Abstract Deciphering the genetic architecture of complex quantitative phenotypes remains challenging in quantitative genetics. These traits not only depend of multiple genetic factors but are also established over time and environments. Although quantitative genetics has investigated the genetic determinism of phenotypic plasticity in contrasted environmental conditions, the time related phenotypic plasticity has received less attention. Here we proposed a multivariate Bayesian framework, the Bayesian Varying Coefficient Model, designed for analysing the genetic architecture of the time related phenotypic plasticity by a multilocus approach. We applied the BVCM to time series phenotypes measured at various time scales (daily, monthly, yearly) across a diverse set of biological species. We included in this study: yeast ( Saccharomyces cerevisiae ), fungi ( Fusarium graminearum ), eucalyptus ( Eucalyptus urophylla × E. grandis ), and sweet cherry tree ( Prunus avium ). The BVCM results were compared with those obtained with a known genome-wide association method carried out time by time. For all species and traits, the BVCM was able to detect the major QTL identified by marker-trait association methods and revealed additional genetic regions of weak effect. It also increased the phenotypic variance explained for most of the phenotypes considered. It revealed dynamic QTLs with transitory, increasing or decreasing effects over time. By considering both the temporal and genetic multivariate structures in a single statistical model, we increased our understanding of the genetic architecture of complex traits notably by reducing the issue of missing heritability. More broadly, this work raises the foundation for extended applications in functional genomics, evolutionary ecology, and crop breeding programs, in which time-related phenotypic plasticity remains crucial for predicting and selecting key quantitative complex traits. Key message By capturing the genetic factors influencing the time related phenotypic plasticity, our approach contributes to a deeper understanding of the dynamic nature of genotype–phenotype relationships.

Prunus subgenus Cerasus contains numerous species with ornamental, edible, and medicinal value. However, limited genomic resources have constrained systematic analyses of structural variation and the genetic basis of key phenological traits in this group. Here, we assemble eight genomes from diverse Cerasus species. Together with 13 published genomes, we construct a pangenome of 21 accessions representing 17 species. Phenological observations reveal substantial variation in flowering time. Integrating comparative genomics, transcriptomics, and population genetic analyses highlight candidate regulators of flowering time. We find that AGAMOUS-LIKE 9 (AGL9) is strongly associated with flowering progression. Both ectopic expression and transient overexpression of PavAGL9 can accelerate post-dormancy flowering progression. We reveal that PavBPC6 binds the PavAGL9 promoter and represses its transcription, indicating a negative regulatory role. Furthermore, PavAGL9 interacts physically with PavSEP1 and PavPMADS2, suggesting synergistic roles in floral organ development. Our pangenome resource establishes a comprehensive genomic framework for Cerasus and provides insights into the regulation of flowering progression.

Background Structural variation represents a substantial fraction of genetic diversity but remains incompletely characterized using single-reference genomes. In sweet cherry ( Prunus avium L.), breeding has focused on a few loci controlling fruit quality, yet the associated structural variation and genome-wide patterns of diversity remain poorly understood. Results We constructed a chromosome-scale haplotype-resolved graph pangenome from 27 high-quality genome assemblies representing diverse breeding levels and geographic origins. The pangenome comprised 1.42 Gb of graph sequence with a small core and large accessory fraction, indicating an open and diverse genome despite chromosome-scale collinearity among accessions. We identified over 150,000 structural variants along with millions of SNPs and short indels, revealing extensive polymorphism across chromosomes. Integrating SNPs and structural variants within the graph framework enabled genome-wide association analyses for major fruit traits, including fruit weight and maturity date, identifying candidate loci on chromosomes 2 and 4 overlapping previously reported QTL hotspots. Several associated structural variants were located near genes involved in cellular growth and ripening processes. Genome-wide differentiation between landrace and modern germplasm revealed localized differences in genome architecture in regions enriched for defense and specialized metabolism genes, with limited overlap between differentiation signals and trait-associated loci. Conclusions These findings suggest that modern breeding may have contributed to differentiation in a limited subset of genomic regions, while substantial diversity persists elsewhere in the genome. This haplotype-resolved pangenome provides a comprehensive framework for studying genome and trait architectures, and genomic differentiation in sweet cherry, illustrating the value of graph-based genomic resources for crop breeding.

