Cultivated strawberry ( Fragaria × ananassa), a perennial herb belonging to the family Rosaceae, is a complex octoploid with high heterozygosity at most loci. However, there is no research on the haplotype of the octoploid strawberry genome. Here we aimed to obtain a high-quality genome of the cultivated strawberry cultivar, “Yanli”, using single molecule real-time sequencing and high-throughput chromosome conformation capture technology. The “Yanli” genome was 823 Mb in size, with a long terminal repeat assembly index of 14.99. The genome was phased into two haplotypes, Hap1 (825 Mb with contig N50 of 26.70 Mb) and Hap2 (808 Mb with contig N50 of 27.51 Mb). Using the combination of Hap1 and Hap2, we obtained for the first time a haplotype-resolved genome with 56 chromosomes for the cultivated octoploid strawberry. We identified a ∼10 Mb inversion and translocation on chromosome 2-1. 104 957 and 102 356 protein-coding genes were annotated in Hap1 and Hap2, respectively. Analysis of the genes related to the anthocyanin biosynthesis pathway revealed the structural diversity and complexity in the expression of the alleles in the octoploid F. × ananassa genome. In summary, we obtained a high-quality haplotype-resolved genome assembly of F. × ananassa, which will provide the foundation for investigating gene function and evolution of the genome of cultivated octoploid strawberry.

Fruit color is an important horticultural trait, which greatly affects consumer preferences. In tomato, fruit color is determined by the accumulation of different pigments, such as carotenoids in the pericarp and flavonoids in the peel, along with the degradation of chlorophyll during fruit ripening. Since fruit color is a multigenic trait, it takes years to introgress all color-related genes in a single genetic background via traditional crossbreeding, and the avoidance of linkage drag during this process is difficult. Here, we proposed a rapid breeding strategy to generate tomato lines with different colored fruits from red-fruited materials by CRISPR/Cas9-mediated multiplex gene editing of three fruit color-related genes ( PSY1, MYB12, and SGR1). Using this strategy, the red-fruited cultivar ‘Ailsa Craig’ has been engineered to a series of tomato genotypes with different fruit colors, including yellow, brown, pink, light-yellow, pink-brown, yellow-green, and light green. Compared with traditional crossbreeding, this strategy requires less time and can obtain transgene-free plants with different colored fruits in less than 1 year. Most importantly, it does not alter other important agronomic traits, like yield and fruit quality. Our strategy has great practical potential for tomato breeding and serves as a reference for improving multigene-controlled traits of horticultural crops.

Gibberellins (GAs) play crucial roles in a wide range of developmental processes and stress responses in plants. However, the roles of GA-responsive genes in tomato ( Solanum lycopersicum) fruit development remain largely unknown. Here, we identify 17 GASA ( Gibberellic Acid-Stimulated Arabidopsis) family genes in tomato. These genes encode proteins with a cleavable signal peptide at their N terminus and a conserved GASA domain at their C terminus. The expression levels of all tomato GASA family genes were responsive to exogenous GA treatment, but adding ethylene eliminated this effect. Comprehensive expression profiling of SlGASA family genes showed that SlGASA1 follows a ripening-associated expression pattern, with low expression levels during fruit ripening, suggesting it plays a negative role in regulating ripening. Overexpressing SlGASA1 using a ripening-specific promoter delayed the onset of fruit ripening, whereas SlGASA1-knockdown fruits displayed accelerated ripening. Consistent with their delayed ripening, SlGASA1-overexpressing fruits showed significantly reduced ethylene production and carotenoid contents compared to the wild type. Moreover, ripening-related genes were downregulated in SlGASA1-overexpressing fruits but upregulated in SlGASA1-knockdown fruits compared to the wild type. Yeast two-hybrid, co-immunoprecipitation, transactivation, and DNA pull-down assays indicated that SlGASA1 interacts with the key ripening regulator FRUITFULL1 and represses its activation of the ethylene biosynthesis genes ACS2 and ACO1. Our findings shed new light on the role and mode of action of a GA-responsive gene in tomato fruit ripening.

Branch number is an important agronomic trait in peach ( Prunus persica) trees because plant architecture affects fruit yield and quality. Although breeders can select varieties with different tree architecture, the biological mechanisms underlying architecture remain largely unclear. In this study, a pillar peach (‘Zhaoshouhong’) and a standard peach (‘Okubo’) were compared. ‘Zhaoshouhong’ was found to have significantly fewer secondary branches than ‘Okubo’. Treatment with the synthetic strigolactone (SL) GR24 decreased branch number. Transcriptome analysis indicated that PpTCP18 (a homologous gene of Arabidopsis thaliana BRC1) expression was negatively correlated with strigolactone synthesis gene expression, indicating that PpTCP18 may play an important role in peach branching. Yeast one-hybrid, electrophoretic mobility shift, dual-luciferase assays and PpTCP18-knockdown in peach leaf buds indicated that PpTCP18 could increase expression of PpLBO1, PpMAX1, and PpMAX4. Furthermore, transgenic Arabidopsis plants overexpressing PpTCP18 clearly exhibited reduced primary rosette-leaf branches. Moreover, lncRNA sequencing and transient expression analysis revealed that lncRNA5 targeted PpTCP18, significantly increasing PpTCP18 expression. These results provide insights into the mRNA and lncRNA network in the peach SL signaling pathway and indicate that PpTCP18, a transcription factor downstream of SL signaling, is involved in positive feedback regulation of SL biosynthesis. This role of PpTCP18 may represent a novel mechanism in peach branching regulation. Our study improves current understanding of the mechanisms underlying peach branching and provides theoretical support for genetic improvement of peach tree architecture.

