High Resolution Melting (HRM) analysis on VviDXS to reveal muscats or non-muscats among autochthonous Greek wine producing grape varieties

摘要

Muscat flavor in grapes is associated with a substitution of a Single Nucleotide Polymorphism (SNP) located at position 1822 (SNP1822G>T) within the coding sequence of the VviDXS gene. Various methods, including the use of High Resolution Melting (HRM) analysis, have been suggested to discriminate different SNP allelic states including the molecular discrimination of the muscat from the non-muscat grape varieties, thus providing the ability to minimize lengthy grape breeding programs when selecting for grape muscat flavor before the fruit maturity stage. HRM analysis on the SNP1822 was performed on a group of 128 wine producing grape varieties in order to separate the muscat from the non-muscat genotypes before they are used for further breeding activities. This approach could be used either as a single-step prescreening method or in accordance with recently published methodology to elucidate on varietal characterization and authentication as these are important requirements concerning nurseries, growers and winemakers. IntroductionWine aroma is the final and combinatorial outcome of many genetic and environmental factors, and represents one of the most important and complex characters that highly contribute to the sensory experience and the market value of the product.The muscat aroma of many grapevine varieties is due to the occurrence of high levels of terpenoid compounds, such as linalool, nerol, α-terpineol, and geraniol. The 1-deoxy-D-xylulose-5-phospate synthase (VviDXS) gene that converts glyceraldehyde 3-phosphate and pyruvate into 1-deoxy-D-xylulose-5-phosphate (DXP) has recently been identified as a functional molecular marker associated with the occurrence of muscat flavor in grapevine (Battilana et al. 2009). Three alternative methods have been developed allowing for large-scale screening and facilitating prompt germplasm characterization in grapevine breeding based on the occurrence of four functional SNPs (SNP1784T>C, SNP1822G>T, SNP1917A>G, and SNP1922C>T) on the VviDXS gene (Emanuelli et al. 2014).High-resolution melting analysis (HRM) is a cost-effective and less time-consuming method that could detect polymorphisms even at the single nucleotide level measuring the rate of double-stranded DNA dissociation to single-stranded DNA with increasing temperature. HRM has been performed in the successful identification of plant varieties in other species (Ganopoulos et al. 2013).Here, we focused on SNP1822G>T, introducing an HRM approach in order to molecularly predict the muscat flavor and also detect hidden muscat potential of wine producing varieties. We suggest that this approach could be used either as a single-step pre-screening method or as an auxiliary method to discriminate the muscat from the non-muscat varieties within the Greek grape germplasm.Materials and methodsYoung leaves from a total of 122 autochthonous wine producing Greek varieties were collected and stored at -80°C until analyzed (Table 1). In addition, samples from 6 international varieties were collected and treated similarly, totaling 128 samples. Genetic material is maintained in three collections: i) the ampelographic collection of the Greek Gene Bank, at the Institute of Plant Breeding and Genetic Resources (GGB) of the Hellenic Agricultural Organization 'Demeter' (hereafter HAO-D) at Thermi (Thessaloniki, Greece), ii) the ampelographic collection of the School of Agriculture of the Aristotle University of Thessaloniki (hereafter AUTh collection) (Merkouropoulos et al. 2015), and iii) the grapevine collection of HAO-D at Ampelouzos (Crete, Greece). All DNA isolations were performed using the NucleoSpin Plant II Kit (Macherey Nagel, Duren, Germany) according to the manufacturer’s instructions.Table 1. HRM genotyping data relating to the 128 Muscat and non-Muscat grapevine varieties analyzed used in this study.View popupExpand inlineCollapse inlineNoAccession numberVariety nameMaintained atEmpirically known muscatsHRM profileSNP1822G>TSequencing confirmationof position 182211501NegoskaHAO-D Thermi-Non-muscat-?21502RoditisHAO-D Thermi-Non-muscat-31503AtesaHAO-D Thermi-Non-muscat-41504PamidiHAO-D Thermi-Non-muscat-51505FartsaloHAO-D Thermi-Non-muscat-61506XanthofileroHAO-D Thermi-Non-muscat-71507Moschofilero CampaHAO-D Thermi-Non-muscat-81508Moschofilero ProskinitariHAO-D Thermi-Non-muscat-91509Mavro KalavritinoHAO-D Thermi-Non-muscat-101510ZalovitikoHAO-D Thermi-Non-muscat-111511MavrokorakasHAO-D Thermi-Non-muscat-121512KoumariHAO-D Thermi-Non-muscat-131513TsampatoHAO-D Thermi-Non-muscat-141514PronikoHAO-D Thermi-Non-muscat-151515Mavro TraganoHAO-D Thermi-Muscat-161516Aidani MavroHAO-D Thermi-Non-muscat-171517PiperionosHAO-D Thermi-Non-muscat-181518KarapapasHAO-D Thermi-Non-muscat-191519ParginoHAO-D Thermi-Non-muscat-201520Kahpetsi OoidesHAO-D Thermi-Non-muscat-211521AraklinosHAO-D Thermi-Non-muscat-221522TenedioHAO-D Thermi-Non-muscat-231523RomeikoHAO-D Thermi-Non-muscat-241524Roditis GalanosHAO-D Thermi-Non-muscat-25152