Table-grape breeding in the genomic era

Genome editing has the power to revolutionise cultivar development, but South African growers are at risk of being left behind.

By Anna Mouton

Dr Justin Lashbrooke of the Department of Genetics at Stellenbosch University calls himself a molecular physiologist. “I care about the plant’s physiology, but at the level of DNA and proteins and what happens inside the cell,” he explained. He has been teasing out the genetics underlying cuticular waxes in the hope of decoding the problem of berry cracking. Although environmental conditions and viticultural practices contribute to berry cracking, the variation in cultivar susceptibility suggests that genetics play a significant role. For Lashbrooke, berry cracking illustrates the potential of new genome editing technologies to transform the table-grape industry. But he warned that current South African legislation impedes growers’ access to genome-edited cultivars.

On the surface
The cuticle is a water-resistant layer covering the plant’s surface. It consists of wax crystals embedded in a mesh-like polymer framework of waxy cutin. “Waterproofing was the main reason why the cuticle evolved. But subsequently, so did many other features,” said Lashbrooke. “Because the surface is where the plant interacts with its environment, many of these features are to combat pests and diseases.” Unfortunately, grapevine domestication has often prioritised eating quality at the expense of this protection. Hence, tasty cultivars tend to be more vulnerable to fungal infections than unpalatable wild grapes. The rapid fruit expansion of modern large-berried cultivars probably also predisposes them to cracking. “Berries, and fruits in general, must be able to deal with rapid fruit expansion,” said Lashbrooke. “But we’ve forced grapevines to change, and we keep forcing them to be the ideal plants for our growers and markets, and this often has consequences.” His research group assessed cuticle strength and elasticity to determine a berry-cracking score for 73 table-grape cultivars. They found that elasticity and water resistance played important roles in cracking, whereas skin strength and berry size weren’t as important. Further experimentation demonstrated that elasticity increased, and cracking susceptibility decreased when berries had more structural cutin with less embedded wax. Cultivars differed in their cuticular composition, providing more evidence for a link between berry cracking and genetics.

Inside the cell
Once Lashbrooke identified the importance of cuticle elasticity, he started hunting for the genes that control wax production. “The cuticle is much more complex than just a wax layer,” he said. “It’s hugely diverse.” His group collaborated with Phyllis Burger of the ARC Infruitec-Nietvoorbij, who has crossed a waxy wine grape with a glossy table grape. Lashbrooke analysed the resulting population to identify genes involved in synthesising 30 different types of cuticular waxes. “These compounds are made in metabolic pathways inside the cell,” he said. “Each step in the pathway is controlled by a specific gene. We must find those genes if we want to modulate their effects.” Interestingly, he reported that a waxier appearance doesn’t mean a berry has more wax in its cuticle. The difference between a waxy and a glossy berry lies in the type rather than the amount of cuticular waxes. So far, his group has located 31 regions across the grapevine genome where key genes for wax synthesis are likely to be found. They have identified a previously unknown gene crucial for producing triterpenes, the most common substances in waxy berry coatings. “We’ve characterised this gene to quite a large extent,” said Lashbrooke, “so that it can now be used in breeding.” The gene’s relationship to berry cracking and other traits can be explored by studying gene variations in different cultivars.

A genomics crash course
Lashbrooke explained genomics using the metaphor of the genome as a library. Each bookshelf in the library represents a chromosome, and each book represents a gene. “The books are manuals – instructions of how to build the tools that are proteins,” he said. “There are also other manuals that provide instructions on which tools to use.” Grapevines have 19 chromosomes, which are duplicated, so there are two copies of each gene. The two copies are manuals for building the same, but not identical, tools. Usually, although both tools work, one might be better than the other under specific conditions. Breeders try to equip new cultivars with valuable tools such as mildew resistance by crossing parents that have manuals for making those tools. Their offspring inherit one set of manuals from each parent, and breeding continues with progeny that inherited advantageous characteristics. “The problem with crosses is that you may only have wanted one book, but you brought the whole library,” said Lashbrooke. “You wanted a mildew-killing protein, but you also get an undesirable trait.” Breeders counter this by back-crossing to try and dilute the bad genes while keeping the good ones. Consequently, conventional breeding takes a long time. Genomics can facilitate conventional breeding through techniques such as marker-assisted selection, which helps identify genetically desirable parents and offspring based on genetic analysis. However, this still requires making crosses to obtain progeny. Modern technology offers a better route to optimising cultivar genetics: genome editing.

Designer grapevines
 “Genome editing can be used for precise modification of the genome,” said Lashbrooke. The dominant technology for genome editing is an enzyme called CRISPR-Cas9 that recognises specific DNA sequences. “With CRISPR, you can edit the specific gene you want to change, and you can do it once and have the cultivar you want,” elaborated Lashbrooke. “You don’t have to make backcrosses for multiple generations.” For example, you could theoretically change a seeded cultivar into a seedless one without changing any of the cultivar’s other properties. Genome editing differs from genetic modification. To return to the library analogy, genome editing involves tweaking a book already in the library, whereas genetic modification involves introducing a book from an entirely different library. To create a genetically modified organism or GMO, researchers transfer DNA from a different species, thereby incorporating a desirable trait that couldn’t be obtained through conventional breeding. With genome editing, as with traditional breeding, researchers work with the DNA already present in a species. Worldwide, products from genome-edited crops are already on supermarket shelves. Nearly everywhere, legislation is either open to gene editing or becoming more so – except in SA. “In South Africa, if you develop a crop through genome editing, it will have to go through the same regulatory process as a GMO,” said Lashbrooke. “That is a huge financial burden for products developed here, and our market isn’t big enough to justify doing all the regulatory trials for products developed in, for example, the US.” Lashbrooke’s concern is that the restrictive legislation in SA will block our table-grape growers from accessing new cultivars that are the lifeblood of the industry. “Industry bodies are petitioning our government,” he concluded. “We’ve been at the mercy of an inactive government, but we have a new agricultural minister, so things may move more quickly.”