Fall dormancy (FD) is an essential trait to overcome winter damage and for alfalfa ( Medicago sativa) cultivar selection. The plant regrowth height after autumn clipping is an indirect way to evaluate FD. Transcriptomics, proteomics, and quantitative trait locus mapping have revealed crucial genes correlated with FD; however, these genes cannot predict alfalfa FD very well. Here, we conducted genomic prediction of FD using whole-genome SNP markers based on machine learning-related methods, including support vector machine (SVM) regression, and regularization-related methods, such as Lasso and ridge regression. The results showed that using SVM regression with linear kernel and the top 3000 genome-wide association study (GWAS)-associated markers achieved the highest prediction accuracy for FD of 64.1%. For plant regrowth height, the prediction accuracy was 59.0% using the 3000 GWAS-associated markers and the SVM linear model. This was better than the results using whole-genome markers (25.0%). Therefore, the method we explored for alfalfa FD prediction outperformed the other models, such as Lasso and ElasticNet. The study suggests the feasibility of using machine learning to predict FD with GWAS-associated markers, and the GWAS-associated markers combined with machine learning would benefit FD-related traits as well. Application of the methodology may provide potential targets for FD selection, which would accelerate genetic research and molecular breeding of alfalfa with optimized FD.

Annual rings from 30 year old vines in a California rootstock trial were measured to determine the effects of 15 different rootstocks on Chardonnay and Cabernet Sauvignon scions. Viticultural traits measuring vegetative growth, yield, berry quality, and nutrient uptake were collected at the beginning (1995 to 1999) and end (2017 to 2020) of the lifetime of a vineyard initially planted in 1991 and removed in 2021. X-ray Computed Tomography (CT) was used to measure ring widths in 103 vines. Ring width was modeled as a function of ring number using a negative exponential model. Early and late wood ring widths, cambium width, and scion trunk radius were correlated with 27 traits. Modeling of annual ring width shows that scions alter the width of the first rings but that rootstocks alter the decay of later rings, consistently shortening ring width throughout the lifetime of the vine. Ravaz index, juice pH, photosynthetic assimilation and transpiration rates, and instantaneous water use efficiency are correlated with scion trunk radius. Ultimately, our research indicates that rootstocks modulate secondary growth over years, altering physiology and agronomic traits. Rootstocks act in similar but distinct ways from climate to modulate ring width, which borrowing techniques from dendrochronology, can be used to monitor both genetic and environmental effects in woody perennial crop species.

Tomato ( Solanum lycopersicum) is among the most important vegetables across the world, but cold stress usually affects its yield and quality. The wild tomato species Solanum habrochaites is commonly utilized as rootstock for enhancing resistance against abiotic stresses in cultivated tomato, especially cold resistance. However, the underlying molecular mechanism remains unclear. In this research, we confirmed that S. habrochaites rootstock can improve the cold tolerance of cultivated tomato scions, as revealed by growth, physiological, and biochemical indicators. Furthermore, transcriptome profiling indicated significant differences in the scion of homo- and heterografted seedlings, including substantial changes in jasmonic acid (JA) biosynthesis and signaling, which were validated by RT–qPCR analysis. S. habrochaites plants had a high basal level of jasmonate, and cold stress caused a greater amount of active JA- isoleucine in S. habrochaites heterografts. Moreover, exogenous JA enhanced while JA inhibitor decreased the cold tolerance of tomato grafts. The JA biosynthesis-defective mutant spr8 also showed increased sensitivity to cold stress. All of these results demonstrated the significance of JA in the cold tolerance of grafted tomato seedlings with S. habrochaites rootstock, suggesting a future direction for the characterization of the natural variation involved in S. habrochaites rootstock-mediated cold tolerance.

Momordica charantia L. var. abbreviata Ser. (Mca), known as bitter gourd or bitter melon, is a Momordica variety with medicinal value and belongs to the Cucurbitaceae family. In view of the lack of genomic information on bitter gourd and other Momordica species and to promote Mca genomic research, we assembled a 295.6-Mb telomere-to-telomere (T2T) high-quality Mca genome with six gap-free chromosomes after Hi-C correction. This genome is anchored to 11 chromosomes, which is consistent with the karyotype information, and comprises 98 contigs (N50 of 25.4 Mb) and 95 scaffolds (N50 of 25.4 Mb). The Mca genome harbors 19 895 protein-coding genes, of which 45.59% constitute predicted repeat sequences. Synteny analysis revealed variations involved in fruit quality during the divergence of bitter gourd. In addition, assay for transposase-accessible chromatin by high-throughput sequencing and metabolic analysis showed that momordicosides and other substances are characteristic of Mca fruit pulp. A combined transcriptomic and metabolomic analysis revealed the mechanisms of pigment accumulation and cucurbitacin biosynthesis in Mca fruit peels, providing fundamental molecular information for further research on Mca fruit ripening. This report provides a new genetic resource for Momordica genomic studies and contributes additional insights into Cucurbitaceae phylogeny.

