Dual domestications and origin of traits in grapevine evolution.

We elucidate grapevine evolution and domestication histories with 3525 cultivated and wild accessions worldwide. In the Pleistocene, harsh climate drove the separation of wild grape ecotypes caused by continuous habitat fragmentation. Then, domestication occurred concurrently about 11,000 years ago in Western Asia and the Caucasus to yield table and wine grapevines. The Western Asia domesticates dispersed into Europe with early farmers, introgressed with ancient wild western ecotypes, and subsequently diversified along human migration trails into muscat and unique western wine grape ancestries by the late Neolithic. Analyses of domestication traits also reveal new insights into selection for berry palatability, hermaphroditism, muscat flavor, and berry skin color. These data demonstrate the role of the grapevines in the early inception of agriculture across Eurasia.

Morphological Traits Evaluated with Random Forest Method Explains Natural Classification of Grapevine (Vitis vinifera L.) Cultivars.

There are hundreds of morphologic and morphometric traits available to classify and identify grapevine (Vitis vinifera L.) genotypes, while statistical evaluation of those has certain limitations, especially when we have no information about the traits that are discriminative to a certain sample set. High numbers of investigated characters could cause redundancy, while reducing those numbers may result in data loss. Grapevine is one of the most important horticultural crops, with many cultivars in production. The characterization of the genotypes is of undeniably high importance. In this study, we analyzed a dataset of scientific and historical importance with 125 morphological traits of 97 grapevine cultivars described by Németh in 1966. However, the traits are not independent in a set of a large number of categorical traits with too few cultivars. Therefore, the number of traits was first reduced using a simple and effective algorithm to eliminate traits with redundant information content using the asymmetric measure of association Goodman and Kruskal's λ. We reduced the number of traits from 125 to 59 without any information loss. For the classification, we applied a random forest (RF) method. In this way, 93% of the cultivars were correctly classified using only four traits of the data set. To our knowledge, only a few studies applied a trait elimination algorithm similar to ours in ampelography that can be used for other biological data sets of similar structure. The classification results give a morphological explanation to several cultivars from the Carpathian Basin, a territory where all three Vitis vinifera L. geographical groups, occidentalis, orientalis and pontica, are represented. We found that the information-loss-avoiding data reduction method we applied in our study solved the redundancy-caused interdependencies and provided a suitable dataset for classifying grapevine genotypes. For example, this method may successfully be applied in digital image analysis-based traditional morphometric investigations in ampelography.

Differentiation of grapevine (Vitis vinifera L.) conculta members based on molecular tools.

Twenty-seven grapevine (Vitis vinifera L.) varieties within 12 putative berry colour variation groups (conculta) were genotyped with 14 highly polymorphic microsatellite (simple sequence repeats (SSR)) markers. Three additional oligonucleotide primers were applied for the detection of the Gret1 retroelement insertion in the promoter region of VvMybA1 transcription factor gene regulating the UFGT (UDP-glucose: flavonoid 3-O-glucosyltransferase) activity. UFGT is the key enzyme of the anthocyanin biosynthetic pathway. SSR results proved that the analysed cultivars can be grouped only into nine concultas, the other three putative berry colour variant groups consist of homonyms as a consequence of misnaming. In the case of Sárfehér-Sárpiros, Delaware red-Delaware white and Járdovány fekete-Járdovány fehér, it was attested that they are not bud sports, but homonyms. Some conculta members could be differentiated according to the presence or the absence of the Gret1 retroelement (Chasselas, Furmint and Lisztes), while others, Bajor, Bakator, Gohér and Traminer conculta members, remained indistinguishable either by the microsatellites or the Gret1-based method.

Functional analysis of the grapevine paralogs of the SMG7 NMD factor using a heterolog VIGS-based gene depletion-complementation system.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that identifies and eliminates transcripts having a premature translation termination codon (PTC). NMD is also involved in the control of several wild-type mRNAs. The NMD core machinery consists of three highly conserved NMD factors (UPF1, UPF2 and UPF3) and at least one less conserved 14-3-3-like domain containing protein (SMG7). A PTC is identified by UPF factors, and then SMG7 triggers rapid transcript decay. UPF factors are generally encoded by a single gene, whereas SMG7 has duplicated several times during evolution. Recently it was reported that the plant SMG7 is autoregulated through NMD and that SMG7 has two relatively divergent paralogs in dicots, SMG7 and SMG7L. In mammals all three SMG7 related genes (SMG5, SMG6 and SMG7) are essential in NMD, so we hypothesized that in plants the SMG7 and SMG7L duplicates may also play distinct roles in NMD. To test this possibility, we have analyzed the evolution and the function of plant SMG7 homologs. We show that SMG7L is not required for plant NMD. Interestingly, we found that the grapevine and poplar genomes contain two quite divergent SMG7 paralogs which may have derived from an ancient duplication event. Using heterolog depletion/complementation assays we demonstrate that both grapevine SMG7 copies retained the complete NMD activity and both of them are under NMD control, whilst SMG7L has lost NMD activity and NMD control.

