Low pollen viability may limit grapevine yield under certain conditions, causing relevant economic losses to grape-growers. It is usually evaluated by the quantification of the number of viable and non-viable pollen grains that are present in a sample after an adequate pollen grain staining procedure. Although the manual counting of both types of grains is the simplest and most sensitive approach, it is a laborious and time-demanding process. In this regard, novel image-based approaches can assist in the objective, accurate and cost-effective phenotyping of this trait. Here, we introduce PollenCounter, an open-source macro implemented as a customizable Fiji tool for the high-throughput phenotyping of pollen viability. This tool splits RGB images of stained pollen grains into its primary channels, retaining red and green color fractionated images (which contain information on total and only viable pollen grains, respectively) for the subsequent isolation and counting of the regions of interest (pollen grains). This framework was successfully used for the analysis of pollen viability of a high number of samples collected in a large collection of grapevine cultivars. Results revealed a great genetic variability, from cultivars having very low pollen viability (like Corinto Bianco; viability: 14.1 ± 1.3%) to others with a very low presence of sterile pollen grains (Cuelga; viability: 98.2 ± 0.5%). A wide range of variability was also observed among several clones of cv. Tempranillo Tinto (from 97.9 ± 0.9 to 60.6 ± 5.9%, in the first season). Interestingly, the evaluation of this trait in a second season revealed differential genotype-specific sensitivity to environment. The use of PollenCounter is expected to aid in different areas, including genetics research studies, crop improvement and breeding strategies that need of fast, precise and accurate results. Considering its flexibility, it can be used not only in grapevine, but also in other species showing a differential staining of viable and non-viable pollen grains. The wide phenotypic diversity observed at a species level, together with the identification of specific cultivars and clones largely differing in this trait, pave the way of further analyses aimed to understand the physiological and genetic causes driving to male sterility in grapevine.

Global viticulture has evolved following market trends, causing loss of cultivar diversity and traditional practices. In Montenegro, modern viticulture co-exists with a traditional viticulture that still maintains ancient practices and exploits local cultivars. As a result, this region provides a unique opportunity to explore processes increasing genetic diversity. To evaluate the diversity of Montenegrin grapevines and the processes involved in their diversification, we collected and analyzed 419 samples in situ across the country (cultivated plants from old orchards and vines growing in the wild), and 57 local varieties preserved in a grapevine collection. We obtained 144 different genetic profiles, more than 100 corresponding to cultivated grapevines, representing a surprising diversity for one of the smallest European countries. Part of this high diversity reflects historical records indicating multiple and intense introduction events from diverse viticultural regions at different times. Another important gene pool includes many autochthonous varieties, some on the edge of extinction, linked in a complex parentage network where two varieties (Razaklija and Kratošija) played a leading role on the generation of indigenous varieties. Finally, analyses of genetic structure unveiled several putative proto-varieties, likely representing the first steps involved in the generation of new cultivars or even secondary domestication events.