Tomato ( Solanum lycopersicum) is the most valuable fruit and horticultural crop species worldwide. Compared with the fruits of their progenitors, those of modern tomato cultivars are, however, often described as having unsatisfactory taste or lacking flavor. The flavor of a tomato fruit arises from a complex mix of tastes and volatile metabolites, including sugars, acids, amino acids, and various volatiles. However, considerable differences in fruit flavor occur among tomato varieties, resulting in mixed consumer experiences. While tomato breeding has traditionally been driven by the desire for continual increases in yield and the introduction of traits that provide a long shelf-life, consumers are prepared to pay a reasonable premium for taste. Therefore, it is necessary to characterize preferences of tomato flavor and to define its underlying genetic basis. Here, we review recent conceptual and technological advances that have rendered this more feasible, including multi-omics-based QTL and association analyses, along with the use of trained testing panels, and machine learning approaches. This review proposes how the comprehensive datasets compiled to date could allow a precise rational design of tomato germplasm resources with improved organoleptic quality for the future.

Genome duplication events, comprising whole-genome duplication and single-gene duplication, produce a complex genomic context leading to multiple levels of genetic changes. However, the characteristics of m 6A modification, the most widespread internal eukaryotic mRNA modification, in polyploid species are still poorly understood. This study revealed the characteristics of m 6A methylomes within the early formation and following the evolution of allopolyploid Brassica napus. We found a complex relationship between m 6A modification abundance and gene expression level depending on the degree of enrichment or presence/absence of m 6A modification. Overall, the m 6A genes had lower gene expression levels than the non-m 6A genes. Allopolyploidization may change the expression divergence of duplicated gene pairs with identical m 6A patterns and diverged m 6A patterns. Compared with duplicated genes, singletons with a higher evolutionary rate exhibited higher m 6A modification. Five kinds of duplicated genes exhibited distinct distributions of m 6A modifications in transcripts and gene expression level. In particular, tandem duplication-derived genes showed unique m 6A modification enrichment around the transcript start site. Active histone modifications (H3K27ac and H3K4me3) but not DNA methylation were enriched around genes of m 6A peaks. These findings provide a new understanding of the features of m 6A modification and gene expression regulation in allopolyploid plants with sophisticated genomic architecture.

Gynostemma pentaphyllum (Thunb.) Makino is a perennial creeping herbaceous plant in the family Cucurbitaceae, which has great medicinal value and commercial potential, but urgent conservation efforts are needed due to the gradual decreases and fragmented distribution of its wild populations. Here, we report the high-quality diploid chromosome-level genome of G. pentaphyllum obtained using a combination of next-generation sequencing short reads, Nanopore long reads, and Hi-C sequencing technologies. The genome is anchored to 11 pseudo-chromosomes with a total size of 608.95 Mb and 26 588 predicted genes. Comparative genomic analyses indicate that G. pentaphyllum is estimated to have diverged from Momordica charantia 60.7 million years ago, with no recent whole-genome duplication event. Genomic population analyses based on genotyping-by-sequencing and ecological niche analyses indicated low genetic diversity but a strong population structure within the species, which could classify 32 G. pentaphyllum populations into three geographical groups shaped jointly by geographic and climate factors. Furthermore, comparative transcriptome analyses showed that the genes encoding enzyme involved in gypenoside biosynthesis had higher expression levels in the leaves and tendrils. Overall, the findings obtained in this study provide an effective molecular basis for further studies of demographic genetics, ecological adaption, and systematic evolution in Cucurbitaceae species, as well as contributing to molecular breeding, and the biosynthesis and biotransformation of gypenoside.

Vegetable crops are greatly appreciated for their beneficial nutritional and health components. Hybrid seeds are widely used in vegetable crops for advantages such as high yield and improved resistance, which require the participation of male (stamen) and female (pistil) reproductive organs. Male- or female-sterile plants are commonly used for production of hybrid seeds or seedless fruits in vegetables. In this review we will focus on the types of genic male sterility and factors affecting female fertility, summarize typical gene function and research progress related to reproductive organ identity and sporophyte and gametophyte development in vegetable crops [mainly tomato ( Solanum lycopersicum) and cucumber ( Cucumis sativus)], and discuss the research trends and application perspectives of the sterile trait in vegetable breeding and hybrid production, in order to provide a reference for fertility-related germplasm innovation.

