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Flowering date is an important trait in Prunus fruit species, especially for their adaptation in a global warming context. Numerous quantitative trait loci (QTLs) have been identified and a major one was previously located on LG4. The objectives of this study were to fine-map this QTL in sweet cherry, to identify robust candidate genes by using the new sweet cherry genome sequence of the cultivar “Regina” and to define markers usable in marker-assisted selection (MAS). We performed QTL analyses on two populations derived from crosses using cultivars “Regina” and “Garnet” as parents. The first one (n = 117) was phenotyped over ten years, while the second one (n = 1386) was evaluated during three years. Kompetitive allele specific PCR (KASP) markers located within the QTL region on LG4 were developed and mapped within this region, consisting in the first fine mapping in sweet cherry. The QTL interval was narrowed from 380 kb to 68 kb and candidate genes were identified by using the genome sequence of “Regina”. Their expression was analyzed from bud dormancy period to flowering in cultivars “Regina” and “Garnet”. Several genes, such as PavBOI-E3, PavSR45a and PavSAUR71, were differentially expressed in these two cultivars and could be then considered as promising candidate genes. Two KASP markers were validated using a population derived from a cross between cultivars “Regina” and “Lapins” and two collections, including landraces and modern cultivars. Thanks to the high synteny within the Prunus genus, these results give new insights into the control of flowering date in Prunus species and pave the way for the development of molecular breeding strategies., This work was supported by the “Région Nouvelle-Aquitaine” in the project “CerGEn” reference 2018-1R20203, numbers 00019006 and 00019007. INRAE BAP division and the “Région Nouvelle-Aquitaine” co-funded the PhD scholarship of C.B and funded the Postdoctoral fellowship of A.D. The “Groupement d’Intérêt Scientifique Fruits”, GIS Fruits (https://www.gis-fruits.org/), funded the Master thesis scholarship of N.H.Z.E.

The preservation of maximum diversity within the smallest number of accessions is one of the challenges of germplasm management, and the assessment of the population structure, the relationships between the accessions, and the construction of core collections are the key steps. The choice of suitable molecular markers is often the starting point. In this study, we analyzed a part of the INRAE walnut germplasm collection, which is highly diverse and unique in Europe, consisting of 150 accessions from American, European, and Asian continents. Based on genotypic data already available, using 13 SSRs or 364,275 SNPs, we showed that the first level of structure is detected equally. We also highlighted empirically that approximately 100 SNPs are needed to obtain similar clustering to 13 SSRs in Principal Coordinate Analysis (PCoA). We constructed eight core collections following two strategies (percentage of total allelic diversity or number of accessions) and two construction methods based on different algorithms (“maximum length subtree” and “entry-to-nearest-entry” methods). We showed that core collections based on few SSR markers are able to capture at least 99.5% of the SNP allelic diversity, irrespective of the construction method used. Then, core collections based on each marker type are highly similar, using both construction methods. All these steps are crucial to identify the suitable tools and methods to improve plant genetic resources management.

Additional file 11: Figure S4. Density plots showing the two-year data related to phenological traits in Julian days for the 78 accessions of the F1 progeny.

Additional file 15: Figure S8. Level plot showing the Identical-By-State (IBS) values for the 170 accessions of the GWAS panel.

Additional file 14: Figure S7. Scatter plot showing the estimated kinship coefficients by the proportion of zero Identical-By-State (IBS0) in the F1 progeny.

Additional file 16: Figure S9. Genetic maps and QTLs detected using two-year data. F(X) and U(X) are the linkage groups of ‘Franquette’ and ‘UK 6–2’ parental maps respectively. Legend of the QTLs: black for budbreak date, red for beginning female flowering date, deep green for full female flowering date, blue for end female flowering date, yellow for beginning male flowering date, pink for full female flowering date, and light green for end female flowering date. Solid bars indicate the 95% confidence interval of the QTL, and terminal bars indicate the 99.9% confidence interval of the QTL. The marker names were changed with the corresponding chromosome number and its physical position for a better visualization.

Additional file 8: Figure S1. Scatter plots showing the two-year data related to phenological traits in Julian days for the 170 accessions of the GWAS panel.

