Getting deep into the science of Chenin Blanc clones

By Jamie Goode | 21st February 2023

Chenin Blanc is remarkable grape variety, and it’s one that’s hugely important to the South African wine industry. Genetics show that Chenin is a cross between two less-known grape varieties, Savagnin and Sauvignonasse, but this cross was made a long time ago. A new grape variety is produced when vines have sex and a seed is produced, which is then planted and grows up to make a vine. And Chenin was first mentioned in France in 845 CE. This means that every single Chenin Blanc vine has some from a single seed that was planted at least 1100 years ago, and probably earlier.

By the 16th Century it had become a major grape in France. It was first planted in South Africa in 1655, and became successful there, to the point that it now has a lot more Chenin Blanc than France. Worldwide there are some 35 000 hectares of Chenin, with 9700 hectares in the Loire (of which two-thirds is used to make sparkling wine), while South Africa has 18 600 hectares, and it represents 23% of the harvest.

This separation is interesting, because it gives an opportunity to compare the genetics of what for a long time have been two largely separated populations of the same variety. It also raises the issue of ‘what is a clone?’ It’s a term widely used in the wine world to describe stable phenotypic differences among plants of the same variety, that have been selected for and then propagated. But it’s a slightly misleading term. ‘I’m a professor of genetics at Stellenbosch University,’ says Professor Johan Burger who has been involved in a joint Loire-South Africa project looking at clonal differences in Chenin, ‘and the word clone implies genetically identical: to use clone for differences is contradictory.’

Grape varieties that have been around for a long time tend to generate many clones. Some think that varieties such as Pinot Noir must be genetically more prone to mutation because there are many Pinot Noir clones: instead, it’s much more likely to be because Pinot Noir is an old variety, and has had more time to mutate, coupled with the fact that there’s a lot of it grown (the more individuals, the more likely mutations occur). Some clones may also be a result of mistaken identity: there might be four names for the same one.

One of the problems in this field is that scientists don’t have reproducible and accurate methods to identify clones. ‘This is a problem,’ says Burger, ‘especially for viticulturists and nursery people.’ With the current unofficial moratorium on genetic modification in grape vines, clones remain the only acceptable and realistic way to introduce genetic variability into wine cultivars. ‘Nowadays there are amazing technologies for doing precise genetic modification, but in the wine world when will this be acceptable?’ asks Burger.

Every time a cell divides by mitosis it produces a new copy of its genetic material. There are mechanisms in place so that this copying is accurate, but it isn’t perfect, and because there are so many cell divisions taking place in a plant, occasionally errors (mutations) creep in. There are also mutations induced by environmental influences: for example, ultraviolet light is able to cause mutations in DNA. The vast majority of these mutations are either neutral (for example, falling in genetic code outside of genes) or harmful, but very occasionally a mutation might be positive. This is incredibly rare, but we are dealing with huge numbers, so it can occur. And not all of the mutations or changes are about changing the genetic code itself. The sorts of mutations that induce clonal differences include single nucleotide polymorphisms (SNPs, where one base pair is changed; these are also called sequence variations), indels (insertions or deletions of bits of DNA, also known as structural variations) or the insertion or excision of mobile genetic entities called transposable elements (such as retrotransposons or transposons). There are also non-genetic changes such as epigenetic modifications, which alter the way DNA is read and which can also be inherited. This all sounds quite technical, but genetics is a complicated science!

In France, 95% of plantings of Chenin are from clonal selections. There are 14 clones certified. South Africa has 12 registered clones, and the two countries share four of these.

How can different varieties be separated? The first is by the old science of ampelography – the appearance of the vines. This is how Professor CJ Orffer, Head of Viticulture at the University of Stellenbosch, was finally able to show that South Africa’s Steen was actually Chenin Blanc, in 1963. It’s strange to think that before this South Africa didn’t know the actual identity of its key grape. More recently, molecular biology techniques such as SSRs (simple sequence repeats) and microsatellites have been used to tell varieties apart. But when it comes to separating clones of the same variety, the genetic differences are so minute that the marker-based DNA techniques just don’t work.

Burger’s study looking at Chenin clones, in conjunction with a French research group led by Patrice This, is instead using next generation sequencing to try to tease apart clones, by looking at the entire genome without the use of predefined markers. Similar work has been done with Chardonnay in Australia, where 1620 markers can distinguish 15 Chardonnay clones and have confirmed Gouais Blanc and Pinot Noir as the parents. With Zinfandel in California researchers there have assembled the genome and are sequencing 15 clones. In Spain they are working on Malbec in a similar way. The aims of this Chenin project are to study the accumulated mutations in the different clones, and then develop a sort of genetic assay.

Patrice This, the French partner in the project, started working on grapevine genetic diversity almost 30 years ago, and he’s keen to be involved in a project comparing the evolution of clonal diversity in both France and South Africa. ‘Clonal variation only occurs after extreme replication of material,’ he says, ‘when you can see morphological differences due to sequence variation or epigenetic modifications.’

The grapevine reference genome is PN40024 which was sequenced in 2007 at great expense. Fortunately, sequencing techniques have moved on a lot since then, and now it is much cheaper and easier to sequence the entire DNA of the grapevine. The way this is being done in this project is to extract DNA from the leaves (this is easier), and then cut it into long fragments using Sequel II (mean size 20 000 base pairs). These fragments are then sequenced, and are aligned to their correct spots using the reference genome.

This is still quite a computational task, because the grapevine genome consists of 500 million base pairs spread over 19 chromosomes. The chromosomes come in pairs, and so there are two haplotypes that have to be aligned. This is because genes come from both parents, Savagnin and Sauvignonasse. This creates a reference genome to compare clones with. The next step is to sequence the clones for comparison, and this time the DNA is extracted and then sequenced in short fragments using Illumina. The mean size of these is 150 base pairs, so there are a lot of them. These are then aligned on the reference that has just been made, and the clones are compared at different positions.

The first clone that they sequenced is the most important one, 220. This is work still ongoing. They need to finish this reference genome, then annotate the genes (mark where they are). When this is done, one thing that could be done is to look in the berries at which genes are expressed, and then look at the variation in these genes in the clones. This variation, in the grapes themselves, is likely to be significant. And then, after looking at the genetic differences, it will be time to begin exploring epigenetic differences, too. It’s a complex business, but it’s important work. Understanding clones at this level helps with finding new vine material that may help to future proof South Africa’s vineyards in a time of climate change.