In sweet cherry ( Prunus avium L.), quantitative trait loci have been identified for fruit maturity, colour, firmness, and size to develop markers for marker-assisted selection. However, resolution is usually too low in those analyses to directly target candidate genes, and some associations are missed. In contrast, genome-wide association studies are performed on broad collections of accessions, and assemblies of reference sequences from Tieton and Satonishiki cultivars enable identification of single nucleotide polymorphisms after whole-genome sequencing, providing high marker density. Two hundred and thirty-five sweet cherry accessions were sequenced and phenotyped for harvest time and fruit colour, firmness, and size. Genome-wide association studies were used to identify single nucleotide polymorphisms associated with each trait, which were verified in breeding material consisting of 64 additional accessions. A total of 1 767 106 single nucleotide polymorphisms were identified. At that density, significant single nucleotide polymorphisms could be linked to co-inherited haplotype blocks (median size ∼10 kb). Thus, markers were tightly associated with respective phenotypes, and individual allelic combinations of particular single nucleotide polymorphisms provided links to distinct phenotypes. In addition, yellow-fruit accessions were sequenced, and a ∼90-kb-deletion on chromosome 3 that included five MYB10 transcription factors was associated with the phenotype. Overall, the study confirmed numerous quantitative trait loci from bi-parental populations using high-diversity accession populations, identified novel associations, and genome-wide association studies reduced the size of trait-associated loci from megabases to kilobases and to a few candidate genes per locus. Thus, a framework is provided to develop molecular markers and evaluate and characterize genes underlying important agronomic traits.

Sweetpotato is an important crop that exhibits hexaploidy and high heterozygosity, which limits gene mining for important agronomic traits. Here, 314 sweetpotato germplasm resources were deeply resequenced, and 4 599 509 SNPs and 846 654 InDels were generated, among which 196 124 SNPs were nonsynonymous and 9690 InDels were frameshifted. Based on the Indels, genome-wide marker primers were designed, and 3219 of 40 366 primer pairs were selected to construct the core InDel marker set. The molecular ID of 104 sweetpotato samples verified the availability of these primers. The sweetpotato population structures were then assessed through multiple approaches using SNPs, and diverse approaches demonstrated that population stratification was not obvious for most Chinese germplasm resources. As many as 20 important agronomic traits were evaluated, and a genome-wide association study was conducted on these traits. A total of 19 high-confidence loci were detected in both models. These loci included several candidate genes, such as IbMYB1, IbZEP1, and IbYABBY1, which might be involved in anthocyanin metabolism, carotenoid metabolism, and leaf morphogenesis, respectively. Among them, IbZEP1 and IbYABBY1 were first reported in sweetpotato. The variants in the promoter and the expression levels of IbZEP1 were significantly correlated with flesh color (orange or not orange) in sweetpotato. The expression levels of IbYABBY1 were also correlated with leaf shape. These results will assist in genetic and breeding studies in sweetpotato.

Tomato cultivars show wide variation in nutraceutical folate in ripe fruits, yet the loci regulating folate levels in fruits remain unexplored. To decipher regulatory points, we compared two contrasting tomato cultivars: Periyakulam-1 (PKM-1) with high folate and Arka Vikas (AV) with low folate. The progression of ripening in PKM-1 was nearly similar to AV but had substantially lower ethylene emission. In parallel, the levels of phytohormones salicylic acid, ABA, and jasmonic acid were substantially lower than AV. The fruits of PKM-1 were metabolically distinct from AV, with upregulation of several amino acids. Consistent with higher ◦Brix, the red ripe fruits also showed upregulation of sugars and sugar-derived metabolites. In parallel with higher folate, PKM-1 fruits also had higher carotenoid levels, especially lycopene and β-carotene. The proteome analysis showed upregulation of carotenoid sequestration and folate metabolism-related proteins in PKM-1. The deglutamylation pathway mediated by γ-glutamyl hydrolase (GGH) was substantially reduced in PKM-1 at the red-ripe stage. The red-ripe fruits had reduced transcript levels of GGHs and lower GGH activity than AV. Conversely, the percent polyglutamylation of folate was much higher in PKM-1. Our analysis indicates the regulation of GGH activity as a potential target to elevate folate levels in tomato fruits.

Homeodomain-leucine zipper (HD-Zip) transcription factors are only present in higher plants and are involved in plant development and stress responses. However, our understanding of their participation in the fruit ripening of economical plants, such as tomato ( Solanum lycopersicum), remains largely unclear. Here, we report that VAHOX1, a member of the tomato HD-Zip I subfamily, was expressed in all tissues, was highly expressed in breaker+4 fruits, and could be induced by ethylene. RNAi repression of VAHOX1 ( VAHOX1-RNAi) resulted in accelerated fruit ripening, enhanced sensitivity to ethylene, and increased total carotenoid content and ethylene production. Conversely, VAHOX1 overexpression ( VAHOX1-OE) in tomato had the opposite effect. RNA-Seq results showed that altering VAHOX1 expression affected the transcript accumulation of a series of genes involved in ethylene biosynthesis and signal transduction and cell wall modification. Additionally, a dual-luciferase reporter assay, histochemical analysis of GUS activity and a yeast one-hybrid (Y1H) assay revealed that VAHOX1 could activate the expression of AP2a. Our findings may expand our knowledge about the physiological functions of HD-Zip transcription factors in tomato and highlight the diversities of transcriptional regulation during the fruit ripening process.