Intravaginal sponges impregnated with the progesterone (P4) analogue fluorogestone acetate (FGA) induce synchronous oestrous behaviour and normal ovulatory cycle in goats. To explore alternatives using natural P4 from plants, we developed a method of ethanolic extraction and a specific enzyme immunoassay (EIA) to measure P4 in the different parts of the walnut tree Juglans regia. We found a very high concentration of P4, specifically in the leaves of the three most common French varieties (similar to 100 mg/kg of DM) but not in flowers, fruits, septa, husk, oil or cake. High concentrations of P4-and to a lesser extent its reduction metabolites and phytosterols-were also measured by Gas Chromatography-Mass Spectrometry/Mass Spectrometry in leaf extracts. P4 concentrations were five times higher in October than in June. P4 was detected in 182 varieties of Juglans regia ranging from 35 to 287 mg of P4 per kg of leaf DM. We collected large quantities of leaves over 6 years, which were used to manufacture feed pellets containing 32% of dry leaf for distribution to female goats. To determine their dietary acceptance and their efficacy in terms of P4 blood plasma concentration, three trials in ovariectomised goats and four trials in ovary-intact goats were performed (N = 83). The distribution of 600 g of pellets per day per ovary-intact goat over 3 days, 6 and 4 days before the introduction of males in April allowed us to achieve our objective of a significant increase of P4 plasma concentration to similar to 1.5 ng/mL measured by EIA from 24 to 72 h after the first distribution in the walnut pellet group (n = 13). The two control groups of goats (FGA, n = 12 and control, n = 10) showed no increase in plasma P4. However, despite this high P4 plasma concentration, goats of the walnut group had the same percentages of goats in oestrus at the first ovulation and of goats experiencing short ovulatory cycles after introduction of males (54 and 77%, respectively) as the group of control goats (80 and 90%), whereas the FGA goats showed very different percentages (100 and 0%, P < 0.01). It was concluded that whereas walnut leaves contain a high concentration of P4-and its reduction metabolites and phytosterols-the pellet feeding mode does not allow for restoration of oestrus behaviour and duration of the induced cycle consistently achieved with FGA-impregnated intravaginal sponges.

Cherries are one of the economically important fruit crops in the Rosaceae family,Prunus genus.As the first fruits of the spring season in the northern hemisphere,their attractive appearance,intensely desirable tastes,high nutrients content,and consumer-friendly size captivate consumers worldwide.In the past 30 years,although cherry geneticists and breeders have greatly progressed in understanding the genetic and molecular basis underlying fruit quality,adaptation to climate change,and biotic and abiotic stress resistance,the utilization of cherry genomic data in genetics and molecular breeding has remained limited to date.Here,we thoroughly investigated recent discoveries in constructing genetic linkage maps,identifying quantitative trait loci(QTLs),genome-wide association studies(GWAS),and validating functional genes of edible cherries based on available de novo genomes and genome resequencing data of edible cherries.We further comprehensively demonstrated the genetic architecture of the main agronomic traits of edible cherries by methodically integrating QTLs,GWAS loci,and functional genes into the identical reference genome with improved annotations.These collective endeavors will offer new perspectives on the availability of sequence data and the construction of an interspecific pangenome of edible cherries,ultimately guiding cherry breeding strategies and genetic improvement programs,and facilitating the exploration of similar traits and breeding innovations across Prunus species.

Persian walnut ( Juglans regia L.) is a widespread cultivated nut tree species in temperate regions. Advances in genomic tools, such as the high‐density Axiom J. regia 700K single nucleotide polymorphism (SNP) genotyping array, enable the exploration of genomic prediction (GP) for this crop. This study is the first to evaluate GP accuracy and several influencing factors in walnut for traits related to phenology and nut quality. A core‐collection of 170 accessions was phenotyped for 25 traits over 1 or 2 years. Highly heritable traits, such as budbreak date and female flowering date, were predicted with high accuracy (∼0.75) using ridge regression best linear unbiased prediction (rrBLUP). Three key factors influencing GP performance were examined: marker density, prediction model, and training set size. Selecting the top 1% of 364,275 SNPs based on their variance (∼3600 SNPs) was sufficient to achieve accurate predictions. Bayesian models slightly improved prediction accuracy for some traits when using this reduced SNP set, but rrBLUP provided robust results, balancing accuracy, simplicity, and computational efficiency. Training population size also influenced accuracy, with a subset comprising 50% of the population still yielding reliable predictions. Optimization of training set was assessed using coefficient of determination mean, prediction error variance mean, and mean relatedness (MeanRel) parameters, with MeanRel performing best for shell traits. However, incorporating SNPs identified in genome‐wide association study into the prediction models did not enhance accuracy. In summary, this study demonstrates the feasibility and potential of GPs for walnut breeding programs using a core‐collection, offering valuable insights for optimizing GP approaches in this crop.

