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Pollination is a key step of plant reproduction, allowing individual plants to produce offspring as father, mother or both. However, few studies exist that consider together male and female pollination success. This implies studying both mating system, through paternity analyses, and seed set, by measuring the percentage of flowers giving a seed. Studying these two processes together is needed as they are not independent: gaining fitness advantage through one sex can incur fitness costs through the other due to various tradeoffs including direct sexual interference. Hence, we developed the first spatially explicit mixed-mating model integrating these two interactive processes, by coupling a mating model with a fruit set model, therefore jointly exploring pollen export and import. We used as model an insect-pollinated tree species, chestnut. We carried out a paternity analysis based on nearly exhaustive sampling of potential pollen donors in an intensively studied plot of 273 trees belonging to three interfertile chestnut species and including both male-fertile and male-sterile individuals. We collected a large dataset of 1924 mating events. We further performed fruit set measurements for 216 trees. Our process-based model predicts fruit set with great accuracy, but only if we account for self-pollen interference and associated ovule discounting, a form of sexual interference. This model represents an important step forward for fundamental pollination studies aiming at comprehensively exploring pollen emission, transport and reception in a single study, thus clarifying the consequences of pollination on male and female fitness.

SummaryGynodioecy is a rare sexual system in which two genders, cosexuals and females, coexist. It provides the opportunity to compare individuals having both sexual functions with individuals lacking the male function, an ideal situation to understand how sexes interact within individual plants.We report gynodioecy in the European chestnut, an outcrossing self-incompatible Fagaceae tree species. This finding was unexpected because gynodioecy is often interpreted as an outbreeding mechanism. To understand how female chestnuts compensate for their lack of siring capacity, we compared key female fitness components between genders and performed emasculation experiments.Genders have similar basal area and number of flowers but different fruit set. Following the removal of nectar-producing catkins on branches or entire trees, fruit set increased in cosexual trees but decreased in female trees.These results show that self-pollination impairs fruit set in cosexual trees, a likely effect of self-pollen interference caused by late-acting self-incompatibility and by early inbreeding depression. Female trees escape from self-pollen interference but continue to attract pollinators thanks to their sterile but rewarding male catkins, resulting in a much higher fruit set than cosexuals. This demonstrates that even entirely outcrossed plants can benefit from the cessation of self-pollination.

Key message Pollination is a key step for fruit production. To provide a tool for future in-depth analysis of pollination in chestnut, we describe in detail a chestnut orchard (location, genotype, phenotype and seed-set of all trees). Context Chestnuts, which are insect-pollinated trees, have been massively planted around the world for nut production. Orchards are planted with clonal varieties selected from crosses between the European chestnuts (Castanea sativa) and Japanese chestnuts (C. crenata) or Chinese chestnuts (C. mollissima) because these two last species are tolerant to blight and ink diseases. Aims To characterize chestnut genetic resources and accurately model male and female fitness as well as pollen exchanges in orchards, we characterized all chestnuts of the INRAE chestnut germplasm collection located near Bordeaux (France). Methods All chestnut trees were geolocated and genotyped using 79 SNP and 98 SSR loci. We scored their flowering phenology using chestnut BBCH scale and precisely described their phenotype (height, diameter a breast height (DBH), canopy diameter…), their capacity to produce pollen (flower type, catkins length…) and their fruit production (number of burrs, seed-set…). Results We geolocated 275 trees and genotyped 273 of them. We identified 115 unique genotypes and assigned each genotype to species. To assess phenology, we evaluated 244 trees twice a week, for 6 weeks from early June to mid-July. We also described tree phenotypes with 11 variables, pollen production with 5 variables and fruit production with 3 variables. All measures were recorded in 2018 except seed set that was measured two consecutive years, in 2018 and 2019. Conclusion The data collected is very detailed and allows modelling precisely pollen exchanges between trees. Parts of this data have been successfully published in scientific articles. Data are available at: https://data.inrae.fr/dataset.xhtml?persistentId=doi:10.15454/GSJSWW Associated metadata are available at:https://metadata-afs.nancy.inra.fr/geonetwork/srv/fre/catalog.search#/metadata/02c5ca07-1536-4f89-9a0c-9e8d44a91287

International audience; To better study and manage chestnut trees and species, we identified nuclear single nucleotide polymorphism (SNP) markers using restriction-associated DNA sequencing. Out of 343 loci tested, 68 SNP markers were selected that withhold stringent quality criteria such as quasi-systematic amplification across species and Mendelian segregation in both purebred and hybrid individuals. They provide sufficient power for species, hybrids and backcross characterization as well as for clonal identification, as shown by a comparison with single sequenced repeat (SSR) loci.

