’s the finest vintage ever: Last year, two European teams published high-quality drafts of two Pinot Noir–derived genomes.

Plant biologists are toasting the genomic double-header. This is the first fleshy fruit and just the fourth flowering plant to have its genome decoded. And in economic terms, grapes top the world’s fruit crops: We consume them fresh or dried, crush them into juice, and use them to make wine that can sell for many thousands of dollars a bottle. "The contributions of these sequencing efforts are enormous and historical," says grape researcher Steven Lund of the University of British Columbia in Vancouver, Canada.

The story behind the grape genome is one in which a worldwide scientific community came together, then partially splintered into rival camps; money to support sequencing was hard to come by; and success has brought both new insights and delicious questions. The rivalry provided the drama of the story. For a while, a French-Italian grape genome alliance called Vigna/Vigne looked like it was going to be beaten by a disgruntled researcher who started his own genome effort. "Undoubtedly, competition was a driver here, perhaps in a microcosm of the human genome sequence drama of years past," says Lund, referring to the bitter contest between public and private programs to decipher our genetic code. Recently, however, at a workshop in Udine, Italy, the two grape genome groups began to put aside their rivalry. "I’m hopeful there will be more collaboration now," says Vigna/Vigne member David Horner of the University of Milan in Italy. "It’s cool there are two cultivars done. It allows more comparative work."

A key motivation for deciphering the grape genome is to prevent a repeat of the economic devastation that struck the European wine industry in the late 1800s. At that time, phylloxera, sap-sucking insects from North America, ravaged European grapevines. Today, winemakers and grape researchers are struggling to combat new threats, particularly downy and powdery mildew, diseases that have made their way to Europe from the United States over the past century. These fungi are an environmental as well as an economic nightmare: Although only about 5% of Europe’s farmland is dedicated to wine vineyards, they account for about 70% of the region’s fungicide use.

The new genome information should speed the creation of hardier vines, which has been slow going. "The target now is clearly resistance genes," says Vigna/Vigne member Michele Morgante from the Institute of Genomic Applications (IGA) in Udine. New insights into the locations of these genes can assist breeders as they try to develop better varieties, for example. And identifying genes in the few grapes that are resistant to drought or pests may pave the way for genetically modifying common wine grapes to have the same attributes. But as viticulturists enter the genomic age, many wonder whether the wine industry, particularly the conservative European sector, would dare bypass conventional breeding for genetically modified (GM) grapevines. Scientists have for years been experimenting with GM grapes—usually putting nongrape genes into the fruit’s genome—but most winemakers have shied away from public association with such efforts. "For a lot of consumers of wine, especially high-end ones, history and tradition is a very important part of their experience. If you produce wine from a genetically engineered grape, you strike at the heart of that," says Carole Meredith, a former grape geneticist who now runs a vineyard.

Taking root

Although there’s a dazzling variety of wines produced around the world, the great majority flow from the juices of a single species, Vitis vinifera, commonly called vinifera. Indeed, in Europe, this grape is the only source of fine wines—other grapes are limited to fruit, juice, or so-called table wines. Thanks to centuries of breeding, 7000 cultivated varieties, or cultivars, of vinifera now provide an incredible diversity of flavors, from hearty reds to light whites.

A vinifera genome project began to take root in the mid-1990s, largely through the instigation of Meredith, who was then conducting grape research at the University of California, Davis. Meredith and others wanted to use genetics to identify various grape cultivars— Chardonnay, Cabernet Sauvignon, and so on—that can be tricky to distinguish in other ways. In a business in which wines made from different cultivars can vary enormously in price, correct identification is critical.

The researchers proposed to "fingerprint" grapes based on variable DNA sequences called microsatellite markers. Back then, however, identifying such markers "was expensive and laborious, and no one lab was ever going to develop enough to make their efforts worthwhile," Meredith recalls. "So I approached all the people I knew and suggested working together."

From that suggestion arose an international consortium that amassed hundreds of such markers within several years. The researchers were thirsty for more. In 2001, many of them, including Meredith, formed the International Grape Genome Program, arguing for a fullfledged sequencing of a vinifera cultivar.

But who would pay for it? It was wellknown that grapes weren’t on the U.S. genomics menu. In 2001, Enrico Pé of the Sant’Anna School of Advanced Studies in Pisa proposed a grape genome program in Italy, sponsored by the private sector. But bank foundations and winemakers declined.

Finally, in 2005, the French research agencies INRA and Genoscope joined with various Italian groups, including IGA and universities in Verona and Udine, to form Vigna/Vigne, which means vine in French and vineyard in Italian, respectively. This time, researchers did not ask winemakers for money, only political support, says Pé, who now leads the Italian side of the collaboration. France ultimately contributed about €8.5 million, Italy, about €12 million.

The race is on

Except when discussing European football— their national teams are bitter rivals— the French and Italian groups meshed smoothly. Still, agreeing on what to sequence wasn’t easy. Compared with many plants, vinifera grapevines are extremely heterozygous: The female and male versions of the plant’s chromosomes differ significantly. This complicates the sequencing and assembly of an accurate genome. The Italian scientists first considered a Sangiovese grape, then a Pinot Noir. Their French counterparts lobbied for a Cabernet Sauvignon grape, but after studying its DNA further, the team decided it was too challenging as well.

