Grapevine has a major economical and cultural importance since antiquity. A key step 27! in domestication was the transition from separate sexes (dioecy) in wild Vitis vinifera ssp. 28! sylvestris (V. sylvestris) to hermaphroditism in cultivated Vitis vinifera ssp. vinifera. 29! While the grapevine sex locus is known to be small, its precise boundaries, gene content 30! and the sex-determining genes are unknown. Here we obtained a high-quality de novo 31! reference genome for V. sylvestris and whole-genome resequencing data of a cross.
32!Studying SNP segregation patterns, gene content and expression in wild and cultivated 33! accessions allowed us to build a model for sex determination in grapevine. In this model, 34! up-and down-regulation of a cytokinin regulator is sufficient to cause female sterility 35! and reversal to hermaphroditism, respectively. This study highlights the importance of 36! neo-functionalization of Y alleles in sex determination and provides a resource for 37! studying genetic diversity in V. sylvestris and the genomic processes of grapevine 38! domestication. 39! 40! Dioecy is rare in flowering plants (~6%) but over-represented among crops (~20%) 1 . In some 41! cases, both wild and cultivated plants are dioecious (e.g. date palm, asparagus, persimmons). 42! Other crops, such as grapevine, papaya, and strawberry, derive from dioecious progenitors 43! and switched to hermaphroditism during domestication. The genes underlying this switch are 44! currently not known in any crop. In Vitis sylvestris, wild females produce morphologically 45! bisexual flowers, with retracted anthers that produce few and infertile pollen 2 , while male 46! flowers undergo early ovule abortion 3 . Genetic analyses identified a 143-kb haplotype 47! responsible for sex-determination in the grapevine reference genome 4,5 . Two candidate genes 48! for female sterility have been proposed, in particular APRT3 6 , a putative cytokinin regulator. 49! ! 3! ! However, the sequence of the sex locus was not available in V. sylvestris and no causative 50! mutations have been identified. A genetic and evolutionary model to explain sex 51!determination and switch to hermaphroditism in grapevine is currently lacking.
52!We sequenced a female individual of V. sylvestris using SMRT-sequencing (120X). Contigs
53!were assembled with falcon-unzip 7 and the grapevine reference genome 8 (PN40024 -version 54! 12X.2) was used to build pseudomolecules. We obtained a high-quality diploid assembly of 55! 469 Mb with a contig N50 of 1.7 Mb, 98% of the gene content anchored on chromosomes and 56! a BUSCO evaluation of 95% (Supplementary Table 1, Supplementary Figure 1), comparing 57! favorably to other recently published plant genomes 9,10 . We annotated 39,031 protein-coding 58! genes on primary contigs.
59!To study sex determination, we localized the sex locus, which was fully included in the 5 th 60! largest contig of the assembly. We also sequenced and assembled bacterial artificial 61! chromosomes (BACs) cover...