Cucumber glossiness is an important visual quality trait that affects consumer choice. Accumulating evidence suggests that glossy trait is associated with cuticular wax accumulation. However, the molecular genetic mechanism controlling cucumber glossiness remains largely unknown. Here, we report the map-based cloning and functional characterization of CsGLF1, a locus that determines the glossy trait in cucumber. CsGLF1 encodes a homolog of the Cys2His2-like fold group (C2H2)-type zinc finger protein 6 (ZFP6) and its deletion leads to glossier pericarp and decreased cuticular wax accumulation. Consistently, transcriptomic analysis demonstrated that a group of wax biosynthetic genes were downregulated when CsZFP6 was absent. Further, transient expression assay revealed that CsZFP6 acted as a transcription activator of cuticular wax biosynthetic genes. Taken together, our findings demonstrated a novel regulator of fruit glossiness, which will provide new insights into regulatory mechanism of fruit glossiness in cucumber.

Tanshinone and phenolic acids are the most important active substances of Salvia miltiorrhiza, and the insight into their transcriptional regulatory mechanisms is an essential process to increase their content in vivo. SmMYB36 has been found to have important regulatory functions in the synthesis of tanshinone and phenolic acid; paradoxically, its mechanism of action in S. miltiorrhiza is not clear. Here, we demonstrated that SmMYB36 functions as a promoter of tanshinones accumulation and a suppressor of phenolic acids through the generation of SmMYB36 overexpressed and chimeric SmMYB36-SRDX (EAR repressive domain) repressor hairy roots in combination with transcriptomic-metabolomic analysis. SmMYB36 directly down-regulate the key enzyme gene of primary metabolism, SmGAPC, up-regulate the tanshinones biosynthesis branch genes SmDXS2, SmGGPPS1, SmCPS1 and down-regulate the phenolic acids biosynthesis branch enzyme gene, SmRAS. Meanwhile, SmERF6, a positive regulator of tanshinone synthesis activating SmCPS1, was up-regulated and SmERF115, a positive regulator of phenolic acid biosynthesis activating SmRAS, was down-regulated. Furthermore, the seven acidic amino acids at the C-terminus of SmMYB36 are required for both self-activating domain and activation of target gene expression. As a consequence, this study contributes to reveal the potential relevance of transcription factors synergistically regulating the biosynthesis of tanshinone and phenolic acid.

Apple ( Malus) and pear ( Pyrus) are economically important fruit crops well known for their unique textures, flavours, and nutritional qualities. Both genera are characterised by a distinct pattern of secondary metabolites, which directly affect not only resistance to certain diseases, but also have significant impacts on the flavour and nutritional value of the fruit. The identical chromosome numbers, similar genome size, and their recent divergence date, together with DNA markers have shown that apple and pear genomes are highly co-linear. This study utilized comparative genomic approaches, including simple sequence repeats, high resolution single nucleotide polymorphism melting analysis, and single nucleotide polymorphism chip analysis to identify genetic differences among hybrids of Malus and Pyrus, and F2 offspring. This research has demonstrated and validated that these three marker types, along with metabolomics analysis are very powerful tools to detect and confirm hybridity of progeny derived from crosses between apple and pear in both cross directions. Furthermore, this work analysed the genus-specific metabolite patterns and the resistance to fire blight ( Erwinia amylovora) in progeny. The findings of this work will enhance and accelerate the breeding of novel tree fruit crops that benefit producers and consumers, by enabling marker assisted selection of desired traits introgressed between pear and apple.

CRISPR/Cas9 genome editing technology can overcome many limitations of traditional breeding, offering enormous potential for crop improvement and food production. Although the direct delivery of Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes to grapevine ( Vitis vinifera) protoplasts has been shown before, the regeneration of edited protoplasts into whole plants has not been reported. Here, we describe an efficient approach to obtain transgene-free edited grapevine plants by the transfection and subsequent regeneration of protoplasts isolated from embryogenic callus. As proof of concept, a single-copy green fluorescent protein reporter gene ( GFP) in the grapevine cultivar Thompson Seedless was targeted and knocked out by the direct delivery of RNPs to protoplasts. CRISPR/Cas9 activity, guided by two independent sgRNAs, was confirmed by the loss of GFP fluorescence. The regeneration of GFP − protoplasts into whole plants was monitored throughout development, confirming that the edited grapevine plants were comparable in morphology and growth habit to wild-type controls. We report the first highly efficient protocol for DNA-free genome editing in grapevine by the direct delivery of preassembled Cas9-sgRNA RNP complexes into protoplasts, helping to address the regulatory concerns related to genetically modified plants. This technology could encourage the application of genome editing for the genetic improvement of grapevine and other woody crop plants.

