Using Microsatellite Markers to Determine Genetic Relatedness of Grape Varieties

A “Who’s Your Daddy” post on a whole other level (the Daddy of Who’s Your Daddy?), today we’re going to talk a little bit about genetics and genetic relatedness of grapes. Understanding the molecular make-up of grapes can be very helpful and critical in relation to accurately identifying a cultivar for adequate management of the plant as well as proper identification of the cultivar for distribution purposes throughout various wine regions of the world.

The use of molecular markers in determining genetic differences between organisms had been used in other systems, and could also be extremely helpful in examining the genetic differences between grape cultivars that are under some question as to whether or not they are the variety they are assumed to be, or if one doesn’t know which variety it is to

Photo By Maggie Bartlett, NHGRI [Public domain], via Wikimedia Commons

Photo By Maggie Bartlett, NHGRI [Public domain], via Wikimedia Commons

begin with. In fact, the use of microsatellite markers (just a fancy term for molecular markers) have already been shown to be beneficial in determining the identities of some grape genotypes (genetic make-up), as well as the genetic diversity of different cultivars, rootstocks, and hybrids.

The purpose of today’s study (I swear, I’m going to try and keep it brief today!) was to evaluate the unique identities of several different types of grapes (wine, table, and juice grapes) and to determine the genetic diversity of the grape varieties studied.

Brief (….and I mean brief!) Methods

This study was performed at the Punjab Agricultural University in Ludhiana (Punjab), India. As an aside: I love seeing research out of non-traditional grape growing regions, as to me it shows that there are other areas of the world that love wine than the more obvious ones!

The research group studied the genetic diversity of 27 genotypes of table grapes, wine grapes, and grapevine rootstocks. DNA was extracted from each genotype and molecular analysis (microsatellite amplification) was performed.

Results

• Out of the 27 grape genotypes studied, a total of 375 alleles were found. Very basically, this indicates a lot of genetic variability!
o       The wine grape Chardonnay had the most number of alleles present in its DNA, while rootstock 1616C had the fewest number of alleles.
• Two microsatellite markers were found such that when used in combination, they could identify genetic differences between all 27 grape genotype samples.
o       The two markers found that could differentiate between grape genotypes were: VMC6G8 and VMC8G9.
o       These two markers were found to be easy to analyze and showed strong signals for all grape genotypes.
•  When creating the genetic relatedness tree from the microsatellite data, results showed 3 distinct clusters of relatedness.
o       Cluster 1: 10 Vitis vinifera varieties were found in this cluster. This cluster was further subcategorized into two subgroups of relatedness (i.e. those in the same subgroup or cluster were more related to one another than those in different clusters or subgroups).
                 Subgroup I: 4 table grape varieties made up this group (“Cardinal”, “Muscat Hamburg”, “A35-1”, and “Convent Large Black”), as well as 3 wine grape varieties (“Chasan B”, “Portan”, and “Madeline Angevine”).
                 Subgroup II: The juice grapes “H27” and “Pusa Navrang” were found along with the wine grape “Cinsault”.
o       Cluster 2: This was the largest of the clusters (i.e. the most genetic diversity stemming from common ancestry) and contained two subgroups.
                 12 of the 27 grape genotypes were found in this group.
                 Subgroup I: All rootstocks were found in this subgroup.
                 Subgroup II: Both table and wine grapes were found in this subgroup.
o       Cluster 3: There were 3 wine grape varieties and 2 table grape varieties found in this cluster, and all had Vitis vinifera origins and contained two subgroups.
                 Subgroup I: This subgroup contained all the wine grapes from this cluster (“Shiraz”, “Chardonnay”, and “Cabernet Sauvignon”).
                 Subgroup II: This subgroup contained all of the table grapes from this cluster (“Beauty Seedless”, and “Sharad Seedless”).

Figure 8 from Singh, et al, 2013.  Roman numerals on the right side indicate the three clusters as described in the text.

Figure 8 from Singh, et al, 2013. Roman numerals on the right side indicate the three clusters as described in the text. (Click to enlarge)

What does this all mean?

The results of this study were able to clearly differential the rootstocks from the grape varieties, however, the methods used were not able to differentiate between wine grapes, table grapes, and juice grapes, with the exception of Cluster 3 which did successfully differentiate between wine and table grapes. In general, the genetic relatedness tree was shaped based on the parental Vitis species of the grape variety in question. In other words, an individual grapes place on the genetic relatedness tree was more or less determined by who its parents were in relation to the parents of all the other grape varieties.

Based on the methods used in this study, and likely due to the small number of samples compared, it seems as though the technique was not sensitive enough to detect major differences in genetic make-up between wine, table, and juice grapes. It is somewhat surprising that there was not a clear separation, as all three types of grapes have been artificially selected for and thus a particular genetic make-up was, for a lack of a better phrase, “forced” upon them. For wine grapes, those grapes with higher sugar levels were artificially selected for, while for table grapes, those grapes that had

Photo By not identified, creators were Mary (1817-1893) and Elizabeth (1823-1873) Kirby; publisher Thomas Nelson & Sons [Public domain], via Wikimedia Commons

Photo By not identified, creators were Mary (1817-1893) and Elizabeth (1823-1873) Kirby; publisher Thomas Nelson & Sons [Public domain], via Wikimedia Commons

larger berries were artificially selected for. Theoretically, the genetic make-up of these individuals should be separable using Principle Components Analysis as they did in this study. However, since there were only 27 varieties studied, I feel as though perhaps the sample size was simply too low and therefore the experiment did not have enough power to detect any differences in the first place.

It was interesting that Cluster 3 did show complete separation between wine and table grapes, and it showed a clear separation between rootstocks and grapes, which makes me think that using microsatellites as an easy and fast method for identifying grape variety might be a nice way to quickly identify a variety for authenticity or management reasons. I’d like to see the study repeated with many more varieties, as well as varieties from different corners of the wine world. I’m curious to know how the genetic make-up of a Cabernet Sauvignon in Australia has changed from a Cabernet Sauvignon in California (or anywhere really), simply based on geographic isolation. Many other species show genetic drift (i.e. changes in genetic make-up) when separated geographically, so I would be very curious how the movement of grapes around the world over time has diversified the genetic codes of what was originally the same variety from the same source.

What do you all think of this study? How would you like to see it improved upon? Can you think of any other application of microsatellite/molecular markers in viticulture and/or winemaking? Please feel free to leave your comments!

Source: Singh, A., Kumar, K., Gill, M.I.S., Chhuneja, P., Arora, N.K., and Singh, K. 2013. Genotype identification and inference of genetic relatedness among different purpose grape varieties and rootstocks using microsatellite markers. African Journal of Biotechnology 12(2):134-141.

What do you think about this topic?