Haplotype-resolved genome assembly of the diploid Rosa chinensis provides insight into the mechanisms underlying key ornamental traits.

Roses are consistently ranked at the forefront in cut flower production. Increasing demands of market and changing climate conditions have resulted in the need to further improve the diversity and quality of traits. However, frequent hybridization leads to highly heterozygous nature, including the allelic variants. Therefore, the absence of comprehensive genomic information leads to them making it challenging to molecular breeding. Here, two haplotype-resolved chromosome genomes for Rosa chinensis 'Chilong Hanzhu' (2n = 14) which is high heterozygous diploid old Chinese rose are generated. An amount of genetic variation (1,605,616 SNPs, 209,575 indels) is identified. 13,971 allelic genes show differential expression patterns between two haplotypes. Importantly, these differences hold valuable insights into regulatory mechanisms of traits. RcMYB114b can influence cyanidin-3-glucoside accumulation and the allelic variation in its promoter leads to differences in promoter activity, which as a factor control petal color. Moreover, gene family expansion may contribute to the abundance of terpenes in floral scents. Additionally, RcANT1, RcDA1, RcAG1 and RcSVP1 genes are involved in regulation of petal number and size under heat stress treatment. This study provides a foundation for molecular breeding to improve important characteristics of roses.

Molecular and genetic regulation of petal number variation in plants.

Floral forms with an increased number of petals, also known as double flower (DF) with great agronomic and economic values, have been selected and conserved in many domesticated plants, particularly in ornamentals. The molecular and genetic mechanisms that control this trait are therefore a hot topic, not only for scientists, but also for breeders. In this review, we summarize the current knowledge on the gene regulatory networks of flower initiation and development and known mutations that lead to variation of petal number in many species. Besides the well-accepted miR172/AP2-like module, for which many questions remain unanswered, we also discuss the current knowledge of other pathways in which mutations also lead to extra-petals formation, such as those involved in meristem development, hormones signaling, epigenetic regulations, and responses to environmental signals. We discuss how the concept of "natural mutants" and the recent advances in genomics and genome editing makes it possible to explore the molecular mechanisms underlying the DF formation, and how such knowledge could contribute to the future breeding and selection of this trait in more crops.

The scent of roses, a bouquet of fragrance diversity.

Roses have been domesticated since antiquity for their therapeutic, cosmetic, and ornamental properties. Their floral fragrance has great economic value, which has influenced the production of rose varieties. The production of rose water and essential oil is one of the most lucrative activities, supplying bioactive molecules to the cosmetic, pharmaceutical, and therapeutic industries. In recent years, major advances in molecular genetics, genomic, and biochemical tools have paved the way for the identification of molecules that make up the specific fragrance of various rose cultivars. The aim of this review is to highlight current knowledge on metabolite profiles, and more specifically on fragrance compounds, as well as the specificities and differences between rose species and cultivars belonging to different rose sections and how they contribute to modern roses fragrance.

Light-mediated anthocyanin biosynthesis in rose petals involves a balanced regulatory module comprising transcription factors RhHY5, RhMYB114a, and RhMYB3b.

Roses are significant botanical species with both ornamental and economic value, displaying diverse floral traits, particularly an extensive array of petal colors. The red pigmentation of rose petals is predominantly attributed to anthocyanin accumulation. However, the underlying regulatory mechanism of anthocyanin biosynthesis in roses remains elusive. This study presents a novel light-responsive regulatory module governing anthocyanin biosynthesis in rose petals, which involves the transcription factors RhHY5, RhMYB114a, and RhMYB3b. Under light conditions (1000-1500 µmol m-2 s-1), RhHY5 represses RhMYB3b expression and induces RhMYB114a expression, positively regulating anthocyanin biosynthesis in rose petals. Notably, activation of anthocyanin structural genes probably involves an interaction and synergy between RhHY5 and the MYB114a-bHLH3-WD40 complex. Additionally, RhMYB3b is activated by RhMYB114a to prevent excessive accumulation of anthocyanin. Conversely, under low light conditions (<10 µmol m-2 s-1), the degradation of RhHY5 leads to down-regulation of RhMYB114a and up-regulation of RhMYB3b, which in turn inhibits the expression of both RhMYB114a and anthocyanin structural genes. Additionally, RhMYB3b competes with RhMYB114a for binding to RhbHLH3 and the promoters of anthocyanin-related structural genes. Overall, our study uncovers a complex light-mediated regulatory network that governs anthocyanin biosynthesis in rose petals, providing new insights into the molecular mechanisms underlying petal color formation in rose.

