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. An interesting example is found in green berries sampled in Riccione. 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, bato bucket 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 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 grapevine miRNAs. 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.

The outline of miRNA accumulation across samples is different from that of sRNA-producing loci. While the expression of sRNA-generating regions allows distinguishing very well between ripened and green berries and also between cultivars , the accumulation of miRNAs shows a clear distinction only between ripened and green berries, and when the berries were green, we observe a further dichotomy separating the two cultivars and the two green developmental stages. The same pattern of miRNA accumulation among green and ripened berries of grapevine was observed when we described the miRNA expression atlas of Vitis vinifera . Comparing the distribution of miRNAs expressed throughout our samples, we found a set of 39 miRNAs ubiquitous or nearly ubiquitous to all the libraries, and very few miRNAs specific of a cultivar, vineyard or developmental stage. All these 39 miRNAs belong to known vvi-miRNA families. With few exceptions, the same set of miRNAs was also found expressed in all the small RNA libraries constructed with different tissues of the grapevine cv. Corvina , where the population of expressed miRNAs appears highly variable apart from a well-defined group of miRNAs, probably related to the basal metabolism. These findings are also consistent with previous report in grapevine where a small number of known tissue-specific miRNAs was described . Considering the ripening process as shown in the heat maps , and the correlation dendrogram, it is clear that most miRNAs are modulated during the developmental process. For some miRNA families, we observed the same peculiar patterns of miRNA accumulation, previously described in the grapevine miRNA atlas , e.g., an increase of accumulation toward ripening for miR156 f/g/i, and a decrease for miR166c/e, miR172d, miR319, and miR396a/b, but this is not the main focus of our paper. To establish genotype and environmental influence on miRNA modulation, we performed a statistical analysis that revealed a number of miRNAs differentially expressed. Being aware of the fact that we had only two biological replicates, we applied the exact test as implemented in the EdgeR package. This test has been recently judged a very robust tool that can be used in experiments similar to our, because of its low false positive rate and relative high true positive ratein the presence of a fold change higher than 4 . Considering berries at the same developmental stages, we compared Sangiovese vs. Cabernet Sauvignon in a given vineyard and Montalcino vs. Bolgheri, Montalcino vs. Riccione, and Bolgheri vs. Riccione keeping the cultivar fixed. In total we performed 9 pairwise comparisons for each developmental stage. In general, we observed that berries at 19 ◦Brix and at harvest show a higher number of differentially expressed miRNAs. The most interesting examples are represented by two novel miRNAs, whose predicted targets are related to the biosynthesis and accumulation of secondary metabolites, which are of crucial importance in grapevine berries, since its quality depends mainly on its metabolites . The candidate grapem1191 is differentially expressed in Sangiovese between Riccione and Bolgheri and was predicted to target the transparent-testa 12 gene that encodes a multidrug secondary transporter-like protein involved in the vacuolar accumulation of the flavonoid proanthocyanidin in different species including grapevine . Also, dutch bucket hydroponic in grapevine some studies provide evidences that the intracellular transport of acylated anthocyanins is catalyzed by a MATE transporter . The grape-m1355 seems to be involved in four different pathways, all related to secondary metabolites. It is differentially expressed in Montalcino between the two varieties and was predicted to target a cinnamoyl reductase-like protein , which is part of the of the polyphenol biosynthetic pathway ; a cinnamyl alcohol dehydrogenase involved in the lignin biosynthesis ; a phenylacetaldehyde reductase , which catalyzes, in tomato, the last step in the synthesis of the volatile 2-phenylethanol, important for the aroma and flavor of many foods ; and different bifunctional dihydroflavonol 4-reductases . DFR catalyzes the first step in the conversion of dihydroflavonols to anthocyanins and are responsible for the production of colored anthocyanins . The same miRNA candidate was described in the grape miRNA atlas also predicted to target several genes of DFR-like and one CCR. As for known miRNAs, several members of the miR395 family are differentially expressed at 19 ◦Brix and at harvest in Bolgheri and in both Bolgheri and Riccione, respectively, when comparing the two cultivars.

Moreover, miR395f is differentially expressed also in CS at harvest between Montalcino and Bolgheri. This miRNA has been shown to target genes involved in Sulphate assimilation and metabolism , and hence it could be connected to flavonoid and stilbene pathways as suggested by Tavares et al. . miR399 family members are also differentially expressed in several comparisons: at 19 ◦Brix between Riccione and Bolgheri in CS and between Riccione and Montalcino in SG, plus in Montalcino between CS and SG. At harvest, miR399 are differentially expressed in SG in all the three comparisons among vineyards and in Riccione between CS and SG. miR399 is implicated in Phosphate homeostasis being rapidly up-regulated upon Pi starvation . miR399 regulatory network has been shown to be important in flowering time and was identified as a temperature-sensitive miRNA , however its characterization in fruit ripening is lacking, although intriguing. miR396 family members are known to be regulated during organ development, targeting Growth Regulating Factors and also in berry development , and we observed their modulation during berry ripening in our data as well, but more interestingly, they are also differentially expressed between CS and SG in berries sampled in Bolgheri at 19 ◦Brix. Finally, the investigation of the global relationships of different small RNA classes and miRNAs expressed in different grapevine cultivars, collected in different vineyards and developmental stages, suggests that although the vineyard may influence their profile of abundance it probably does in less proportion than developmental stage and cultivar. Somehow, this behavior would be expected because although the epigenetic state is dynamic and responsive to both developmental and environmental signals, small RNAs in general and even more miRNAs are well known to play numerous crucial roles at each major stage of plants development . The results here described are in agreement with those reported in the grapevine miRNA atlas , especially with respect to the clustering of berries according to their developmental stage, sustaining the idea that miRNAs influence organ identity and clearly separate green and ripened berries. Also, in the study of the grapevine transcriptome performed by Dal Santo et al. , they observed that other factors such as year and developmental stage had more influence on the gene expression, rather than the environment. AUTHOR CONTRIBUTIONS DPP prepared small RNA libraries, performed the in silico analysis and wrote the paper. LB conceived the experimental plan and sampled biological material. SDS prepared plant material for RNA extraction, read critically the paper. GDL prepared plant material for RNA extraction, sampled the biological material, read critically the paper. MP conceived the work. MEP supported the lab work, contributed to data analysis and read critically the paper. BM gave a substantial contribution to in silico analysis. EM wrote the paper, prepared plant material for RNA extraction, supported small RNA libraries preparation and helped data analysis.Although RRBs are a defining feature of ASD, they are not exclusive to the disorder and are present in typically developing children and in children with developmental delays . Repetitive behaviors exhibited in early development are thought to be important mechanisms for learning processes in the acquisition and mastery of skills such as motor control and actions with objects . Typically developing infants and toddlers exhibit a variety of RRBs such as repetitive motor actions, rigidity in routines, repetitive manipulation of objects and significant attachment to certain items . Understanding what forms and functions RRBs serve in the typically developing population will better inform our understanding of how these behaviors may differ for individuals with ASD. RRBs are now understood to be a continuum of behaviors that serve various functions throughout development and may occur frequently within typical infant and toddler development and vary based on children’s skill acquisition and mastery level .