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.

Identification and Characterization of DAMs Mutations Associated With Early Blooming in Sweet Cherry, and Validation of DNA-Based Markers for Selection.

Dormancy release and bloom time of sweet cherry cultivars depend on the environment and the genotype. The knowledge of these traits is essential for cultivar adaptation to different growing areas, and to ensure fruit set in the current climate change scenario. In this work, the major sweet cherry bloom time QTL qP-BT1.1 m (327 Kbs; Chromosome 1) was scanned for candidate genes in the Regina cv genome. Six MADS-box genes (PavDAMs), orthologs to peach and Japanese apricot DAMs, were identified as candidate genes for bloom time regulation. The complete curated genomic structure annotation of these genes is reported. To characterize PavDAMs intra-specific variation, genome sequences of cultivars with contrasting chilling requirements and bloom times (N = 13), were then mapped to the 'Regina' genome. A high protein sequence conservation (98.8-100%) was observed. A higher amino acid variability and several structural mutations were identified in the low-chilling and extra-early blooming cv Cristobalina. Specifically, a large deletion (694 bp) upstream of PavDAM1, and various INDELs and SNPs in contiguous PavDAM4 and -5 UTRs were identified. PavDAM1 upstream deletion in 'Cristobalina' revealed the absence of several cis-acting motifs, potentially involved in PavDAMs expression. Also, due to this deletion, a non-coding gene expressed in late-blooming 'Regina' seems truncated in 'Cristobalina'. Additionally, PavDAM4 and -5 UTRs mutations revealed different splicing variants between 'Regina' and 'Cristobalina' PavDAM5. The results indicate that the regulation of PavDAMs expression and post-transcriptional regulation in 'Cristobalina' may be altered due to structural mutations in regulatory regions. Previous transcriptomic studies show differential expression of PavDAM genes during dormancy in this cultivar. The results indicate that 'Cristobalina' show significant amino acid differences, and structural mutations in PavDAMs, that correlate with low-chilling and early blooming, but the direct implication of these mutations remains to be determined. To complete the work, PCR markers designed for the detection of 'Cristobalina' structural mutations in PavDAMs, were validated in an F2 population and a set of cultivars. These PCR markers are useful for marker-assisted selection of early blooming seedlings, and probably low-chilling, from 'Cristobalina', which is a unique breeding source for these traits.

Fine tuning of hormonal signaling is linked to dormancy status in sweet cherry flower buds.

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.

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.

Genetic determinism of phenological traits highly affected by climate change in Prunus avium: flowering date dissected into chilling and heat requirements.

The present study investigated the genetic determinism of flowering date (FD), dissected into chilling (CR) and heat (HR) requirements. Elucidation of the genetic determinism of flowering traits is crucial to anticipate the increasing of ecological misalignment of adaptative traits with novel climate conditions in most temperate-fruit species. CR and HR were evaluated over 3 yr and FD over 5 yr in an intraspecific sweet cherry (Prunus avium) F1 progeny, and FD over 6 yr in a different F1 progeny. One quantitative trait locus (QTL) with major effect and high stability between years of evaluation was detected for CR and FD in the same region of linkage group (LG) 4. For HR, no stable QTL was detected. Candidate genes underlying the major QTL on LG4 were investigated and key genes were identified for CR and FD. Phenotypic dissection of FD and year repetitions allowed us to identify CR as the high heritable component of FD and a high genotype × environment interaction for HR. QTLs for CR reported in this study are the first described in this species. Our results provide a foundation for the identification of genes involved in CR and FD in sweet cherry which could be used to develop ideotypes adapted to future climatic conditions.