Progesterone (P4) is an endogenous sex steroid hormone involved in the ovulatory cycle and pregnancy of animal species. In sheep and goats, P4 analogues are used to induce synchronized ovulations and oestrus behavior of the females. In humans, P4 from chemical synthesis is used to treat peri-menopausal disorders. However, such molecules are released into aquatic environment and can be a source of pollution, are prohibited in organic farms and go against the trend of “naturality” in animal production as well as in human health. A natural alternative may consist in the extraction and use of P4 in plants. Mammalian hormones were discovered in an increasing number of plant species, including walnut leaves that contain high levels of P4. We compared the content of P4 in leaves of 170 accessions of Juglans regia from the walnut germplasm collection of INRAE Prunus-Juglans Biological Resources Center previously genotyped using the Axiom™ J. regia 700 K SNP array. We conducted a genome-wide association study (GWAS) using multi-locus models. When collected in October, P4 content goes from 34,1 to 287,5 mg/kg dry weight of leaves. The two laciniate accessions have the largest P4 content. We identified seven significant marker-trait associations on chromosomes 1, 2, 3, 6, 7, 15 and 16, and a candidate gene involved in the metabolism of sterols, precursors of plant steroid hormones. Our results raise the huge variability of P4 content within J. regia and propose a candidate gene which may have a role in the control of this variability, opening the way to a potential use of walnut P4 by the pharmaceutical industry towards more natural source of chemical compounds.

Cerasus is a subgenus of Prunus in the family Rosaceae that is popular owing to its ornamental, edible, and medicinal properties. Understanding the evolution of the Cerasus subgenus and identifying selective trait loci in edible cherries are crucial for the improvement of cherry cultivars to meet producer and consumer demands. In this study, we performed a de novo assembly of a chromosome-scale genome for the sweet cherry (Prunus avium L.) cultivar ‘Burlat’, covering 297.55 Mb and consisting of eight chromosomes with 33,756 protein-coding genes. The resequencing and population structural analysis of 384 Cerasus representative accessions revealed that they could be divided into four groups (Group 1, Group 2, Group 3, and Group 4). We inferred that Group 1 was the oldest population and Groups 2, 3, and 4 were clades derived from it. In addition, we found selective sweeps for fruit flavor and improved stress resistance in different varieties of edible cherries (P. avium, P. cerasus, and P. pseudocerasus). Transcriptome analysis revealed significant differential expression of genes associated with key pathways, such as sucrose starch and sucrose metabolism, fructose and mannose metabolism, and the pentose phosphate pathway, between the leaves and fruits of P. avium. This study enhances the understanding of the evolutionary processes of the Cerasus subgenus and provides resources for functional genomics research and the improvement of edible cherries.

In sweet cherry (Prunus avium L.), flowering date is strongly dependent on the environment conditions and, therefore, is a trait of major interest for adaptation to climate change. Such trait can be influenced by genotype-by-environment interaction (G×E), that refers to differences in the response of genotypes to different environments. If not taken into account, G×E can reduce selection accuracy and overall genetic gain. However, little is known about G×E in fruit tree species. Flowering date is a highly heritable and polygenic trait for which many quantitative trait loci (QTLs) have been identified. As for the overall genetic performance, differential expression of QTLs in response to environment (QTL-by-environment interaction, QTL×E) can occur. The present study is based on the analysis of a multi-environment trial (MET) suitable for the study of G×E and QTL×E in sweet cherry. It consists of a sweet cherry F1 full-sib family (n = 121) derived from the cross between cultivars ‘Regina’ and ‘Lapins’ and planted in two copies in five locations across four European countries (France, Italy, Slovenia and Spain) covering a large range of climatic conditions. The aim of this work was to study the effect of the environment on flowering date and estimate G×E, to carry QTL detection in different environments in order to study the QTL stability across environments and to estimate QTL×E. A strong effect of the environment on flowering date and its genetic control was highlighted. Two large-effect and environment-specific QTLs with significant QTL×E were identified on linkage groups (LGs) 1 and 4. This work gives new insights into the effect of the environment on a trait of main importance in one of the most economically important fruit crops in temperate regions. Moreover, molecular markers were developed for flowering date and a strategy consisting in using specific markers for warm or cold regions was proposed to optimize marker-assisted selection (MAS) in sweet cherry breeding programs.