The genus Rhododendron (Ericaceae), with more than 1000 species highly diverse in flower color, is providing distinct ornamental values and a model system for flower color studies. Here, we investigated the divergence between two parental species with different flower color widely used for azalea breeding. Gapless genome assembly was generated for the yellow-flowered azalea, Rhododendron molle. Comparative genomics found recent proliferation of long terminal repeat retrotransposons (LTR-RTs), especially Gypsy, has resulted in a 125 Mb (19%) genome size increase in species-specific regions, and a significant amount of dispersed gene duplicates (13 402) and pseudogenes (17 437). Metabolomic assessment revealed that yellow flower coloration is attributed to the dynamic changes of carotenoids/flavonols biosynthesis and chlorophyll degradation. Time-ordered gene co-expression networks (TO-GCNs) and the comparison confirmed the metabolome and uncovered the specific gene regulatory changes underpinning the distinct flower pigmentation. B3 and ERF TFs were found dominating the gene regulation of carotenoids/flavonols characterized pigmentation in R. molle, while WRKY, ERF, WD40, C2H2, and NAC TFs collectively regulated the anthocyanins characterized pigmentation in the red-flowered R simsii. This study employed a multi-omics strategy in disentangling the complex divergence between two important azaleas and provided references for further functional genetics and molecular breeding.

The ubiquitous lipid-derived molecules N-acylethanolamines (NAEs) have multiple immune functions in mammals, but their roles and mechanisms in plant defense response during changing environment remain largely unclear. Here, we found that exogenous NAE18:0 and NAE18:2 promoted defense against the necrotrophic pathogen Botrytis cinerea but suppressed defense to the hemi-biotrophic pathogen Pseudomonas syringae pv. tomato ( Pst) DC3000 in tomato. The knocking-down and overexpression function analysis of the pathogen-responsive NAE synthetic gene PHOSPHOLIPASE Dγ ( PLDγ) and hydrolytic gene FATTY ACID AMID HYDROLASE 1 ( FAAH1) revealed that the NAE pathway is crucial for plant defense response. Using exogenous applications and SA-abolished NahG plants, we unveiled the antagonistic relationship between NAE and SA in plant defense response. Elevated CO₂ and temperature significantly changed the NAE pathway in response to pathogens, while inhibition of the NAE pathway led to the alternation of environment-mediated defense variations against Pst DC3000 in tomato, indicating that NAE pathway is associated with plant defense variations in response to elevated CO₂ and temperature. The results herein reveal a new function of NAE in plant defense, and its involvement in environment-mediated defense variation in tomato. These findings shed light on the NAE-based plant defense, which may have relevance to crop disease management in future changing climate.

Mitogen-activated protein kinase (MAPK/MPK) cascades play crucial parts in plant growth, development processes, immune ability, and stress responses; however, the regulatory mechanism by which MAPK affects fruit ripening remains largely unexplored. Here, we reported that MaMPK14 cooperated with MaMYB4 to mediate postharvest banana fruit ripening. Transient overexpression of individual MaMPK14 and MaMYB4 in banana fruit delayed fruit ripening, confirming the negative roles in the ripening. The ripening negative regulator MaMYB4 could repress the transcription of genes associated with ethylene biosynthesis and fruit softening, such as MaACS1, MaXTH5, MaPG3, and MaEXPA15. Furthermore, MaMPK14 phosphorylated MaMYB4 at Ser160 via a direct interaction. Mutation at Ser160 of MaMYB4 reduced its interaction with MaMPK14 but did not affect its subcellular localization. Importantly, phosphorylation of MaMYB4 by MaMPK14 enhanced the MaMYB4-mediated transcriptional inhibition, binding strength, protein stability, and the repression of fruit ripening. Taken together, our results delineated the regulation pathway of MAPK module during banana fruit ripening, which involved the phosphorylation modification of MaMYB4 mediated by MaMPK14.

Increasing soil salinization seriously impairs plant growth and development, resulting in crop loss. The Salt-Overly-Sensitive (SOS) pathway is indispensable to the mitigation of Na⁺ toxicity in plants under high salinity. However, whether natural variations of SOS2 contribute to salt tolerance has not been reported. Here a natural variation in the SlSOS2 promoter region was identified to be associated with root Na⁺/K⁺ ratio and the loss of salt resistance during tomato domestication. This natural variation contains an ABI4-binding cis-element and plays an important role in the repression of SlSOS2 expression. Genetic evidence revealed that SlSOS2 mutations increase root Na⁺/K⁺ ratio under salt stress conditions and thus attenuate salt resistance in tomato. Together, our findings uncovered a critical but previously unknown natural variation of SOS2 in salt resistance, which provides valuable natural resources for genetic breeding for salt resistance in cultivated tomatoes and other crops.