Additional file 12: Figure S5. Detection of the number of clusters using a) Prob(K), and b) deltaK method (Evanno et al., 2005) in the GWAS panel.

Additional file 9: Figure S2. Density plots showing the two-year data related to phenological traits in Julian days for the 170 accessions of the GWAS panel.

Additional file 13: Figure S6. Principal Component Analysis performed on the GWAS panel. The two first principal components show accessions colored according to fastSTRUCTURE results with EEAs for Eastern Europe and Asia, and WEAm for Western Europe and America.

Additional file 10: Figure S3. Scatter plots showing the two-year data related to phenological traits in Julian days for the 78 accessions of the F1 progeny.

Additional file 9: Figure S2. Density plots showing the two-year data related to phenological traits in Julian days for the 170 accessions of the GWAS panel.

Additional file 8: Figure S1. Scatter plots showing the two-year data related to phenological traits in Julian days for the 170 accessions of the GWAS panel.

The dating of diversification events, including transitions between biomes, is key to elucidate the processes that underlie the assembly and evolution of tropical biodiversity. Afzelia is a widespread genus of tropical trees, threatened by exploitation for its valuable timber, that presents an interesting system to investigate diversification events in Africa. Africa hosts diploid Afzelia species in the savannahs north and south of the Guineo-Congolian rainforest and autotetraploid species confined to the rainforest. Species delimitation and phylogenetic relationships among the diploid and tetraploid species remained unresolved in previous studies using small amounts of DNA sequence data. We used genotyping-by-sequencing in the five widespread Afzelia species in Africa, the savannah species A. africana and A. quanzensis and the rainforest species A. bipindensis, A. pachyloba, and A. bella. Maximum likelihood and coalescent approaches resolved all species as monophyletic and placed the savannah and rainforest taxa into two separate clades corresponding to contrasted ploidy levels. Our data are thus compatible with a single biome shift in Afzelia in Africa, although we were unable to conclude on its direction. SNAPP calibrated species trees show that the savannah diploids started to diversify early, at 12 (9.09–14.89) Ma, which contrasts with a recent and rapid diversification of the rainforest tetraploid clade, starting at 4.22 (3.12 – 5.36) Ma. This finding of older diversification in a tropical savannah clade vs. its sister rainforest clade is exceptional; it stands in opposition to the predominant observation of young ages for savannahs lineages in tropical regions during the relatively recent expansion of the savannah biome.

Background: Unravelling the genetic architecture of agronomic traits in walnut such as budbreak date and bearing habit, is crucial for climate change adaptation and yield improvement. A Genome-Wide Association Study (GWAS) using multi-locus models was conducted in a panel of 170 walnut accessions genotyped using the Axiom™ J. regia 700K SNP array, with phenological data from several years. These accessions come from the INRAE walnut germplasm collection which is the result of important prospecting work performed in many countries around the world. In parallel, an F1 progeny of 78 individuals segregating for phenology-related traits, was genotyped with the same array and phenotyped for the same traits, to construct linkage maps and perform Quantitative Trait Loci (QTLs) detection. Results: Using GWAS, we found strong associations of SNPs located at the beginning of chromosome 1 with both budbreak and female flowering dates. These findings were supported by QTLs detected in the same genomic region. Highly significant associated SNPs were also detected using GWAS for heterodichogamy and lateral bearing habit, both on chromosome 11. We developed a Kompetitive Allele Specific PCR (KASP) marker for budbreak date in walnut, and validated it using plant material from the Walnut Improvement Program of the University of California, Davis, demonstrating its effectiveness for marker-assisted selection in Persian walnut. We found several candidate genes involved in flowering events in walnut, including a gene related to heterodichogamy encoding a sugar catabolism enzyme and a cell division related gene linked to female flowering date. Conclusions: This study enhances knowledge of the genetic architecture of important agronomic traits related to male and female flowering processes and lateral bearing in walnut. The new marker available for budbreak date, the first key trait for good fruiting, will facilitate the selection and development of new walnut cultivars suitable for specific climates.