Deep sequencing analysis of viral short RNAs from an infected Pinot Noir grapevine.

Virus-derived short interfering RNAs (vsiRNAs) isolated from grapevine V. vinifera Pinot Noir clone ENTAV 115 were analyzed by high-throughput sequencing using the Illumina Solexa platform. We identified and characterized vsiRNAs derived from grapevine field plants naturally infected with different viruses belonging to the genera Foveavirus, Maculavirus, Marafivirus and Nepovirus. These vsiRNAs were mainly of 21 and 22 nucleotides (nt) in size and were discontinuously distributed throughout Grapevine rupestris stem-pitting associated virus (GRSPaV) and Grapevine fleck virus (GFkV) genomic RNAs. Among the studied viruses, GRSPaV and GFkV vsiRNAs had a 5' terminal nucleotide bias, which differed from that described for experimental viral infections in Arabidopsis thaliana. VsiRNAs were found to originate from both genomic and antigenomic GRSPaV RNA strands, whereas with the grapevine tymoviruses GFkV and Grapevine Red Globe associated virus (GRGV), the large majority derived from the antigenomic viral strand, a feature never observed in other plant-virus interactions.

Deep sequencing of viroid-derived small RNAs from grapevine provides new insights on the role of RNA silencing in plant-viroid interaction.

BACKGROUND: Viroids are circular, highly structured, non-protein-coding RNAs that, usurping cellular enzymes and escaping host defense mechanisms, are able to replicate and move through infected plants. Similarly to viruses, viroid infections are associated with the accumulation of viroid-derived 21-24 nt small RNAs (vd-sRNAs) with the typical features of the small interfering RNAs characteristic of RNA silencing, a sequence-specific mechanism involved in defense against invading nucleic acids and in regulation of gene expression in most eukaryotic organisms. METHODOLOGY/PRINCIPAL FINDINGS: To gain further insights on the genesis and possible role of vd-sRNAs in plant-viroid interaction, sRNAs isolated from Vitis vinifera infected by Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1) were sequenced by the high-throughput platform Solexa-Illumina, and the vd-sRNAs were analyzed. The large majority of HSVd- and GYSVd1-sRNAs derived from a few specific regions (hotspots) of the genomic (+) and (-) viroid RNAs, with a prevalence of those from the (-) strands of both viroids. When grouped according to their sizes, vd-sRNAs always assumed a distribution with prominent 21-, 22- and 24-nt peaks, which, interestingly, mapped at the same hotspots. CONCLUSIONS/SIGNIFICANCE: These findings show that different Dicer-like enzymes (DCLs) target viroid RNAs, preferentially accessing to the same viroid domains. Interestingly, our results also suggest that viroid RNAs may interact with host enzymes involved in the RNA-directed DNA methylation pathway, indicating more complex scenarios than previously thought for both vd-sRNAs genesis and possible interference with host gene expression.

Aureusvirus P14 is an efficient RNA silencing suppressor that binds double-stranded RNAs without size specificity.

RNA silencing is a conserved eukaryotic gene regulatory system in which sequence specificity is determined by small RNAs. Plant RNA silencing also acts as an antiviral mechanism; therefore, viral infection requires expression of a silencing suppressor. The mechanism and the evolution of silencing suppression are still poorly understood. Tombusvirus open reading frame (ORF) 5-encoded P19 is a size-selective double-stranded RNA (dsRNA) binding protein that suppresses silencing by sequestering double-stranded small interfering RNAs (siRNAs), the specificity determinant of the antiviral silencing system. To better understand the evolution of silencing suppression, we characterized the suppressor of the type member of Aureusviruses, the closest relatives of the genus Tombusvirus. We show that the Pothos latent virus (PoLV) ORF 5-encoded P14 is an efficient suppressor of both virus- and transgene-induced silencing. Findings that in vitro P14 binds dsRNAs and double-stranded siRNAs without obvious size selection suggest that P14, unlike P19, can suppress silencing by sequestering both long dsRNA and double-stranded siRNA components of the silencing machinery. Indeed, P14 prevents the accumulation of hairpin transcript-derived siRNAs, indicating that P14 inhibits inverted repeat-induced silencing by binding the long dsRNA precursors of siRNAs. However, viral siRNAs accumulate to high levels in PoLV-infected plants; therefore, P14 might inhibit virus-induced silencing by sequestering double-stranded siRNAs. Finally, sequence analyses suggest that P14 and P19 suppressors diverged from an ancient dsRNA binding suppressor that evolved as a nested protein within the common ancestor of aureusvirus-tombusvirus movement proteins.