Microsatellite DNA Analysis, Shaping Genetic Understanding of Grapes
By Rob Tebeau
Originally published in Palate Press: The online wine magazine
Many wine enthusiasts are aware that cabernet sauvignon is the result of a crossing that happened long ago between sauvignon blanc and cabernet franc, but have you ever wondered just how we know that? Why do we know the parents of some grapes and not others? Many are also aware that the grape known as zinfandel in California is genetically the same as the grape known as primitivo in southern Italy. Given how similar many vines look even to expert viticulturists, how are we able to make a claim like that with such certainty?
Most wine books and articles gloss over the method itself and inform the reader that this knowledge was obtained using “DNA analysis,” which is true, but I’ve never really found this to be a satisfactory explanation. When I started to look into it, I found that the techniques that scientists were using for grapes were the same as the techniques used by crime labs and hospitals for human beings. I think that the interested wine drinker should have some option for learning about this stuff between the phrase “DNA analysis” and an article in a scientific journal, and I hope the information below can help to fill that gap.
DNA, the genetic material found in all organisms, is composed of intertwined chains of molecules in a twisting pattern known as a double helix. The individual molecules that make up a strand of DNA are known as nucleotide bases and there are four of them. There’s guanine (G), cytosine (C), adenine (A), and thymine (T), and the various patterns that these four molecules form are what make up the genetic fingerprint of every living thing on earth. One function of DNA is to encode the information needed for cells to synthesize proteins, but only certain segments of cellular DNA perform this function. Those segments are called genes and their action or inaction forms the basis for all life on Earth.
Some of the DNA that does not encode proteins has other, more complicated functions, but there are some regions of DNA whose purpose is unclear. There is quite a bit of “filler” throughout the DNA of an organism, but there are certain sections where a repeating pattern of nucleotides develops, and these sections are known as microsatellites. Imagine that the series of letters below is a part of a DNA strand:
That repetitive section in the middle with all the “CT” segments is a microsatellite, and there are thousands of them spread throughout an individual’s DNA. Microsatellites tend to be located in the same place on the genome across individuals, but vary between them in the number of repeat sequences. For example, one person might have 140 repetitions of CT at this spot, but another might have 150. The possibility exists, though, that you might have 140 repetitions at that spot as well, so in order to differentiate between us, we would need to look at several different microsatellite sites. If you look at enough sites, eventually the chances that any two individuals would match at every single site get obscenely remote, meaning the only reasonable conclusion you can draw is that they’re from the same individual.
Sequencing, or reading the DNA in these microsatellite positions, is how we know that zinfandel and primitivo are the same grape. When analyzing the DNA of each plant, scientists found that the two plants matched at site after site and concluded that they were the same. This is the same technique that forensic scientists use to analyze crime scene data. If police and forensic investigators find DNA at a crime scene, they can look at the microsatellite data of different suspects to determine which, if any, of them match up at the different microsatellite regions.
The same technique is used to determine grape parentage as well. Humans and grapes are diploid organisms, which means they have two sets of chromosomes. Each individual, then, has two different copies of each microsatellite site as well, and on one copy the repeat may happen 165 times while on the other copy it may happen 170 times. One of the purported parent grapes must then have that same repeat of 165 on one of their sets of DNA, while the other parent must have 170. The same rules of probability apply, so multiple microsatellite regions are used (usually at least 25) to identify the ancient parents of grapes. If it is found that cabernet sauvignon matches cabernet franc at every site on one of its sets of chromosomes and cabernet sauvignon matches sauvignon blanc at every site on the other set, then cabernet franc and sauvignon blanc are the parents of cabernet sauvignon. This is exactly the same technique that is used in the paternity suit cases made so famous on trashy daytime talk shows.
Determining grape pedigrees isn’t easy work. For starters, many of the grapes that we currently enjoy have been around for hundreds of years and in that time period, a lot of different grape varieties have disappeared. Many of these extinct varieties were certainly parents to some of the grapes we know today, and once they’re gone we have no way to access their genetic information to see where they might fit in a grape’s family tree. Secondly, in order to fully construct a pedigree, you need all three members of the family. If you have two grapes that each share half of their genetic material, there’s no way to tell which one passed those genes on to the other. The third member provides the information needed to confirm which is the parent and which is the offspring.
Genetic analysis is revolutionizing the way that we think about grapes and how they relate to one another. Before this technology emerged, it was impossible to know for sure about the parentage of a grape unless it was the result of a deliberate crossing and the breeder responsible took reliable notes. DNA analysis gives us incredible insight into the connections between vines that we might not usually think of together. Further, in the past the differentiation of grape varieties and the assignment of synonymies was accomplished by examining individual vines and comparing their leaf sizes and shapes, grape cluster sizes and shapes and various other physiological characteristics, a practice known as ampelography. It is an inexact science at best, but modern DNA analysis gives precise answers to questions about identity for vines. We can see how geographical differences can cause physiological changes in vines that are genetically identical, giving us a sense of how vines can adapt to new places over time. It’s a wonderful example of how science can expand and deepen our understanding without taking any of the magic away from the process of enjoying wine.
Sefc, K.M., Pejic, I., Maletic, E., Thomas, M.R., & Lefort, F. (2009). Microsatellite markers for grapevine: tools for cultivar identification & pedigree reconstruction. In K.A. Roubelakis-Angelakis (ed.), Grapevine Molecular Physiology & Biotechnology, 2nd ed. pp. 565-596.
Pinder, R.M., & Meredith, C.P. (2003). The identity and parentage of wine grapes. In M. Sandler & R. Pinder (eds.) Wine: A Scientific Exploration. pp 260-273.