Synergistic approach of PCR-based fragment length analysis and amplicon deep sequencing reveals rich diversity of S-alleles in sweet cherries from the Caucasian region of origin.

Introduction: The self-incompatibility system in sweet cherry (Prunus avium L.) prevents fertilization with own or genetically related pollen, and is genetically determined by the multi-allelic S-locus. Therefore, determining S-alleles is crucial for plant breeding and fruit production, as it enables the selection of compatible combinations of S-genotypes for successful pollination. Methods: In this study, S-alleles were identified in a total of 260 genotypes from the Caucasian region, the species' center of origin. S-allele genotyping was conducted using PCR fragment length analysis with the standard marker PaConsI-F/R2 and reference genotypes, complemented by sequence analysis through amplicon deep sequencing. Results and discussion: The genotypes collected from Azerbaijan and Turkey exhibit a high allelic richness at the S-locus, particularly compared to modern sweet cherry cultivars worldwide. Nine previously undescribed S-alleles were identified and designated as S45, S46, S47, S48, S49, S50, S51, S52 and S53. Given the expected high diversity for other traits, this plant material represents a valuable resource for further breeding research and introgression of new traits in future breeding programs. Furthermore, our results underscore that fragment length alone may not be sufficient for unambiguous assignment of S-alleles due to minimal length differences between different alleles. To address this issue, an S-allele reference ladder was developed using the rich diversity for precise assignment of the S-alleles. This tool can be applied in future experiments as a robust and cost-effective method for accurate S-genotyping across different runs and laboratories. Additionally, several selected S-genotypes were planted in a trial field and will be maintained as an S-allele reference collection.

Genotype-by-environment and QTL-by-environment interactions in sweet cherry (Prunus avium L.) for flowering date.

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.

Genome-wide association mapping in a sweet cherry germplasm collection (Prunus avium L.) reveals candidate genes for fruit quality 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.

New insights into flowering date in Prunus: fine mapping of a major QTL in sweet cherry.

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.

SSR-Based Analysis of Genetic Diversity and Structure of Sweet Cherry (Prunus avium L.) from 19 Countries in Europe.

Sweet cherry (Prunus avium L.) is a temperate fruit species whose production might be highly impacted by climate change in the near future. Diversity of plant material could be an option to mitigate these climate risks by enabling producers to have new cultivars well adapted to new environmental conditions. In this study, subsets of sweet cherry collections of 19 European countries were genotyped using 14 SSR. The objectives of this study were (i) to assess genetic diversity parameters, (ii) to estimate the levels of population structure, and (iii) to identify germplasm redundancies. A total of 314 accessions, including landraces, early selections, and modern cultivars, were monitored, and 220 unique SSR genotypes were identified. All 14 loci were confirmed to be polymorphic, and a total of 137 alleles were detected with a mean of 9.8 alleles per locus. The average number of alleles (N = 9.8), PIC value (0.658), observed heterozygosity (Ho = 0.71), and expected heterozygosity (He = 0.70) were higher in this study compared to values reported so far. Four ancestral populations were detected using STRUCTURE software and confirmed by Principal Coordinate Analysis (PCoA), and two of them (K1 and K4) could be attributed to the geographical origin of the accessions. A N-J tree grouped the 220 sweet cherry accessions within three main clusters and six subgroups. Accessions belonging to the four STRUCTURE populations roughly clustered together. Clustering confirmed known genealogical data for several accessions. The large genetic diversity of the collection was demonstrated, in particular within the landrace pool, justifying the efforts made over decades for their conservation. New sources of diversity will allow producers to face challenges, such as climate change and the need to develop more sustainable production systems.

Comparison of cell wall chemical evolution during the development of fruits of two contrasting quality from two members of the Rosaceae family: Apple and sweet cherry.