Comprehensive Genome-Wide Analysis of Histone Acetylation Genes in Roses and Expression Analyses in Response to Heat Stress.

Roses have high economic values as garden plants and for cut-flower and cosmetics industries. The growth and development of rose plants is affected by exposure to high temperature. Histone acetylation plays an important role in plant development and responses to various stresses. It is a dynamic and reversible process mediated by histone deacetylases (HDAC) and histone acetyltransferases (HAT). However, information on HDAC and HAT genes of roses is scarce. Here, 23 HDAC genes and 10 HAT genes were identified in the Rosa chinensis 'Old Blush' genome. Their gene structures, conserved motifs, physicochemical properties, phylogeny, and synteny were assessed. Analyses of the expression of HDAC and HAT genes using available RNAseq data showed that these genes exhibit different expression patterns in different organs of the three analyzed rose cultivars. After heat stress, while the expression of most HDAC genes tend to be down-regulated, that of HAT genes was up-regulated when rose plants were grown at high-temperature conditions. These data suggest that rose likely respond to high-temperature exposure via modification in histone acetylation, and, thus, paves the way to more studies in order to elucidate in roses the molecular mechanisms underlying rose plants development and flowering.

Tissue-Specific Expression of the Terpene Synthase Family Genes in Rosa chinensis and Effect of Abiotic Stress Conditions.

Rose (Rosa chinensis) is one of the most famous ornamental plants worldwide, with a variety of colors and fragrances. Terpene synthases (TPSs) play critical roles in the biosynthesis of terpenes. In this work, we report a comprehensive study on the genome-wide identification and characterization of the TPS family in R. chinensis. We identified 49 TPS genes in the R. chinensis genome, and they were grouped into five subfamilies (TPS-a, TPS-b, TPS-c, TPS-g and TPS-e/f). Phylogenetics, gene structure and conserved motif analyses indicated that the RcTPS genes possessed relatively conserved gene structures and the RcTPS proteins contained relatively conserved motifs. Multiple putative cis-acting elements involved in the stress response were identified in the promoter region of RcTPS genes, suggesting that some could be regulated by stress. The expression profile of RcTPS genes showed that they were predominantly expressed in the petals of open flowers, pistils, leaves and roots. Under osmotic and heat stresses, the expression of most RcTPS genes was upregulated. These data provide a useful foundation for deciphering the functional roles of RcTPS genes during plant growth as well as addressing the link between terpene biosynthesis and abiotic stress responses in roses.

Integrated multi-omic data and analyses reveal the pathways underlying key ornamental traits in carnation flowers.

Carnation (Dianthus caryophyllus) is one of the most popular ornamental flowers in the world. Although numerous studies on carnations exist, the underlying mechanisms of flower color, fragrance, and the formation of double flowers remain unknown. Here, we employed an integrated multi-omics approach to elucidate the genetic and biochemical pathways underlying the most important ornamental features of carnation flowers. First, we assembled a high-quality chromosome-scale genome (636 Mb with contig N50 as 14.67 Mb) of D. caryophyllus, the 'Scarlet Queen'. Next, a series of metabolomic datasets was generated with a variety of instrumentation types from different parts of the flower at multiple stages of development to assess spatial and temporal differences in the accumulation of pigment and volatile compounds. Finally, transcriptomic data were generated to link genomic, biochemical, and morphological patterns to propose a set of pathways by which ornamental traits such as petal coloration, double flowers, and fragrance production are formed. Among them, the transcription factors bHLHs, MYBs, and a WRKY44 homolog are proposed to be important in controlling petal color patterning and genes such as coniferyl alcohol acetyltransferase and eugenol synthase are involved in the synthesis of eugenol. The integrated dataset of genomics, transcriptomics, and metabolomics presented herein provides an important foundation for understanding the underlying pathways of flower development and coloration, which in turn can be used for selective breeding and gene editing for the development of novel carnation cultivars.

Cantaloupe melon genome reveals 3D chromatin features and structural relationship with the ancestral cucurbitaceae karyotype.

