Term for a branch of molecular biology for researching the structure of the genome (the entirety of genetic material in an organism) and the function and interaction of genes (the hereditary factors localised on the DNA strand in the chromosomes of each cell nucleus). Genetic engineering or genetic engineering is the application of genetic methods in practice and usually involves the artificial manipulation of genes with the aim of introducing desired, previously non-existent characteristics, such as resistance to fungal diseases, into an existing organism through the technically feasible implantation of individual replicated foreign genes from foreign organisms into the genome of a variety or breed.
Since the invention of the PCR method (polymerase chain reaction) for the amplification of the smallest amounts of DNA in the 1990s, grape varieties can now also be clearly characterised, defined and recognised genotypically as varieties with the help of molecular genetic characterisation methods. This introduced the genotype into ampelography, which can help to clarify many unanswered questions as a supplement to traditional, visual phenotype-based ampelography. Each independent grape variety germinated from a seed is represented by an unmistakable genotype, which is characterised by the unique new combination of maternal and paternal genetic material during fertilisation and is present in the nucleus of each plant cell. Each grape variety resulting from sexual reproduction has an individual genetic pattern that distinguishes it from all other grape varieties.
For genotypic comparisons or the identification of grape varieties and the determination of the parent pair, the so-called genetic fingerprint is used, which can be created with a microsatellite analysis. Microsatellites are areas within the long DNA chain that are defined by molecular markers and can be found again, which have a different number of repeating base sequences. In a given double set of chromosomes (2 x 19 = 38), these sequences, which repeat twice, can vary in length between two homologous chromosomes as well as from grape variety to grape variety. These sequences can be copied and duplicated as individual fragments so that a specific characteristic is obtained from the length of these genome fragments.
Many such fragments from several gene loci, always present in pairs, result in a fragment pattern that is typical of the variety and characterises this variety, which can be compared with the fragment patterns of other grape varieties. This fragment pattern is, so to speak, a genetic fingerprint. The probability of two grape varieties having the same pattern is statistically around 1:6 million, which is a negligible probability given that there are only around 14,000 existing grape varieties and breeding strains. There is an agreement within the European grapevine breeding institutes that it is therefore sufficient for the genotypic characterisation of grapevine varieties to determine the genotype at six defined gene loci (microsatellites) in order to clearly define the profile of a variety.
Before the method of microsatellite analysis, it was only possible to compare and differentiate varieties by comparing the morphology (external appearance) of the vine. The characteristics typical of the variety were described and attempts were made to filter out features that allowed the varieties to be identified and differentiated. These morphological catalogues of characteristics describe the phenotype of the grape variety. Both approaches have advantages and disadvantages, but complement each other perfectly. Genetic analyses in the laboratory require only a small amount of genetic material as starting material, and the investigations are not dependent on external conditions such as season, plant age, state of health, virus status or growing conditions at the location, which can distort the phenotype (the appearance) beyond recognition.
Laboratory tests cannot simply be carried out blindly on thousands of vines in the field, as can be done quickly and at no extra cost with the trained eye of an ampelograph. It is also still easier to differentiate phenotypically between varieties that have emerged as mutant clones or somatic chimeras from vegetative propagation. This is because the colour mutants of Pinot Noir, namely Pinot Gris and Pinot Blanc, are strictly speaking not separate but different berry skin colour variants of the same grape variety. This is a specific mutation process that leads to the loss of the dark colour. Somatic chimeras such as Pinot Meunier (Black Riesling) or Garnacha Peluda are much easier and quicker to differentiate visually from the original Pinot Noir or Garnacha Tinta varieties due to their strong leaf hairiness than through complex genetic analyses.
Experience has shown that these six defined markers are sufficient for characterisation using microsatellite analysis. For the clarification of relationships or even the differentiation of clones and somatic chimeras within a variety, significantly more markers must be used. However, there is the rare exception that two externally different phenotypes (varieties) have the same genotypic profile of six markers. This can occur when a somatic chimera has branched off from a grape variety through mutations in specific cell layers. Varieties that have been created by self-crossing or backcrossing (mother with child) can also still be very similar. Especially in the case of sibling varieties with the same parents, this can sometimes lead to failure in purely visual identification attempts.
The genetic characterisation of grape varieties has been underway since the mid-1990s. As a basis for comparison, the fingerprints of several thousand varieties from the world's largest range of grape varieties were analysed at the Domaine de Vassal near Montpellier. A centralised European grape variety database is currently being developed (see also VIVC). In recent years, the parentage of hundreds of grape varieties has been determined or, in many cases, corrected in relation to the information frequently provided incorrectly by the breeders. Important ampelographers with regard to DNA analysis are Alain Bouquet, Dr Jean-Michel Boursiquot, Thierry Lacombe (France), Javier Ibáñez (Spain), Dr Erika Maul (Germany), Dr Carole P. Meredith (USA), Dr Ferdinand Regner (Austria), Anna Schneider (Italy) and Dr José F. Vouillamoz (Switzerland).
