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Seedlessness is an important objective in grape breeding programs being highly appreciated by consumers. Substantial advances in the comprehension of the molecular underlying mechanisms have been achieved. However, most studies inspected the stenospermocarpic variety Sultanine, whereas different sources of seedlessness have been much less investigated. With the aim of providing additional insights into the regulation of seed formation and develop testable candidate gene hypotheses, we explored our grapevine germplasm collections searching for clones with contrasting seed content. In total, we identified nine variant pairs that differ only in those characteristics related to the presence/absence of seeds while showing identical microsatellite marker profile. Here we report their characterization, with special emphasis on Sangiovese and its seedless variant, for which we previously performed a comprehensive transcriptomic comparison. A subset of the paired accessions and some additional genotypes were characterized during 4 years for fruit and seed set in control, self-pollinated and emasculated conditions. Our observations suggest that when pollination is not prevented stenospermocarpy may be responsible for the seedless phenotype and is potentially driven by pollen and/or ovule defects. Two genotypes were unexpectedly found to develop fruits also without pollen contribution, which occasionally showed normal-like seeds. The possible origin of this phenomenon is currently under investigation. In the seek for structural variation each seedless mutant was compared to its seeded reference variety by using the GrapeReSeq_Illumina_20K_SNP_chip. None of the putative SNPs was validated by resequencing, proving they are near-isogenic lines. Conversely, RNA-Seq-based variant calling allowed the identification of 5 nonsynonymous SNPs that were experimentally confirmed in the original and additional Sangiovese accessions. These polymorphisms are suitable to be tested as diagnostic markers in clone identification and as functional candidates for the seedless phenotype.

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