Cucumis melo displays a large diversity of horticultural groups with cantaloupe melon the most cultivated type. Using a combination of single-molecule sequencing, 10X Genomics link-reads, high-density optical and genetic maps, and chromosome conformation capture (Hi-C), we assembled a chromosome scale C. melo var. cantalupensis Charentais mono genome. Integration of RNA-seq, MeDip-seq, ChIP-seq, and Hi-C data revealed a widespread compartmentalization of the melon genome, segregating constitutive heterochromatin and euchromatin. Genome-wide comparative and evolutionary analysis between melon botanical groups identified Charentais mono genome increasingly more divergent from Harukei-3 (reticulatus), Payzawat (inodorus), and HS (ssp. agrestis) genomes. To assess the paleohistory of the Cucurbitaceae, we reconstructed the ancestral Cucurbitaceae karyotype and compared it to sequenced cucurbit genomes. In contrast to other species that experienced massive chromosome shuffling, melon has retained the ancestral genome structure. We provide comprehensive genomic resources and new insights in the diversity of melon horticultural groups and evolution of cucurbits.

Integrative genome-wide analysis reveals the role of WIP proteins in inhibition of growth and development.

In cucurbits, CmWIP1 is a master gene controlling sex determination. To bring new insight in the function of CmWIP1, we investigated two Arabidopsis WIP transcription factors, AtWIP1/TT1 and AtWIP2/NTT. Using an inducible system we showed that WIPs are powerful inhibitor of growth and inducer of cell death. Using ChIP-seq and RNA-seq we revealed that most of the up-regulated genes bound by WIPs display a W-box motif, associated with stress signaling. In contrast, the down-regulated genes contain a GAGA motif, a known target of polycomb repressive complex. To validate the role of WIP proteins in inhibition of growth, we expressed AtWIP1/TT1 in carpel primordia and obtained male flowers, mimicking CmWIP1 function in melon. Using other promoters, we further demonstrated that WIPs can trigger growth arrest of both vegetative and reproductive organs. Our data supports an evolutionary conserved role of WIPs in recruiting gene networks controlling growth and adaptation to stress.

Mapping a double flower phenotype-associated gene DcAP2L in Dianthus chinensis.

The double flower is a highly important breeding trait that affects the ornamental value in many flowering plants. To get a better understanding of the genetic mechanism of double flower formation in Dianthus chinensis, we have constructed a high-density genetic map using 140 F2 progenies derived from a cross between a single flower genotype and a double flower genotype. The linkage map was constructed using double-digest restriction site-associated DNA sequencing (ddRAD-seq) with 2353 single nucleotide polymorphisms (SNPs). Quantitative trait locus (QTL) mapping analysis was conducted for 12 horticultural traits, and major QTLs were identified for nine of the 12 traits. Among them, two major QTLs accounted for 20.7% and 78.1% of the total petal number variation, respectively. Bulked segregant RNA-seq (BSR-seq) was performed to search accurately for candidate genes associated with the double flower trait. Integrative analysis of QTL mapping and BSR-seq analysis using the reference genome of Dianthus caryophyllus suggested that an SNP mutation in the miR172 cleavage site of the A-class flower organ identity gene APETALA2 (DcAP2L) is responsible for double flower formation in Dianthus through regulating the expression of DcAG genes.

The development of a high-density genetic map significantly improves the quality of reference genome assemblies for rose.

Roses are important woody plants featuring a set of important traits that cannot be investigated in traditional model plants. Here, we used the restriction-site associated DNA sequencing (RAD-seq) technology to develop a high-density linkage map of the backcross progeny (BC1F1) between Rosa chinensis 'Old Blush' (OB) and R. wichuraiana 'Basyes' Thornless' (BT). We obtained 643.63 million pair-end reads and identified 139,834 polymorphic tags that were distributed uniformly in the rose genome. 2,213 reliable markers were assigned to seven linkage groups (LGs). The length of the genetic map was 1,027.425 cM in total with a mean distance of 0.96 cM per marker locus. This new linkage map allowed anchoring an extra of 1.21/23.14 Mb (12.18/44.52%) of the unassembled OB scaffolds to the seven reference pseudo-chromosomes, thus significantly improved the quality of assembly of OB reference genome. We demonstrate that, while this new linkage map shares high collinearity level with strawberry genome, it also features two chromosomal rearrangements, indicating its usefulness as a resource for understanding the evolutionary scenario among Rosaceae genomes. Together with the newly released genome sequences for OB, this linkage map will facilitate the identification of genetic components underpinning key agricultural and biological traits, hence should greatly advance the studies and breeding efforts of rose.

TCTP and CSN4 control cell cycle progression and development by regulating CULLIN1 neddylation in plants and animals.