DNA analysis of the chloroplasts can also be used to check the direction of crossing (mother and father), as these special organ cells are only passed on from the mother variety. In 1997, Meredith and Bowers at the University of California succeeded for the first time in determining the parentage of a grape variety: Cabernet Sauvignon originated from Cabernet Franc (mother) x Sauvignon Blanc (father). Subsequently, some important Central European leading varieties such as Gouais Blanc (White Heunisch), Pinot and Savagnin(Traminer) were identified. The great age and widespread distribution of these varieties favoured the spontaneous cross-breeding and thus the emergence of many other grape varieties. Gouais Blanc/Heunisch, which was cultivated throughout Central Europe in the Middle Ages, has crossed into 80 grape varieties alone.
As with paternity testing, genetic fingerprinting can now be used for the first time to reconstruct ancestry, determine relationships or recognise hybrid crosses. However, this requires a comprehensive genetic analysis of at least 15 and up to 50 gene loci (markers). The Swiss biologist Dr José Vouillamoz believes that there should be at least 30 to 60 (35 for the identification of Cabernet Sauvignon). Only if the potential parents are known, for example through information provided by the breeder, would fewer be sufficient. As each child has inherited one chromosome from the father and one from the mother, each grape variety must also have one characteristic in common with the father variety and the other with the mother variety at a specific gene locus. This rule applies strictly, i.e. failure to fulfil the parentage criterion usually means the end of the parentage hypothesis. The more gene loci are included in the investigation, the more certain it is. However, 100% proof is not possible, as the hypothesis can only ever be positively tested at a few selected gene loci, but never at all of them.
If one of the parents cannot be identified because its DNA values have not yet been recorded and are not known, or because the variety may already be extinct), it is still possible to prove the relationships between parent plants and offspring of two different varieties. The phrase "not directly related" is often used in sources. This usually means that there is no parent-offspring relationship between the grape varieties under consideration, i.e. no "mother-father-child relationship". Direct relationships between grape varieties (i.e. "mother-father-child") can be clearly reconstructed with the genetic fingerprint. However, even with siblings or "distant relatives" such as "aunts", "uncles" or "cousins", the relationship is hardly obvious from the genetic profiles.
For some time now, manipulative genetic engineering has also been used to breed modern grape varieties with specific resistance characteristics. The international project IGGP (International Grape Genome Programme) was launched in this regard. In tissue culture suspensions, foreign resistance genes with desired properties can be introduced into the genome of plant cells of certain conventional varieties. These include, for example, resistance to fungal diseases and viruses or to animal pests such as the dreaded phylloxera. The genetically manipulated cells can be used to regenerate grapevine plants, which this time do not represent a new variety, but have essentially retained the genetic characteristics of the selected variety, but with the addition of the "improving" foreign genes.
In practice, however, the hoped-for effects usually failed to materialise or were unsatisfactory. In the case of woody plants, it is obviously not enough to simply insert individual genes into the genome somewhere and hope that such complex and environment-dependent characteristics as fungal resistance will develop. The extent to which this technology will lead to success and practical application in the vine, measured against the effort and costs involved, is not foreseeable at the moment and is also not in demand. In any case, the introduction of genetically manipulated grape variety clones would lead to a further impoverishment of varieties and a further reduction of clone diversity to just a few clones. Whether these artificially manipulated grapevine plants, whose genetic balance has been disturbed, will prove to be stable in the long term when propagated vegetatively on a large scale cannot yet be seriously answered.
Today, DNA analyses in wine are used with great success to detect wine adulteration (pantscherei). In 2002, researchers from the INRAE Institute in Montpellier developed a method for DNA isolation from wine and must that can be used to distinguish between pure quality wine and cheaper blends. The method is so advanced that the varietal fingerprint of unfiltered wine can be used to determine whether a wine has been pressed from the specified grape variety or whether must from other grape varieties has been added. It is not (yet) possible to quantitatively determine the proportions of the different varieties in the wine, but it is possible to determine which varieties are present in the wine.
So far, the INRAE team has identified the genetic profiles of 600 grape variety wines. However, the method does not yet work for bottled wines because a large proportion of the DNA is filtered out during the filtering process and the residual concentration of DNA in the finished wine is too low for genetic analysis. However, research is continuing intensively. The PNA-FISH method can be used to detect microbiological wine faults, such as horse sweat. See also under Nuclear Magnetic Resonance and Certification of vines.
For more information on this topic, see also Ancestry, Flowering, Chromosome, DNA, Vine family tree, Vine systematics, Determination of grape varieties and Taxonomy as well as lists of relevant keywords under Vine area and Grapevine.
Pictures: Ursula Brühl, Doris Schneider, Julius Kühn Institute (JKI)
For my many years of work as an editor with a wine and culinary focus, I always like to inform myself about special questions at Wine lexicon. Spontaneous reading and following links often leads to exciting discoveries in the wide world of wine.
Dr. Christa Hanten
Fachjournalistin, Lektorin und Verkosterin, Wien