Polyploidy has rarely been documented in rain forest trees but it has recently been found in African species of the genus Afzelia (Leguminosae), which is composed of four tetraploid rain forest species and two diploid dry forest species. The genus Afzelia thus provides an opportunity to examine how and when polyploidy and habitat shift occurred in Africa, and whether they are associated. In this study, we combined three plastid markers (psbA, trnL, ndhF), two nuclear markers (ribosomal ITS and the single-copy PEPC E7 gene), plastomes (obtained by High Throughput Sequencing) and morphological traits, with an extensive taxonomic and geographic sampling to explore the evolutionary history of Afzelia. Both nuclear DNA and morphological vegetative characters separated diploid from tetraploid lineages. Although the two African diploid species were well differentiated genetically and morphologically, the relationships among the tetraploid species were not resolved. In contrast to the nuclear markers, plastid markers revealed that one of the diploid species forms a well-supported clade with the tetraploids, suggesting historical hybridisation, possibly in relation with genome duplication (polyploidization) and habitat shift from dry to rain forests. Molecular dating based on fossil-anchored gene phylogenies indicates that extant Afzelia started diverging c. 14.5 or 20 Ma while extant tetraploid species started diverging c. 7.0 or 9.4 Ma according to plastid and nuclear DNA, respectively. Additional studies of tropical polyploid plants are needed to assess whether the ploidy-habitat association observed in African Afzelia would reflect a role of polyploidization in niche divergence in the tropics.

In temperate trees, optimal timing and quality of flowering directly depend on adequate winter dormancy progression, regulated by a combination of chilling and warm temperatures. Physiological, genetic and functional genomic studies have shown that hormones play a key role in bud dormancy establishment, maintenance and release. We combined physiological, transcriptional analyses, quantification of abscisic acid (ABA) and gibberellins (GAs), and modelling to further investigate how these signaling pathways are associated with dormancy progression in the flower buds of two sweet cherry cultivars.Our results demonstrated that GA-associated pathways have distinct functions and may be differentially related with dormancy. In addition, ABA levels rise at the onset of dormancy, associated with enhanced expression of ABA biosynthesisPavNCEDgenes, and decreased prior to dormancy release. Following the observations that ABA levels are correlated with dormancy depth, we identifiedPavUG71B6, a sweet cherryUDP-GLYCOSYLTRANSFERASEgene that up-regulates active catabolism of ABA to ABA-GE and may be associated with low ABA content in the early cultivar. Subsequently, we modelled ABA content and dormancy behavior in three cultivars based on the expression of a small set of genes regulating ABA levels. These results strongly suggest the central role of ABA pathway in the control of dormancy progression and open up new perspectives for the development of molecular-based phenological modelling.

Polyploidy has rarely been documented in rain forest trees but it has recently been found in African species of the genus Afzelia (Leguminosae), which is composed of four tetraploid rain forest species and two diploid dry forest species. The genus Afzelia thus provides an opportunity to examine how and when polyploidy and habitat shift occurred in Africa, and whether they are associated. In this study, we combined three plastid markers (psbA, trnL, ndhF), two nuclear markers (ribosomal ITS and the single-copy PEPC E7 gene), plastomes (obtained by High Throughput Sequencing) and morphological traits, with an extensive taxonomic and geographic sampling to explore the evolutionary history of Afzelia. Both nuclear DNA and morphological vegetative characters separated diploid from tetraploid lineages. Although the two African diploid species were well differentiated genetically and morphologically, the relationships among the tetraploid species were not resolved. In contrast to the nuclear markers, plastid markers revealed that one of the diploid species forms a well-supported Glade with the tetraploids, suggesting historical hybridisation, possibly in relation with genome duplication (polyploidization) and habitat shift from dry to rain forests. Molecular dating based on fossil-anchored gene phylogenies indicates that extant Afzelia started diverging c. 14.5 or 20 Ma while extant tetraploid species started diverging c. 7.0 or 9.4 Ma according to plastid and nuclear DNA, respectively. Additional studies of tropical polyploid plants are needed to assess whether the ploidy-habitat association observed in African Afzelia would reflect a role of polyploidization in niche divergence in the tropics.