Translationally Controlled Tumor Protein (TCTP) controls growth by regulating the G1/S transition during cell cycle progression. Our genetic interaction studies show that TCTP fulfills this role by interacting with CSN4, a subunit of the COP9 Signalosome complex, known to influence CULLIN-RING ubiquitin ligases activity by controlling CULLIN (CUL) neddylation status. In agreement with these data, downregulation of CSN4 in Arabidopsis and in tobacco cells leads to delayed G1/S transition comparable to that observed when TCTP is downregulated. Loss-of-function of AtTCTP leads to increased fraction of deneddylated CUL1, suggesting that AtTCTP interferes negatively with COP9 function. Similar defects in cell proliferation and CUL1 neddylation status were observed in Drosophila knockdown for dCSN4 or dTCTP, respectively, demonstrating a conserved mechanism between plants and animals. Together, our data show that CSN4 is the missing factor linking TCTP to the control of cell cycle progression and cell proliferation during organ development and open perspectives towards understanding TCTP's role in organ development and disorders associated with TCTP miss-expression.

Biosynthesis of 2-Phenylethanol in Rose Petals Is Linked to the Expression of One Allele of RhPAAS.

Floral scent is one of the most important characters in horticultural plants. Roses (Rosa spp.) have been cultivated for their scent since antiquity. However, probably by selecting for cultivars with long vase life, breeders have lost the fragrant character in many modern roses, especially the ones bred for the cut flower market. The genetic inheritance of scent characters has remained elusive so far. In-depth knowledge of this quantitative trait is thus very much needed to breed more fragrant commercial cultivars. Furthermore, rose hybrids harbor a composite genomic structure, which complexifies quantitative trait studies. To understand rose scent inheritance, we characterized a segregating population from two diploid cultivars, Rosa × hybrida cv H190 and Rosa wichurana, which have contrasting scent profiles. Several quantitative trait loci for the major volatile compounds in this progeny were identified. One among these loci contributing to the production of 2-phenylethanol, responsible for the characteristic odor of rose, was found to be colocalized with a candidate gene belonging to the 2-phenylethanol biosynthesis pathway: the PHENYLACETALDEHYDE SYNTHASE gene RhPAAS An in-depth allele-specific expression analysis in the progeny demonstrated that only one allele was highly expressed and was responsible for the production of 2-phenylethanol. Unexpectedly, its expression was found to start early during flower development, before the production of the volatile 2-phenylethanol, leading to the accumulation of glycosylated compounds in petals.

A miR172 target-deficient AP2-like gene correlates with the double flower phenotype in roses.

One of the well-known floral abnormalities in flowering plants is the double-flower phenotype, which corresponds to flowers that develop extra petals, sometimes even containing entire flowers within flowers. Because of their highly priced ornamental value, spontaneous double-flower variants have been found and selected for in a wide range of ornamental species. Previously, double flower formation in roses was associated with a restriction of AGAMOUS expression domain toward the centre of the meristem, leading to extra petals. Here, we characterized the genomic region containing the mutation associated with the switch from simple to double flowers in the rose. An APETALA2-like gene (RcAP2L), a member of the Target Of EAT-type (TOE-type) subfamily, lies within this interval. In the double flower rose, two alleles of RcAP2L are present, one of which harbours a transposable element inserted into intron 8. This insertion leads to the creation of a miR172 resistant RcAP2L variant. Analyses of the presence of this variant in a set of simple and double flower roses demonstrate a correlation between the presence of this allele and the double flower phenotype. These data suggest a role of this miR172 resistant RcAP2L variant in regulating RcAGAMOUS expression and double flower formation in Rosa sp.

Genome-Wide Identification and Characterization of Aquaporins and Their Role in the Flower Opening Processes in Carnation (Dianthus caryophyllus).