Cell wall composition was studied during the development of apple cultivars (14-161/182 days after full bloom, DAA) maintaining firm fruit (Ariane) or evolving to mealy texture (Rome Beauty) when ripe and in sweet cherry cultivars (21/26-70/75 DAA) to assess their skin-cracking susceptibility (tolerant Regina and susceptible Garnet). Pectin sugar composition and hemicellulose fine structure assessed by enzymatic degradation coupled to MALDI-TOF MS analysis were shown to vary markedly between apples and cherries during fruit development. Apple showed decreasing rhamnogalacturonan I (RGI) and increasing homogalacturonan (HG) pectic domain proportions from young to mature fruit. Hemicellulose-cellulose (HC) sugars peaked at the beginning of fruit expansion corresponding to the maximum cell wall content of glucose and mannose. In contrast, HG peaked very early in the cell wall of young developing cherries and remained constant until ripening whereas RGI content continuously increased. HC content decreased very early and remained low in cell walls. Only the low content of mannose and to a lesser extent fucose increased and then slowly decreased from the beginning of the fruit expansion phase. Hemicellulose structural profiling showed strong varietal differences between cherry cultivars. Both apples and cherries demonstrated a peak of glucomannan oligomers produced by ß-glucanase hydrolysis of the cell wall at the onset of cell expansion. The different glucomannan contents and related oligomers released from cell walls are discussed with regard to the contribution of glucomannan to cell wall mechanical properties. These hemicellulose features may prove to be early markers of apple mealiness and cherry skin-cracking susceptibility.

Multi-year analyses on three populations reveal the first stable QTLs for tolerance to rain-induced fruit cracking in sweet cherry (Prunus avium L.).

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.

A fruit firmness QTL identified on linkage group 4 in sweet cherry (Prunus avium L.) is associated with domesticated and bred germplasm.

Fruit firmness is an important market driven trait in sweet cherry (Prunus avium L.) where the desirable increase in fruit firmness is associated with landrace and bred cultivars. The aim of this work was to investigate the genetic basis of fruit firmness using plant materials that include wild cherry (syn. mazzard), landrace and bred sweet cherry germplasm. A major QTL for fruit firmness, named qP-FF4.1, that had not previously been reported, was identified in three sweet cherry populations. Thirteen haplotypes (alleles) associated with either soft or firm fruit were identified for qP-FF4.1 in the sweet cherry germplasm, and the "soft" alleles were dominant over the "firm" alleles. The finding that sweet cherry individuals that are homozygous for the "soft" alleles for qP-FF4.1 are exclusively mazzards and that the vast majority of the bred cultivars are homozygous for "firm" alleles suggests that this locus is a signature of selection. Candidate genes related to plant cell wall modification and various plant hormone signaling pathways were identified, with an expansin gene being the most promising candidate. These results advance our understanding of the genetic basis of fruit firmness and will help to enable the use of DNA informed breeding for this trait in sweet cherry breeding programs.

Prediction of genetic value for sweet cherry fruit maturity among environments using a 6K SNP array.

The timing of fruit maturity is an important trait in sweet cherry production and breeding. Phenotypic variation for phenology of fruit maturity in sweet cherry appears to be under strong genetic control, but that control might be complicated by phenotypic instability across environments. Although such genotype-by-environment interaction (G × E) is a common phenomenon in crop plants, knowledge about it is lacking for fruit maturity timing and other sweet cherry traits. In this study, 1673 genome-wide SNP markers were used to estimate genomic relationships among 597 weakly pedigree-connected individuals evaluated over two seasons at three locations in Europe and one location in the USA, thus sampling eight 'environments'. The combined dataset enabled a single meta-analysis to investigate the environmental stability of genomic predictions. Linkage disequilibrium among marker loci declined rapidly with physical distance, and ordination of the relationship matrix suggested no strong structure among germplasm. The most parsimonious G × E model allowed heterogeneous genetic variance and pairwise covariances among environments. Narrow-sense genomic heritability was very high (0.60-0.83), as was accuracy of predicted breeding values (>0.62). Average correlation of additive effects among environments was high (0.96) and breeding values were highly correlated across locations. Results indicated that genomic models can be used in cherry to accurately predict date of fruit maturity for untested individuals in new environments. Limited G × E for this trait indicated that phenotypes of individuals will be stable across similar environments. Equivalent analyses for other sweet cherry traits, for which multiple years of data are commonly available among breeders and cultivar testers, would be informative for predicting performance of elite selections and cultivars in new environments.

Yield potential definition of the chilling requirement reveals likely underestimation of the risk of climate change on winter chill accumulation.