Prunus pusilliflora is a wild cherry germplasm resource distributed mainly in Southwest China. Despite its ornamental and economic value, a high-quality assembled P. pusilliflora genome is unavailable, hindering our understanding of its genetic background, population diversity, and evolutionary processes. Here, we de novo assembled a chromosome-scale P. pusilliflora genome using Oxford Nanopore, Illumina, and chromosome conformation capture sequencing. The assembled genome size was 309.62 Mb, with 76 scaffolds anchored to eight pseudochromosomes. We predicted 33 035 protein-coding genes, functionally annotated 98.27% of them, and identified repetitive sequences covering 49.08% of the genome. We found that P. pusilliflora is closely related to Prunus serrulata and Prunus yedoensis, having diverged from them ~41.8 million years ago. A comparative genomic analysis revealed that P. pusilliflora has 643 expanded and 1128 contracted gene families. Furthermore, we found that P. pusilliflora is more resistant to Colletotrichum viniferum, Phytophthora capsici, and Pseudomonas syringae pv. tomato (Pst) DC3000 infections than cultivated Prunus avium. P. pusilliflora also has considerably more nucleotide-binding site-type resistance gene analogs than P. avium, which explains its stronger disease resistance. The cytochrome P450 and WRKY families of 263 and 61 proteins were divided into 42 and 8 subfamilies respectively in P. pusilliflora. Furthermore, 81 MADS-box genes were identified in P. pusilliflora, accompanying expansions of the SVP and AGL15 subfamilies and loss of the TM3 subfamily. Our assembly of a high-quality P. pusilliflora genome will be valuable for further research on cherries and molecular breeding.

Flowering and fruit production in sweet cherry (Prunus avium) are strongly environment-dependent. Climate change, by inducing increased temperatures in winter and spring, can lead to strong yield and economic losses. In Winter, chilling requirements for dormancy release are not satisfied, which causes a delay in the release of dormancy and flowering disorders. In the Spring, it induces earlier blooming and a higher frost risk. Thus, increasing our knowledge about the genetic control of flowering and fruit-related traits in sweet cherry is a key step to obtain new cultivars well adapted to changing environment and to ensure the production of cherry in the future. The genetic determinism of flowering date and main agronomic and fruit quality traits such as maturity date, productivity, fruit weight, firmness and sugar/acidity content were evaluated. QTL detections were conducted on a sweet cherry F-1 population derived from the cross between cultivars 'Regina' and 'Lapins'. The population, composed of 122 individuals, was planted in five locations across four European countries (France, Italy, Slovenia and Spain), constituting a multi environment trial (MET) suitable for the study of genotype-by-environment interactions (GxE). Detection for flowering date confirmed the presence of a major QTL on linkage group 4 (LG4), explaining up to 40% of the phenotypic variation. This QTL is highly significant in all environments but Murcia, the only location characterized by a mild winter. In Murcia, another region was identified at the end of LG1 and is the major one, demonstrating the high effect of environmental conditions on the flowering process. Factor-analytical models were applied to the analysis of the MET and patterns of GxE were explored for all traits.

In sweet cherry (Prunus avium L.), large variability exists for various traits related to fruit quality. There is a need to discover the genetic architecture of these traits in order to enhance the efficiency of breeding strategies for consumer and producer demands. With this objective, a germplasm collection consisting of 116 sweet cherry accessions was evaluated for 23 agronomic fruit quality traits over 2-6 years, and characterized using a genotyping-by-sequencing approach. The SNP coverage collected was used to conduct a genome-wide association study using two multilocus models and three reference genomes. We identified numerous SNP-trait associations for global fruit size (weight, width, and thickness), fruit cracking, fruit firmness, and stone size, and we pinpointed several candidate genes involved in phytohormone, calcium, and cell wall metabolisms. Finally, we conducted a precise literature review focusing on the genetic architecture of fruit quality traits in sweet cherry to compare our results with potential colocalizations of marker-trait associations. This study brings new knowledge of the genetic control of important agronomic traits related to fruit quality, and to the development of marker-assisted selection strategies targeted towards the facilitation of breeding efforts.

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.

Due to the important economic development of the walnut, the current and future varietal choice in France does not seem sufficient to respond to the new constraints. It is necessary to adapt to global competition, climate change that is probably already leading to increased sanitary problems, and the reduction of plant protection products and inputs. The goal of the Ctifl, in collaboration with INRA and under support of the French walnut industry, is to initiate a new and more efficient breeding program. The walnut genetic resources and an intra specific progeny of Juglans regia will be studied. The objectives of the project are firstly to assess the genetic diversity of the walnut collection, and secondly to identify the genetic determinism of agronomic traits such as those related to phenology, tree architecture, susceptibility to different diseases and the quality of the fruit. The complete plant material will be phenotyped for the previous agronomic traits and genotyped using SSR and SNP markers. The study of the INRA walnut collection will allow the creation of a core collection and the identification of promising genitors. This collection and the progeny mapping population will be exploited for the study of the genetic determinism of the characters of interest by two complementary approaches: the quantitative trait loci and the association genetics (GWAS). Ultimately, the results will provide the information needed to implement a new marker-assisted breeding program by Ctifl.