Theanine, a unique and the most abundant non-proteinogenic amino acid in tea plants, endows tea infusion with the umami taste and anti-stress effects. Its content in tea correlates highly with green tea quality. Theanine content in new shoots of tea plants is high in mid-spring and greatly decreases in late spring. However, how the decrease is regulated is largely unknown. In a genetic screening, we observed that a yeast mutant, glutamate dehydrogenase 2 ( gdh2), was hypersensitive to 40 mM theanine and accumulated more theanine. This result implied a role of CsGDH2s in theanine accumulation in tea plants. Therefore, we identified the two homologs of GDH2, CsGDH2.1 and CsGDH2.2, in tea plants. Yeast complementation assay showed that the expression of CsGDH2.1 in yeast gdh2 mutant rescued the theanine hypersensitivity and hyperaccumulation of this mutant. Subcellular localization and tissue-specific expression showed CsGDH2.1 localized in the mitochondria and highly expressed in young tissues. Importantly, CsGDH2.1 expression was low in early spring, and increased significantly in late spring, in the new shoots of tea plants. These results all support the idea that CsGDH2.1 regulates theanine accumulation in the new shoots. Moreover, the in vitro enzyme assay showed that CsGDH2.1 had glutamate catabolic activity, and knockdown of CsGDH2.1 expression increased glutamate and theanine accumulation in the new shoots of tea plants. These findings suggested that CsGDH2.1-mediated glutamate catabolism negatively regulates theanine accumulation in the new shoots in late spring, and provides a functional gene for improving late-spring green tea quality.

Chimeric plants composed of green and albino tissues have great ornamental value. To unveil the functional genes responsible for albino phenotypes in chimeric plants, we inspected the complete plastid genomes (plastomes) in green and albino leaf tissues from 23 ornamental chimeric plants belonging to 20 species, including monocots, dicots, and gymnosperms. In nine chimeric plants, plastomes were identical between green and albino tissues. Meanwhile, another 14 chimeric plants were heteroplasmic, showing a mutation between green and albino tissues. We identified 14 different point mutations in eight functional plastid genes related to plastid-encoded RNA polymerase (rpo) or photosystems which caused albinism in the chimeric plants. Among them, 12 were deleterious mutations in the target genes, in which early termination appeared due to small deletion-mediated frameshift or single nucleotide substitution. Another was single nucleotide substitution in an intron of the ycf3 and the other was a missense mutation in coding region of the rpoC2 gene. We inspected chlorophyll structure, protein functional model of the rpoC2, and expression levels of the related genes in green and albino tissues of Reynoutria japonica. A single amino acid change, histidine-to-proline substitution, in the rpoC2 protein may destabilize the peripheral helix of plastid-encoded RNA polymerase, impairing the biosynthesis of the photosynthesis system in the albino tissue of R. japonica chimera plant.

Sweet orange originated from the introgressive hybridizations of pummelo and mandarin resulting in a highly heterozygous genome. How alleles from the two species cooperate in shaping sweet orange phenotypes under distinct circumstances is unknown. Here, we assembled a chromosome-level phased diploid Valencia sweet orange (DVS) genome with over 99.999% base accuracy and 99.2% gene annotation BUSCO completeness. DVS enables allele-level studies for sweet orange and other hybrids between pummelo and mandarin. We first configured an allele-aware transcriptomic profiling pipeline and applied it to 740 sweet orange transcriptomes. On average, 32.5% of genes have a significantly biased allelic expression in the transcriptomes. Different cultivars, transgenic lineages, tissues, development stages, and disease status all impacted allelic expressions and resulted in diversified allelic expression patterns in sweet orange, but particularly citrus Huanglongbing (HLB) shifted the allelic expression of hundreds of genes in leaves and calyx abscission zones. In addition, we detected allelic structural mutations in an HLB-tolerant mutant (T19) and a more sensitive mutant (T78) through long-read sequencing. The irradiation-induced structural mutations mostly involved double-strand breaks, while most spontaneous structural mutations were transposon insertions. In the mutants, most genes with significant allelic expression ratio alterations (≥1.5-fold) were directly affected by those structural mutations. In T19, alleles located at a translocated segment terminal were upregulated, including CsDnaJ, CsHSP17.4B, and CsCEBPZ. Their upregulation is inferred to keep phloem protein homeostasis under the stress from HLB and enable subsequent stress responses observed in T19. DVS will advance allelic level studies in citrus.

Camellia oil extracted from the seeds of Camellia oleifera Abel. is a popular and high-quality edible oil, but its yield is limited by seed setting, which is mainly caused by self-incompatibility (SI). One of the obvious biological features of SI plants is the inhibition of self-pollen tubes; however, the underlying mechanism of this inhibition in C. oleifera is poorly understood. In this study, we constructed a semi- in vivo pollen tube growth test (SIV-PGT) system that can screen for substances that inhibit self-pollen tubes without interference from the genetic background. Combined with multi-omics analysis, the results revealed the important role of galloylated catechins in self-pollen tube inhibition, and a possible molecular regulatory network mediated by UDP-glycosyltransferase ( UGT) and serine carboxypeptidase-like ( SCPL) was proposed. In summary, galloylation of catechins and high levels of galloylated catechins are specifically involved in pollen tube inhibition under self-pollination rather than cross-pollination, which provides a new understanding of SI in C. oleifera. These results will contribute to sexual reproduction research on C. oleifera and provide theoretical support for improving Camellia oil yield in production.