Evaluation of chilling requirements of cultivars of temperate fruit trees provides key information to assess regional suitability, according to winter chill, for both industry expansion and ongoing profitability as climate change progresses. Traditional methods for calculating chilling requirements use climate-controlled chambers and define chilling requirements (CR) using a fixed bud burst percentage, usually close to 50% (CR-50%). However, this CR-50% definition may estimate chilling requirements that lead to flowering percentages that are lower than required for orchards to be commercially viable. We used sweet cherry to analyse the traditional method for calculating chilling requirements (CR-50%) and compared the results with a more restrictive method, where the chilling requirement was defined by a 90% bud break level (CRm-90%). For sweet cherry, this higher requirement of flowering success (90% as opposed to 50%) better represents grower production needs as a greater number of flowers leads to greater potential yield. To investigate the future risk of insufficient chill based on alternate calculations of the chilling requirement, climate projections of winter chill suitability across Europe were calculated using CR-50% and CRm-90%. Regional suitability across the landscape was highly dependent on the method used to define chilling requirements, and differences were found for both cold and mild winter areas. Our results suggest that bud break percentage levels used in the assessment of chilling requirements for sweet cherry influence production risks of current and future production areas. The use of traditional methods to determine chilling requirements can result in an underestimation of productivity chilling requirements for tree crops like sweet cherry which rely on a high conversion of flowers to mature fruit to obtain profitable yields. This underestimation may have negative consequences for the fruit industry as climate change advances with climate risk underestimated.

A collection of European sweet cherry phenology data for assessing climate change.

Professional and scientific networks built around the production of sweet cherry (Prunus avium L.) led to the collection of phenology data for a wide range of cultivars grown in experimental sites characterized by highly contrasted climatic conditions. We present a dataset of flowering and maturity dates, recorded each year for one tree when available, or the average of several trees for each cultivar, over a period of 37 years (1978-2015). Such a dataset is extremely valuable for characterizing the phenological response to climate change, and the plasticity of the different cultivars' behaviour under different environmental conditions. In addition, this dataset will support the development of predictive models for sweet cherry phenology exploitable at the continental scale, and will help anticipate breeding strategies in order to maintain and improve sweet cherry production in Europe.

Genetic diversity, linkage disequilibrium, population structure and construction of a core collection of Prunus avium L. landraces and bred cultivars.

BACKGROUND: Depiction of the genetic diversity, linkage disequilibrium (LD) and population structure is essential for the efficient organization and exploitation of genetic resources. The objectives of this study were to (i) to evaluate the genetic diversity and to detect the patterns of LD, (ii) to estimate the levels of population structure and (iii) to identify a 'core collection' suitable for association genetic studies in sweet cherry. RESULTS: A total of 210 genotypes including modern cultivars and landraces from 16 countries were genotyped using the RosBREED cherry 6 K SNP array v1. Two groups, mainly bred cultivars and landraces, respectively, were first detected using STRUCTURE software and confirmed by Principal Coordinate Analysis (PCoA). Further analyses identified nine subgroups using STRUCTURE and Discriminant Analysis of Principal Components (DAPC). Several sub-groups correspond to different eco-geographic regions of landraces distribution. Linkage disequilibrium was evaluated showing lower values than in peach, the reference Prunus species. A 'core collection' containing 156 accessions was selected using the maximum length sub tree method. CONCLUSION: The present study constitutes the first population genetics analysis in cultivated sweet cherry using a medium-density SNP (single nucleotide polymorphism) marker array. We provided estimations of linkage disequilibrium, genetic structure and the definition of a first INRA's Sweet Cherry core collection useful for breeding programs, germplasm management and association genetics studies.

Mapping of Candidate Genes Involved in Bud Dormancy and Flowering Time in Sweet Cherry (Prunus avium).

