Among these 209 candidate precursors, 61 belonged to 31 known vvi-miRNA that passed allthe filters and 148 were identified as putatively novel miRNA candidates. To certify that they were novel candidates rather than variants of known vvi-miRNAs we compared their sequences and coordinates with the miRNAs registered in miRBase . In order to reduce false positives and the selection of siRNA-like miRNAs, we considered only 20, 21, and 22 nt candidates whose stemloop structures were manually evaluated . Eventually, 26 miRNAs homologous to other plant species were identified with high confidence. Twenty-two were new members of nine known V. vinifera families, whereas the other four belong to three families not yet described in grapevine . For 16 homologs we were able to retrieve also the complementary sequence. Finally, excluding these 26 miRNAs and other si-RNA like miRNAs, we identified 7 completely novel bona fide miRNAs. Apart from the 61 known vvi-miRNAs identified by the pipeline, we searched the dataset for others known vvi-miRNAs eliminated throughout the pipeline, seedling starter pot looking for isomiRs that were actually more abundant than the annotated sequences. Their complementary 3p or 5p sequence was also retrieved when possible. Hence 89 known vvi-miRNAs were identified in at least one of our libraries . Among the known vvi-miRNAs identified, 24 had an isomiR more abundant than the annotated sequence and 4 have the complementary sequence as the most abundant sequence mapping to their precursor.
We found 16 vvi-miRNA isomiRs that were either longer or shorter than the annotated sequence, 7 vvi-miRNAs that mapped in the precursor in a position shifted with respect to the annotated ones and one miRNA that contains a nucleotide gap when compared to the annotated sequence . An extreme case of shifted position was found in vvi-miRNA169c, where the annotated sequence had only 5 TP4M when summing its individual abundance in the 48 libraries. Another sequence, shifted 16 bp as compared to its annotated position on the precursor had an abundance sum of 1921 TP4M, and was retained together with the annotated sequence, and named vvi-miRNA169c.1. For 36 of the 48 V. vinifera miRNA families deposited in miRBase we found at least one member. An in silico prediction of miRNA targets was performed for the 191 mature miRNAs here identified. Using the miRferno tool , and considering only targets predicted with high stringency, 1192 targets were predicted for 143 miRNAs, including six completely novel vvi-miRNA candidates . Two novel candidates seem to be involved in the regulation of important secondary metabolites biosynthesis. Among the six targets predicted for grape-m1191, the TT12 gene is known to be involved in the vacuolar accumulation of proanthocyanidins in grapevine . For grape-m1355, 12 targets were predicted and all of them are involved in secondary metabolism pathways. Nine targets code a bifunctional dihydroflavonol 4-reductase that is responsible for the production of anthocyanins , catalyzing the first step in the conversion of dihydroflavonols to anthocyanins . Another targeted gene codes a phenylacetaldehyde reductase which, in tomato, was demonstrated to catalyze the last step in the synthesis of the aroma volatile 2-phenylethanol, important for the aroma and flavor . Still this same miRNA candidate was predicted to target with high confidence a cinnamoyl reductaselike protein that is part of polyphenol biosynthetic pathway . The grapem1355 candidate maps on chromosome 3, exactly on thefirst exon of its target , in a region where another two isoforms of the same gene are located .
The last target of this miRNA candidate, codes a cinnamyl alcohol dehydrogenase known to be involved in the lignin biosynthesis . Other novel vvi-miRNA candidates seem to be involved in cell proliferation and in chloroplasts-related functions . Furthermore, for the new vvi-miRC482b candidate, besides the already known involvement of this miRNA family with disease resistance also predicted here, one predicted target encodes an anthocyanin 5-aromatic acyltransferase-like protein known to be involved in the biosynthesis of anthocyanin in different species . As for the conserved known vvi-miRNAs, most of the well-established examples of miR-targets, such as miR156-SPB, miR166-HD-ZIP, miR171-GRAS, miR172-AP2, confirmed in several plant species and already predicted in grapevine, were also predicted here.To gain statistical evidence of miRNA differential expression driven by the environment and/or genotype, we made pairwise comparisons, keeping constant the developmental stage, and evaluating the miRNA modulation among vineyards or between cultivars . The analyses reveal that some miRNAs are differentially expressed between the two genotypes grown in the same environment, but also that a number of miRNAs are modulated by the environment. In particular the number of differentially expressed miRNAs is higher in ripened berries , while no miRNAs are differentially expressed at bunch closure stage . In details, 14 reads are differentially expressed at pea size stage, in at least one comparison, corresponding to 6 distinct miRNA families; 27 reads are modulated at 19 ◦Brix stage, corresponding to 12 miRNA families and 35 reads are differentially expressed in berries at harvest, corresponding to 12 miRNA families.