Aquaporins (AQPs) are associated with the transport of water and other small solutes across biological membranes. Genome-wide identification and characterization will pave the way for further insights into the AQPs' roles in the commercial carnation (Dianthus caryophyllus). This study focuses on the analysis of AQPs in carnation (DcaAQPs) involved in flower opening processes. Thirty DcaAQPs were identified and grouped to five subfamilies: nine PIPs, 11 TIPs, six NIPs, three SIPs, and one XIP. Subsequently, gene structure, protein motifs, and co-expression network of DcaAQPs were analyzed and substrate specificity of DcaAQPs was predicted. qRT-PCR, RNA-seq, and semi-qRTRCR were used for DcaAQP genes expression analysis. The analysis results indicated that DcaAQPs were relatively conserved in gene structure and protein motifs, that DcaAQPs had significant differences in substrate specificity among different subfamilies, and that DcaAQP genes' expressions were significantly different in roots, stems, leaves and flowers. Five DcaAQP genes (DcaPIP1;3, DcaPIP2;2, DcaPIP2;5, DcaTIP1;4, and DcaTIP2;2) might play important roles in flower opening process. However, the roles they play are different in flower organs, namely, sepals, petals, stamens, and pistils. Overall, this study provides a theoretical basis for further functional analysis of DcaAQPs.

The Rosa genome provides new insights into the domestication of modern roses.

Roses have high cultural and economic importance as ornamental plants and in the perfume industry. We report the rose whole-genome sequencing and assembly and resequencing of major genotypes that contributed to rose domestication. We generated a homozygous genotype from a heterozygous diploid modern rose progenitor, Rosa chinensis 'Old Blush'. Using single-molecule real-time sequencing and a meta-assembly approach, we obtained one of the most comprehensive plant genomes to date. Diversity analyses highlighted the mosaic origin of 'La France', one of the first hybrids combining the growth vigor of European species and the recurrent blooming of Chinese species. Genomic segments of Chinese ancestry identified new candidate genes for recurrent blooming. Reconstructing regulatory and secondary metabolism pathways allowed us to propose a model of interconnected regulation of scent and flower color. This genome provides a foundation for understanding the mechanisms governing rose traits and should accelerate improvement in roses, Rosaceae and ornamentals.

Identification and Characterization of the MADS-Box Genes and Their Contribution to Flower Organ in Carnation (Dianthus caryophyllus L.).

Dianthus is a large genus containing many species with high ornamental economic value. Extensive breeding strategies permitted an exploration of an improvement in the quality of cultivated carnation, particularly in flowers. However, little is known on the molecular mechanisms of flower development in carnation. Here, we report the identification and description of MADS-box genes in carnation (DcaMADS) with a focus on those involved in flower development and organ identity determination. In this study, 39 MADS-box genes were identified from the carnation genome and transcriptome by the phylogenetic analysis. These genes were categorized into four subgroups (30 MIKCc, two MIKC*, two Mα, and five Mγ). The MADS-box domain, gene structure, and conserved motif compositions of the carnation MADS genes were analysed. Meanwhile, the expression of DcaMADS genes were significantly different in stems, leaves, and flower buds. Further studies were carried out for exploring the expression of DcaMADS genes in individual flower organs, and some crucial DcaMADS genes correlated with their putative function were validated. Finally, a new expression pattern of DcaMADS genes in flower organs of carnation was provided: sepal (three class E genes and two class A genes), petal (two class B genes, two class E genes, and one SHORT VEGETATIVE PHASE (SVP)), stamen (two class B genes, two class E genes, and two class C), styles (two class E genes and two class C), and ovary (two class E genes, two class C, one AGAMOUS-LIKE 6 (AGL6), one SEEDSTICK (STK), one B sister, one SVP, and one Mα). This result proposes a model in floral organ identity of carnation and it may be helpful to further explore the molecular mechanism of flower organ identity in carnation.

The Arabidopsis thaliana F-box gene HAWAIIAN SKIRT is a new player in the microRNA pathway.

In Arabidopsis, the F-box HAWAIIAN SKIRT (HWS) protein is important for organ growth. Loss of function of HWS exhibits pleiotropic phenotypes including sepal fusion. To dissect the HWS role, we EMS-mutagenized hws-1 seeds and screened for mutations that suppress hws-1 associated phenotypes. We identified shs-2 and shs-3 (suppressor of hws-2 and 3) mutants in which the sepal fusion phenotype of hws-1 was suppressed. shs-2 and shs-3 (renamed hst-23/hws-1 and hst-24/hws-1) carry transition mutations that result in premature terminations in the plant homolog of Exportin-5 HASTY (HST), known to be important in miRNA biogenesis, function and transport. Genetic crosses between hws-1 and mutant lines for genes in the miRNA pathway also suppress the phenotypes associated with HWS loss of function, corroborating epistatic relations between the miRNA pathway genes and HWS. In agreement with these data, accumulation of miRNA is modified in HWS loss or gain of function mutants. Our data propose HWS as a new player in the miRNA pathway, important for plant growth.