Finally, an almost-forgotten grapevine growing in a French greenhouse provided a solution. In the 1980s, a French viticulturist hoping to develop a better vine for winemakers began inbreeding a Pinot Noir. Several generations of selfings produced a few lines with simplified genomes but also stunted their growth and made them unappealing for wine production. Vigna/Vigne decided one of those, PN40024, would offer the best potential for sequencing, even if it was, as Pé jokes, "pathetic, hardly a grape."

Just months after Vigna/Vigne began its PN40024 sequencing effort, Pé and his colleagues were shocked to learn of a rival effort. In March 2006, Riccardo Velasco and his colleagues at the Istituto Agrario San Michele all’Adige (IASMA) in Trentino, Italy, announced that they were almost done sequencing the genome of a Pinot Noir grape used in many countries to make red and sparkling wines. Velasco had been involved in some of the original genome planning efforts, including Pé’s Italian proposal, but he disagreed with the initial decision to switch from sequencing an outbred Pinot Noir to a Cabernet Sauvignon. His institute then lobbied the Trentino regional government for about €10 million to pursue its own grape genome. IASMA initially kept quiet about the true scope of the project, says Velasco, because "the possibility of failure was high." Unlike Vigna/Vigne, which kept sequencing inhouse, IASMA contracted with Myriad Genetics in Salt Lake City, Utah, to sequence and help assemble most of the genome.

After the IASMA bombshell, Anne-Francoise Adam-Blondon of INRA says that the French-Italian effort ground to a halt for several months as members discussed whether to spend their governments’ money on something that had apparently already been accomplished. But IASMA hadn’t published its genome, so Vigna/Vigne decided to press on.

The race ended with Vigna/Vigne winning by a nose. It published the PN40024 genome online 27 August in Nature (sciencenow.sciencemag.org/cgi/content/full/2007/827/1). Velasco’s Pinot Noir genome appeared online 19 December in PLoS One. Some Vigna/Vigne members felt that Velasco initially downplayed their feat, by, for example, reminding the media that PN40024 wasn’t a real Pinot Noir or used for wine. But Velasco attended last month’s workshop, which focused on Vigna/Vigne’s results, and he was greeted warmly. "The two projects are fully complementary," he says.

Decanting the genome

It’s already clear that the two genomes vary significantly. Although both efforts predict that the vinifera genome contains about 30,000 genes, the Pinot Noir sequenced by Velasco’s team has about 270 members of a gene family associated with disease resistance, whereas the inbred PN40024 strain has almost 360, Adam-Blondon reported at the workshop. The two grapes also differ in the number of the many hundreds of genes related to the production of polyphenols, flavenoids, and resveratrols—all of which contribute to a wine’s color, aroma, and taste.

Several of the talks in Udine centered on how genomic data could aid the breeding of disease-resistant grapevines. Gabriele Di Gaspero of IGA, for example, is trying to harness the powers of a Central Asian grape that is used for raisins. It is the first vinifera shown to stand up to powdery mildew, a discovery by a Hungarian group that electrified grape biologists a few years ago. Di Gaspero and colleagues have been crossbreeding the grape with a mildew-vulnerable vinifera and have used DNA markers identified through the genome projects to pinpoint the chromosomal location of a disease-resistance gene.

In theory, researchers can now—even without identifying the exact gene—breed resistance into popular wine grapes such as Chardonnay and Pinot Noir. After crossing the Central Asian grape with their favorite wine grape, they can select and continue to breed just the seedlings whose DNA contains the markers bracketing the mystery resistance gene. Ultimately, such marker-assisted selection could result in a disease-resistant grape that retains most of the qualities needed to make a good wine, instead of raisins. Still, bringing a new grape to market can take decades—and scientists have to be sure the transferred resistance gene is stable in its new genetic surroundings. "When you plant a grape field, it’s for 30 years, so you really need durable resistance," notes Adam-Blondon.

Grape researchers are also using the new genome data to probe the interplay of genes, environment, and wine flavors in a variety of cultivars. In Udine, Mario Pezzotti of the University of Verona detailed genetic studies of the unusual process that produces the Italian red wine Amarone. The grape involved typically produces a sweet wine, but decades ago winemakers realized that if those same grapes dry for several months after harvest, the withered fruit make a more intense and bitter wine that has since become highly valued. Although some of his colleagues predicted that genetics had little to do with the withering process, Pezzotti revealed that large numbers of genes are active during this period, including many that influence a wine’s taste and aroma. Amarone makers are now following the research with intense interest, he says.

The Vigna/Vigne and the IASMA teams are now on more cordial terms, but they do have some scientific disagreements. Both have found evidence of whole-genome duplications in vinifera’s past—a common feature of plant evolution in which new species arose after an ancestral plant accidentally duplicated its genome or hybridized with another to expand its gene set (see p. 481). Velasco and his colleagues argue that such an event happened relatively recently, after grapes had split off from the branches on which Arabidopsis and poplar belong. Vigna/Vigne, on the other hand, has concluded that the grape genome did not undergo any recent expansion. It instead suggests that vinifera derives from an ancient hybrid that once had six sets of chromosomes. Because each team used different strategies for discerning and dating duplications, says Velasco, "who knows who’s right?"

Indeed, Pé ended the Udine workshop with the reminder that having a grape genome—or two—in hand merely provides a foundation for future work. "Most of the data still have to be digested," he notes. A glass or two of vinifera’s valuable juices, perhaps a nicely aged Cognac, should speed the process.

-JOHN TRAVIS
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Science, Vol. 320, 25 april 2008

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