Leaves and flowers are crucial for the growth and development of higher plants. In this study we identified a mutant with narrow leaflets and early flowering ( nlef) in an ethyl methanesulfonate-mutagenized population of woodland strawberry ( Fragaria vesca) and aimed to identify the candidate gene. Genetic analysis revealed that a single recessive gene, nlef, controlled the mutant phenotype. We found that FvH4_1g25470, which encodes a putative DNA polymerase α with a polymerase and histidinol phosphatase domain (PHP), might be the candidate gene, using bulked segregant analysis with whole-genome sequencing, molecular markers, and cloning analyses. A splice donor site mutation (C to T) at the 5 ′ end of the second intron led to an erroneous splice event that reduced the expression level of the full-length transcript of FvePHP in mutant plants. FvePHP was localized in the nucleus and was highly expressed in leaves. Silencing of FvePHP using the virus-induced gene silencing method resulted in partial developmental defects in strawberry leaves. Overexpression of the FvePHP gene can largely restore the mutant phenotype. The expression levels of FveSEP1, FveSEP3, FveAP1, FveFUL, and FveFT were higher in the mutants than those in ‘Yellow Wonder’ plants, probably contributing to the early flowering phenotype in mutant plants. Our results indicate that mutation in FvePHP is associated with multiple developmental pathways. These results aid in understanding the role of DNA polymerase in strawberry development.

Heat stress limits growth and development of crops including grapevine which is a popular fruit in the world. Genetic variability in crops thermotolerance is not well understood. We identified and characterized heat stress transcription factor HSFA2 in heat sensitive Vitis vinifera ‘Jingxiu’ (named as VvHSFA2) and heat tolerant Vitis davidii ‘Tangwei’ (named as VdHSFA2). The transcriptional activation activities of VdHSFA2 are higher than VvHSFA2, the variation of single amino acid (Thr315Ile) in AHA1 motif leads to the difference of transcription activities between VdHSFA2 and VvHSFA2. Based on 41 Vitis germplasms, we found that HSFA2 is differentiated at coding region among heat sensitive V. vinifera, and heat tolerant Vitis davidii and Vitis quinquangularis. Genetic evidence demonstrates VdHSFA2 and VvHSFA2 are positive regulators in grape thermotolerance, and the former can confer higher thermotolerance than the latter. Moreover, VdHSFA2 can regulate more target genes than VvHSFA2. As a target gene of both VdHSFA2 and VvHSFA2, overexpression of MBF1c enhanced the grape thermotolerance whereas dysfunction of MBF1c resulted in thermosensitive phenotype. Together, our results revealed that VdHSFA2 confers higher thermotolerance than VvHSFA2, and MBF1c acts as their target gene to induce thermotolerance. The VdHSFA2 may be adopted for molecular breeding in grape thermotolerance.

The complex leaf senescence process is governed by various levels of transcriptional and translational regulation. Several features of the leaf senescence process are similar across species, yet the extent to which the molecular mechanisms underlying the process of leaf senescence are conserved remains unclear. Currently used experimental approaches permit the identification of individual pathways that regulate various physiological and biochemical processes; however, the large-scale regulatory network underpinning intricate processes like leaf senescence cannot be built using these methods. Here, we discovered a series of conserved genes involved in leaf senescence in a common horticultural crop ( Solanum lycopersicum), a monocot plant ( Oryza sativa), and a eudicot plant ( Arabidopsis thaliana) through analyses of the evolutionary relationships and expression patterns among genes. Our analyses revealed that the genetic basis of leaf senescence is largely conserved across species. We also created a multi-omics workflow using data from more than 10 000 samples from 85 projects and constructed a leaf senescence-associated co-functional gene network with 2769 conserved, high-confidence functions. Furthermore, we found that the mitochondrial unfolded protein response (UPR^mt) is the central biological process underlying leaf senescence. Specifically, UPR^mt responds to leaf senescence by maintaining mitostasis through a few cross-species conserved transcription factors (e.g. NAC13) and metabolites (e.g. ornithine). The co-functional network built in our study indicates that UPR^mt figures prominently in cross-species conserved mechanisms. Generally, the results of our study provide new insights that will aid future studies of leaf senescence.

Telomere to telomere (T2T) assembly relies on the correctness of sequence alignments. However, the existing aligners tend to generate a high proportion of false-positive alignments in repetitive genomic regions which impedes the generation of T2T-level reference genomes for more important species. In this paper, we present an automatic algorithm called RAfilter for removing the false-positives in the outputs of existing aligners. RAfilter takes advantage of rare k-mers representing the copy-specific features to differentiate false-positive alignments from the correct ones. Considering the huge numbers of rare k-mers in large eukaryotic genomes, a series of high-performance computing techniques such as multi-threading and bit operation are used to improve the time and space efficiencies. The experimental results on tandem repeats and interspersed repeats show that RAfilter was able to filter 60%–90% false-positive HiFi alignments with almost no correct ones removed, while the sensitivities and precisions on ONT datasets were about 80% and 50% respectively.