The timing of flowering in perennial plants is crucial for their survival in temperate climates and is regulated by the duration of bud dormancy. Bud dormancy release and bud break depend on the perception of cumulative chilling during endodormancy and heat during the bud development. The objectives of this work were to identify candidate genes involved in dormancy and flowering processes in sweet cherry, their mapping in two mapping progenies 'Regina' × 'Garnet' and 'Regina' × 'Lapins', and to select those candidate genes which co-localized with quantitative trait loci (QTLs) associated with temperature requirements for bud dormancy release and flowering. Based on available data on flowering processes in various species, a list of 79 candidate genes was established. The peach and sweet cherry orthologs were identified and primers were designed to amplify sweet cherry candidate gene fragments. Based on the amplified sequences of the three parents of the mapping progenies, SNPs segregations in the progenies were identified. Thirty five candidate genes were genetically mapped in at least one of the two progenies and all were in silico mapped. Co-localization between candidate genes and QTLs associated with temperature requirements and flowering date were identified for the first time in sweet cherry. The allelic composition of the candidate genes located in the major QTL for heat requirements and flowering date located on linkage group 4 have a significant effect on these two traits indicating their potential use for breeding programs in sweet cherry to select new varieties adapted to putative future climatic conditions.

Construction and comparative analyses of highly dense linkage maps of two sweet cherry intra-specific progenies of commercial cultivars.

Despite the agronomical importance and high synteny with other Prunus species, breeding improvements for cherry have been slow compared to other temperate fruits, such as apple or peach. However, the recent release of the peach genome v1.0 by the International Peach Genome Initiative and the sequencing of cherry accessions to identify Single Nucleotide Polymorphisms (SNPs) provide an excellent basis for the advancement of cherry genetic and genomic studies. The availability of dense genetic linkage maps in phenotyped segregating progenies would be a valuable tool for breeders and geneticists. Using two sweet cherry (Prunus avium L.) intra-specific progenies derived from crosses between 'Black Tartarian' × 'Kordia' (BT×K) and 'Regina' × 'Lapins'(R×L), high-density genetic maps of the four parental lines and the two segregating populations were constructed. For BT×K and R×L, 89 and 121 F(1) plants were used for linkage mapping, respectively. A total of 5,696 SNP markers were tested in each progeny. As a result of these analyses, 723 and 687 markers were mapped into eight linkage groups (LGs) in BT×K and R×L, respectively. The resulting maps spanned 752.9 and 639.9 cM with an average distance of 1.1 and 0.9 cM between adjacent markers in BT×K and R×L, respectively. The maps displayed high synteny and co-linearity between each other, with the Prunus bin map, and with the peach genome v1.0 for all eight LGs (LG1-LG8). These maps provide a useful tool for investigating traits of interest in sweet cherry and represent a qualitative advance in the understanding of the cherry genome and its synteny with other members of the Rosaceae family.

Hybrid performance in taro (Colocasia esculenta) in relation to genetic dissimilarity of parents.

Taro (Colocasia esculenta) breeding, as other root crop breeding, is based on the production and evaluation of large numbers of hybrids. The selection of parents is based on their phenotypic value in the absence of information concerning general combining ability (GCA), specific combining ability (SCA), or genetic distances between varieties. By combining data from heritability trials and from genetic diversity studies conducted with AFLP and SSR markers, we aimed at studying the relationship between hybrid vigour and genetic dissimilarity between parents. The traits studied included number of suckers, corm weight, corm dimensions, and dry matter content. Correlation coefficients between hybrid gain and dissimilarity values were calculated. The prediction of hybrid performance based on the mid-parent value was compared to the prediction based on a modified expression that takes into account the genetic relationships between parents. Correlations were all but one positive but not statistically significant for all traits, with the exception of the number of suckers, when using SSR markers for dissimilarity calculations. Accordingly, the genetic dissimilarities in the prediction of hybrid performances did not increase the correlation between predicted and observed hybrid vigour values. However, large differences were observed among the residual means from the regression between predicted and observed values when using AFLP or SSR markers, mainly due to the much higher polymorphism revealed by the latter. Models need to be further adapted to the type of molecular marker used, since their ability to reveal different rates of polymorphism will have a direct incidence on the calculation of genetic dissimilarities between genotypes. Nevertheless, since SSR markers are more polymorphic and more informative than AFLP markers, they should be preferentially used for these studies. Low genetic dissimilarity of parents yielded weak heterosis effects and future studies need to be conducted by using a broader genetic base. This is the first study assessing the relationship of hybrid vigour with the genetic distances between parents, conducted on a tropical root crop.