Rain-induced fruit cracking is a major problem in sweet cherry cultivation. Basic research has been conducted to disentangle the physiological and mechanistic bases of this complex phenomenon, whereas genetic studies have lagged behind. The objective of this work was to disentangle the genetic determinism of rain-induced fruit cracking. We hypothesized that a large genetic variation would be revealed, by visual field observations conducted on mapping populations derived from well-contrasted cultivars for cracking tolerance. Three populations were evaluated over 7–8 years by estimating the proportion of cracked fruits for each genotype at maturity, at three different areas of the sweet cherry fruit: pistillar end, stem end, and fruit side. An original approach was adopted to integrate, within simple linear models, covariates potentially related to cracking, such as rainfall accumulation before harvest, fruit weight, and firmness. We found the first stable quantitative trait loci (QTLs) for cherry fruit cracking, explaining percentages of phenotypic variance above 20%, for each of these three types of cracking tolerance, in different linkage groups, confirming the high complexity of this trait. For these and other QTLs, further analyses suggested the existence of at least two-linked QTLs in each linkage group, some of which showed confidence intervals close to 5 cM. These promising results open the possibility of developing marker-assisted selection strategies to select cracking-tolerant sweet cherry cultivars. Further studies are needed to confirm the stability of the reported QTLs over different genetic backgrounds and environments and to narrow down the QTL confidence intervals, allowing the exploration of underlying candidate genes.

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 and transcriptional analyses, quantification of abscisic acid (ABA) and gibberellins (GAs), and modeling 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 biosynthesis PavNCED genes, and decreased prior to dormancy release. Following the observations that ABA levels are correlated with dormancy depth, we identified PavUG71B6, a sweet cherry UDP-GLYCOSYLTRANSFERASE gene that up-regulates active catabolism of ABA to ABA glucosyl ester (ABA-GE) and may be associated with low ABA content in the early cultivar. Subsequently, we modeled 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 modeling.

Elucidating the genetic determinants of fruit quality traits in walnut is essential to breed new cultivars meeting the producers and consumers' needs. We conducted a genome-wide association study (GWAS) using multi-locus models in a panel of 170 accessions of Juglans regia from the INRAE walnut germplasm collection, previously genotyped using the AxiomTM J. regia 700K SNP array. We phenotyped the panel for 25 fruit traits related to morphometrics, shape, volume, weight, ease of cracking, and nutritional composition. We found more than 60 marker-trait associations (MTAs), including a highly significant SNP associated with nut face diameter, nut volume and kernel volume on chromosome 14, and 5 additional associations were detected for walnut weight. We proposed several candidate genes involved in nut characteristics, such as a gene coding for a beta-galactosidase linked to several size-related traits and known to be involved in fruit development in other species. We also confirmed associations on chromosomes 5 and 11 with nut suture strength, recently reported by the University of California, Davis. Our results enhance knowledge of the genetic control of important agronomic traits related to fruit quality in walnut, and pave the way for the development of molecular markers for future assisted selection.

In France, walnut crop is the second largest fruit crop after apple, with 36,000 tons of in-shell walnuts produced in 2016, making France the 9th producer in the world with almost 20,000 ha devoted. The INRAE's walnut germplasm collection includes 253 accessions from worldwide thanks to the prospecting work of Eric Germain from 1977 to 2007, the former head of breeding program at INRAE of Bordeaux. Among them, 217 are Juglans regia accessions including 194 cultivars and 23 intraspecific hybrids, coming from the major growing areas such as North-America, Europe and Asia. The germplasm collection includes also 36 Juglans accessions of 14 related species from the two sections Rhysocaryon (J. nigra, J. hindsii, J. microcarpa, J. californica, J. major, J. mollis) and Cardiocaryon (J. sieboldiana, J. cathayensis, J. mandshurica, J. cinerea). In this study, 13 simple sequence repeat (SSR) markers, selected from the literature (10 genomic SSRs from J. nigra and 3 EST-SSRs from J. regia), were used to genotype the 253 accessions. All SSR loci were highly polymorphic with a range from 2 to 17 alleles/locus (mean: 8.92) considering all the J. regia accessions. The results indicate a high diversity among the genotypes which could be useful for the new French walnut improvement program. This analysis permitted to select 170 accessions that will be used for association genetics studies, using the 600K SNP Affymetrix((R)) array, in order to identify the genetic determinism of agronomic traits of interest such as those related to phenology, fruit and kernel quality, and susceptibility to different diseases.