International audience; Chestnuts (Castanea spp.) are ecologically and economically important forest and fruit trees. They are cultivated for their nutritious nuts. To select varieties to be cultivated in chestnut orchards, their phenology needs to be considered. In this work, we adapt the international BBCH system to chestnuts by building on an existing phenological scale used in some European countries for these species. The proposed BBCH scale uses eight of the ten principal growth stages for fruit trees and secondary growth stages that are specific to chestnut trees. We tested it by monitoring chestnut trees phenology during three growing seasons, illustrating its suitability for high-throughput phenotyping studies. Overall, the approach used, despite its inherent limitations, is straightforward, accessible and flexible, allowing particularly precise description of the complex flowering phenology of these trees.

International audience; Wind, insects, or both? The pollination mode of chestnuts, an important genus of nut-producing forest trees of the Fagaceae family, is still unclear. We revisit this old question using an integrated approach, focusing on cultivated Castanea sativa trees and hybrids in South-western France. We first conducted a large-scale insect isolation experiment. We then monitored 16 trees, focusing on flowering phenology, flower abundance and insect visits. Half of these trees are male-sterile, helping explore the role of pollen in insect attraction. Finally, we characterized the pollination syndrome of chestnuts and contrasted it with that of wind-pollinated oaks using original and published data. Chestnut female flowers have erect styles resembling stamens from male flowers, a probable case of intersexual mimicry. The tree's unusual phenology includes two peaks of pollen production. Pollinator exclusion experiments demonstrated a predominant role of insects in chestnut pollination. Flowering trees attract large numbers of beetles, bees and flies. In contrast, the few insects seen on female flowers (66 in 32 h of observation

International audience; Although chestnut mosaic disease (ChMD) was described several decades ago, its etiology is still not clear. Using classical approaches and high-throughput sequencing (HTS) techniques, we identified a novel Badnavirus that is a strong etiological candidate for ChMD. Two disease sources from Italy and France were submitted to HTS-based viral indexing. Total RNAs were extracted, ribodepleted, and sequenced on an Illumina NextSeq500 (2 × 150 nt or 2 × 75 nt). In each source, we identified a single contig of ≈7.2 kb that corresponds to a complete circular viral genome and shares homologies with various badnaviruses. The genomes of the two isolates have an average nucleotide identity of 90.5%, with a typical badnaviral genome organization comprising three open reading frames. Phylogenetic analyses and sequence comparisons showed that this virus is a novel species; we propose the name Chestnut mosaic virus (ChMV). Using a newly developed molecular detection test, we systematically detected the virus in symptomatic graft-inoculated indicator plants (chestnut and American oak) as well in chestnut trees presenting typical ChMD symptoms in the field (100 and 87% in France and Italy surveys, respectively). Datamining of publicly available chestnut sequence read archive transcriptomic data allowed the reconstruction of two additional complete ChMV genomes from two Castanea mollissima sources from the United States as well as ChMV detection in C. dentata from the United States. Preliminary epidemiological studies performed in France and central eastern Italy showed that ChMV has a high incidence in some commercial orchards and low within-orchard genetic diversity.

Chestnut species have large ecological, cultural and economic importance. Developing genetic markers for these species is of interest for conservation, breeding or evolutionary studies. We designed 192 primer pairs targeting microsatellites detected in the Castanea mollissima reference genome and tested them on C. sativa and C. crenata. We PCR amplified 3 × 50 microsatellites in 106 chestnut trees. Microhaplotype calling accounting for all polymorphisms resulted in a total of 98 high confidence polymorphic markers. Mean number of haplotypes per marker was 9.05 with respectively 71%, 12% and 16% of the variation corresponding to microsatellite variation in repeats number, SNP within the repeat motif and SNP or INDEL in the flanking sequence. Overall, the simple protocol described here generated a powerful multilocus genetic dataset for chestnut genetic investigations.