The long generation time and large effective size of widespread forest tree species can result in slow evolutionary rate and incomplete lineage sorting, complicating species delimitation. We addressed this issue with the African timber tree genus Milicia that comprises two morphologically similar and often confounded species: M. excelsa, widespread from West to East Africa, and M. regia, endemic to West Africa. We combined information from nuclear microsatellites (nSSRs), nuclear and plastid DNA sequences, and morphological systematics to identify significant evolutionary units and infer their evolutionary and biogeographical history. We detected five geographically coherent genetic clusters using nSSRs and three levels of genetic differentiation. First, one West African cluster matched perfectly with the morphospecies M. regia that formed a monophyletic clade at both DNA sequences. Second, a West African M. excelsa cluster formed a monophyletic group at plastid DNA and was more related to M. regia than to Central African M. excelsa, but shared many haplotypes with the latter at nuclear DNA. Third, three Central African clusters appeared little differentiated and shared most of their haplotypes. Although gene tree paraphyly could suggest a single species in Milicia following the phylogenetic species concept, the existence of mutual haplotypic exclusivity and nonadmixed genetic clusters in the contact area of the two taxa indicate strong reproductive isolation and, thus, two species following the biological species concept. Molecular dating of the first divergence events showed that speciation in Milicia is ancient (Tertiary), indicating that long-living tree taxa exhibiting genetic speciation may remain similar morphologically.

Background: Species delimitation in closely related plant taxa can be challenging because (i) reproductive barriers are not always congruent with morphological differentiation, (ii) use of plastid sequences might lead to misinterpretation, (iii) rare species might not be sampled. We revisited molecular-based species delimitation in the African genus Milicia, currently divided into M. regia (West Africa) and M. excelsa (from West to East Africa). We used 435 samples collected in West, Central and East Africa. We genotyped SNP and SSR loci to identify genetic clusters, and sequenced two plastid regions (psbA-trnH, trnC-ycf6) and a nuclear gene (At103) to confirm species' divergence and compare species delimitation methods. We also examined whether ecological niche differentiation was congruent with sampled genetic structure. Results: West African M. regia, West African and East African M. excelsa samples constituted three well distinct genetic clusters according to SNPs and SSRs. In Central Africa, two genetic clusters were consistently inferred by both types of markers, while a few scattered samples, sympatric with the preceding clusters but exhibiting leaf traits of M. regia, were grouped with the West African M. regia cluster based on SNPs or formed a distinct cluster based on SSRs. SSR results were confirmed by sequence data from the nuclear region At103 which revealed three distinct ‘Fields For Recombination' corresponding to (i) West African M. regia, (ii) Central African samples with leaf traits of M. regia, and (iii) all M. excelsa samples. None of the plastid sequences provide indication of distinct clades of the three species-like units. Niche modelling techniques yielded a significant correlation between niche overlap and genetic distance. Conclusions: Our genetic data suggest that three species of Milicia could be recognized. It is surprising that the occurrence of two species in Central Africa was not reported for this well-known timber tree. Globally, our work highlights the importance of collecting samples in a systematic way and the need for combining different nuclear markers when dealing with species complexes. Recognizing cryptic species is particularly crucial for economically exploited species because some hidden taxa might actually be endangered as they are merged with more abundant species., SCOPUS: ar.j, info:eu-repo/semantics/published