It is worth noting that 4 of the 6 families modulated in the berries at pea size, are still present among the miRNAs differentially expressed in the berries sampled at 19 ◦Brix and at harvest , even though not always in the same comparisons. Some of the modulated miRNAs, both novel and known are intriguingly connected to berry development and secondary metabolism, even though most of the modulated families are still uncharacterized, or with targets not clearly involved in berry ripening and development, and deserve further studies to fully understand their biological roles.Using high throughput sequencing coupled with robust bio-informatics pipelines we analyzed small RNAs derived from the berries of Cabernet Sauvignon and Sangiovese, grown sideby-side in three vineyards, representative of different grapevine cultivation areas in Italy . We obtained nearly 750 MB reads comprising a significant proportion of small RNAs. The size distribution profiles of our libraries were in general consistent with previous reports in berry grapevine, where the 21-nt class was more abundant than the 24-nt class . Our analysis revealed dynamic features of the regulatory network mediated by miRNAs and other small RNAs, at the basis of genotype-environment interactions.Plants evolved a series of pathways that generate small RNAs of different sizes with dedicated functions . Although the various small RNA classes have been intensively studied, we are still far from understanding how many small RNA pathways exist, and how they are connected . Additionally, new classes of small non-coding RNAs continue to be discovered and many studies demonstrate a substantial redundancy and cross-talk between known small RNA pathways . Estimating the exact percentage of the plant genome covered by small RNA-generating loci still remains a challenge. By applying static cluster analysis, we investigated small RNA abundances across the genome, identifying 4408 small RNAs producing hotspots. We analyzed their expression in different cultivars, environments and developmental stages, highlighting that the majority of the considered small RNA producing regions was modulated in different conditions. This suggests a strong influence of small RNAs in the response to environment in grapevine berries. Only 462 small RNA-generating loci, round nursery pots corresponding to about 10% of the total, were expressed in all the analyzed libraries, possibly involved in essential biological pathways. Comparing the two cultivars, we observed, with few exceptions, that Cabernet Sauvignon berries have a higher number of expressed sRNA-generating loci than Sangiovese berries when collected in the same conditions . Considering the fact that small RNAs are implicated in the regulation of gene expression in several processes , the higher number of small RNAs expressed in Cabernet Sauvignon compared to Sangiovese berries may reflect a buffering effect of small RNAs influencing grapevine response to diverse growing environments. We believe that these characteristics may have contributed to the wide diffusion of Cabernet Sauvignon, allowing its wide cultivation in almost all wine producing countries. This is not the case for Sangiovese whose cultivation is more restricted. It is worth noting that Sangiovese is considered a very unsettled grapevine cultivar , showing a wide range of variability in response to year, clone and bunch exposure . Differently, Cabernet Sauvignon is a cultivars showing less inter-annual differences in terms, for example, of concentration of secondary metabolites . To better evaluate varietal differences in response to the environment, we calculated the CS/SG ratio for the small RNA producing hotspots in the three vineyards.
A region on chromosome 4 showed a 390-fold change in the small RNA abundance, when comparing Cabernet vs. Sangiovese . Most of the reads produced in this region are 21 nt long and are also phased in intervals of 21 nt from both strands, typical of a phased locus . The gene in this locus, also known as VvRD22g, encodes a BURP domain containing protein, involved in an ABA-mediated abiotic stress response, which persists still after long periods of stress . The small RNAs profile suggests that the locus is regulated by phased siRNAs similarly to the mechanisms already described for PPR, NB-LRR, and MYB gene families . This is a clear example of GxE interactions since the BURP domain gene modulates phased siRNAs production in the two cultivars only when grown in Riccione. When removing the threshold of minimum cluster abundance set to 5 HNA, in the CS/SG ratio, a high number of clusters with fold change greater than 50 was found, where one of the libraries has 0 HNA and the other any number greater than 30 HNA. This fact suggests a very strong modulation of the expression of small RNAs between the two cultivars, which is more or less pronounced depending on the vineyard where the berries were cultivated. A similar situation was observed comparing the expression level of small RNAs between reciprocal hybrids of Solanum lycopersicum and S. pimpinellifolium . The ripening process of grapevine berries is highly affected by the environment and we observed the impact of the environment on the ripening process in the expression of small RNAs. The most relevant observation is that Riccione is very peculiar in relation to the activation of sRNA hotspots, as indicated by the high number of Riccionespecific clusters and by the extreme modification it induces in the CS/SG ratio : in Riccione in fact this ratio decreases in green berries and increases in ripened berries, and this is not observed in any other vineyard; in addition to this the already discussed example of BURP domain gene, is observed in Riccione, as well. Riccione is the most diverse environment when compared to Montalcino and Bolgheri. Riccione is located at the Adriatic coast and has a temperate sub-littoral climate, while Montalcino and Bolgheri are both located in Tuscany with typically Mediterranean climate. Moreover, both cultivars show a peculiar profile of small RNA loci during berries ripening, in Riccione. The expression of small RNA loci in Cabernet Sauvignon berries drastically changed during development, especially when collected in Riccione , not only in the number of active loci but also in the different genic or intergenic disposition: ripened berries have a 2.6-fold increase in small RNA loci active in genic regions. Differently, when Sangiovese is grown in Riccione, there is a very high number of small RNA loci active in green berries, mainly associated to transposable elements that remains almost stable during development although the proportion of intergenic loci is reduced. Sangiovese berries collected in Montalcino show a 2.5- fold increase of small RNA producing loci during development.Differences during berry development between the cultivars may explain their different behavior in different environments, and the characteristics of each vineyard may favor one or other variety according to their demands. For example, Sangiovese needs a long growing season with sufficient warmth to fully ripen . Consequently, cooler environments will require a reprograming of Sangiovese gene expression in order to achieve ripening. Other factors such as composition of soil, level of humidity, photoperiod and density of cultivation may be exerting the same influence on the ripening of the berries triggering the activation of different small RNA loci. Applying a conservative pipeline to the analysis of our 48 small RNA libraries, we recognized 89 known and annotated grape vinemiRNAs. In addition, when compared to previous reports in grapevine we identified 7 completely novel miRNAs plus 26 homologous to other plant species, but novel to grapevine. This is a remarkable number considering the stringency of our pipeline and that our study is based only on four developmental stages of berries.