Polymorphic (1) and monomorphic (0) SNP loci (P0040, P0095, etc.) in the six genetic clusters of Milicia (K1 to K6). Table S2. Geographic coordinates of Central African individuals of Milicia that may represent a new species according to SNP and based on the whole Milicia sample of the ITTO Project PD 620/11 Rev.1 (M): “Development and implementation of species identification and timber tracking in Africa with DNA fingerprints and stable isotopes” (1833 individuals). Individuals in bold are those selected for the SNP-SSR comparison (435 individuals in total) and which all turned out to be representatives of a new species as the nuclear gene At103 confirmed. Figure S1. Determination of the number of genetic clusters in Milicia populations based on SNPs genotypes. Figure S2. Determination of the number of clusters in Milicia populations based on SSRs genotypes. Figure S3. (A) Lower surface of leaf for a tree morphologically identified as M. regia in Central Africa and confirmed by molecular markers, compared to a neighbour tree with typical leaves of M. excelsa (B). Figure S4. Graphical membership coefficient of Central African individuals of Milicia (genetic clusters K3 to K6) based on nuclear SSR loci. The six colours (blue, black, yellow, pink, green and red) stand for the six genetic clusters. The West African genetic clusters, K1 and K2, were not illustrated but were represented in some individual genome (blue and dark colors). Figure S5. Haplotype network and geographical distribution of trnH-psbA haplotypes in Milicia populations. Figure S6. Haplotype network and geographical distribution of trnC-ycf6 haplotypes in Milicia populations. Figure S7. Ecological niches of the six genetic clusters of Milicia derived from nuclear SSR loci, in the environmental space produced by the principal component analysis method (PCA). The figure above indicates projection of the climatic variables in the plan formed by the two first axes (71.9% of the total variance). Similarly, the grey-to-black shading in the six small figures (1 = cluster K1; 2 = cluster K2; etc.) represents the grid cell density (black being the highest density) of the concerned genetic cluster in the PCA plan. The first dashed curve represents the 50% of the available environment space whereas the solid line stands for the entirety of the species environment. Figure S8. Potential distribution range of Milicia genetic clusters during the Last Glacial Maximum (LGM) according to niche modelling through Maxent approach. Black areas represent predicted regions with a probability higher than 50%. (DOCX 1353 kb)

Le genre Afzelia Smith est connu pour comporter sept espèces africaines d’arbres dont deux se retrouvent dans la région zambézienne, une en région soudanienne et les quatre autres en région guinéo-congolaise. Ces taxons, à haute valeur commerciale, sont difficiles à distinguer les uns des autres. Ils sont donc commercialisés sous un même nom : « doussié » ou « afzelia ». Ces difficultés de distinction peuvent s’avérer préjudiciables à la gestion durable des populations.Le but de cette thèse de doctorat est de caractériser l’histoire évolutive du genre Afzelia. Plus spécifiquement, cette étude vise à : (i) évaluer le niveau des divergences morphologiques au sein du genre et décrire les relations phylogénétiques, afin de quantifier l’isolement reproductif entre les taxons d’Afzelia en mettant en évidence le rôle des changements climatiques passés et/ou des gradients écologiques dans la spéciation ; (ii) procéder à une analyse approfondie de la diversité et de la structuration génétique spatiale d’Afzelia spp. ; (iii) identifier et décrire les facteurs écologiques, biotiques et abiotiques susceptibles d’influencer les flux géniques à l’échelle des populations d’une espèce d’Afzelia (A. bipindensis).Une analyse morphogénétique des espèces a été effectuée et a confirmé la forte ressemblance botanique entre les taxons. Les espèces de savane se sont avérées être diploïdes et présentent la moitié de la taille du génome des espèces forestières qui sont donc tétraploïdes. Les phylogénies de gènes, nucléaires et chloroplastiques, diffèrent entre elles. De telles différences peuvent être générées à la suite d’épisodes d’hybridation ancestrale entre espèces. Ces hybridations seraient probablement anciennes et seraient survenues entre les lignées des espèces forestières et celles d’A. quanzensis (une espèce des savanes zambéziennes). La polyploïdie serait survenue entre 7 et 9,4 millions d’années au cours de l’histoire évolutive du genre. De plus, une assignation bayésienne et des analyses d’isolement reproductif ont suggéré l’existence de croisements interspécifiques, mais uniquement chez les espèces forestières distribuées en sympatrie. À une échelle spatiale plus limitée, deux groupes génétiques bien différenciés ont été observés en sympatrie chez A. bipindensis. Ceux-ci présentent une différenciation morphologique et un décalage phénologique de la floraison qui peut contribuer à leur isolement reproductif.Cette étude a permis de mettre en évidence quelques points importants que sont : la découverte d’un complexe polyploïde au sein du genre Afzelia ; la confirmation de la délimitation des espèces diploïdes de savane ; et la nécessité de réviser la taxonomie des espèces tétraploïdes forestières.