Modern cultivated caladiums (Caladium × hortulanum) are grown for their long-lasting and colorful leaves. Understanding the mode of inheritance for caladium leaf characteristics is critical for plant breeders to select appropriate parents, predict progeny performance, estimate breeding population sizes needed, and increase breeding efficiencies. This study was conducted to determine the mode of inheritance of two leaf background colors (lemon and green) in caladium and to understand their relationships with four other important leaf characteristics including leaf shape, main vein color, spotting, and rugosity. Seven caladium cultivars and three breeding lines were used as parents in 19 crosses, and their progeny were phenotyped for segregation of leaf traits. Results showed that the two leaf background colors are controlled by a single nuclear locus, with two alleles, LEM and lem, which control the dominant lemon and the recessive green leaf background color, respectively. The lemon-colored cultivar ‘Miss Muffet’ and breeding lines UF-52 and UF-53 have a heterozygous genotype LEMlem. Chi-square tests showed that the leaf background color locus LEM is independent from the leaf shape locus F, but is tightly linked to three loci (S, V and RLF) controlling leaf spotting, main vein color, and rugosity in caladium. A linkage map that consists of four loci controlling major caladium leaf characteristics and extends ~ 15 cM was developed based on the observed recombination frequencies. This is the first report on the mode of inheritance of leaf background colors in caladium and in the Araceae family. The information gained in this study will be very useful for caladium breeding and study of the inheritance of leaf colors in other ornamental aroids, an important group of ornamental plants in the world.

The primary goal of genomic selection is to increase genetic gains for complex traits by predicting performance of individuals for which phenotypic data are not available. The objective of this study was to experimentally evaluate the potential of genomic selection in strawberry breeding and to define a strategy for its implementation. Four clonally replicated field trials, two in each of 2 years comprised of a total of 1628 individuals, were established in 2013–2014 and 2014–2015. Five complex yield and fruit quality traits with moderate to low heritability were assessed in each trial. High-density genotyping was performed with the Affymetrix Axiom IStraw90 single-nucleotide polymorphism array, and 17 479 polymorphic markers were chosen for analysis. Several methods were compared, including Genomic BLUP, Bayes B, Bayes C, Bayesian LASSO Regression, Bayesian Ridge Regression and Reproducing Kernel Hilbert Spaces. Cross-validation within training populations resulted in higher values than for true validations across trials. For true validations, Bayes B gave the highest predictive abilities on average and also the highest selection efficiencies, particularly for yield traits that were the lowest heritability traits. Selection efficiencies using Bayes B for parent selection ranged from 74% for average fruit weight to 34% for early marketable yield. A breeding strategy is proposed in which advanced selection trials are utilized as training populations and in which genomic selection can reduce the breeding cycle from 3 to 2 years for a subset of untested parents based on their predicted genomic breeding values.

The chrysanthemum genome harbors three FT-like genes: CmFTL1 and CmFTL3 are thought to act as regulators of floral induction under long-day (LD) and short-day (SD) conditions, respectively, whereas the function of CmFTL2 is currently unclear. The objective of the present research was to explore the function of CmFTL2 in the determination of flowering time of the photo-insensitive chrysanthemum cultivar ‘Floral Yuuka’, both in response to variation in the photoperiod and to the exogenous provision of sucrose. Spraying leaves of ‘Floral Yuuka’ plants with 50 mM sucrose accelerated flowering and increased the level of CmFTL2 transcription in the leaf more strongly than either CmFTL1 or FTL3 under both long and SD conditions. Transcription profiling indicated that all three CmFTL genes were upregulated during floral induction. The relationship of the CmFTL2 sequence with that of other members of the PEBP family suggested that its product contributes to the florigen rather than to the anti-florigen complex. The heterologous expression of CmFTL2 in the Arabidopsis thaliana ft-10 mutant rescued the mutant phenotype, showing that CmFTL2 could compensate for the absence of FT. These results suggest that CmFTL2 acts as a regulator of floral transition and responds to both the photoperiod and sucrose.

The apple (Malus × domestica) cultivar Honeycrisp has become important economically and as a breeding parent. An earlier study with SSR markers indicated the original recorded pedigree of ‘Honeycrisp’ was incorrect and ‘Keepsake’ was identified as one putative parent, the other being unknown. The objective of this study was to verify ‘Keepsake’ as a parent and identify and genetically describe the unknown parent and its grandparents. A multi-family based dense and high-quality integrated SNP map was created using the apple 8 K Illumina Infinium SNP array. This map was used alongside a large pedigree-connected data set from the RosBREED project to build extended SNP haplotypes and to identify pedigree relationships. ‘Keepsake’ was verified as one parent of ‘Honeycrisp’ and ‘Duchess of Oldenburg’ and ‘Golden Delicious’ were identified as grandparents through the unknown parent. Following this finding, siblings of ‘Honeycrisp’ were identified using the SNP data. Breeding records from several of these siblings suggested that the previously unreported parent is a University of Minnesota selection, MN1627. This selection is no longer available, but now is genetically described through imputed SNP haplotypes. We also present the mosaic grandparental composition of ‘Honeycrisp’ for each of its 17 chromosome pairs. This new pedigree and genetic information will be useful in future pedigree-based genetic studies to connect ‘Honeycrisp’ with other cultivars used widely in apple breeding programs. The created SNP linkage map will benefit future research using the data from the Illumina apple 8 and 20 K and Affymetrix 480 K SNP arrays.

Ornamental plant variety improvement is limited by current phenotyping approaches and neglected use of experimental designs. The present study was conducted to show the benefits of using an experimental design and corresponding analysis in ornamental breeding regarding simulated response to selection in Pelargonium zonale for production-related traits. This required establishment of phenotyping protocols for root formation and stem cutting counts, with which 974 genotypes were assessed in a two-phase experimental design. The present paper evaluates this protocol. The possibility of varietal improvement through indirect selection on secondary traits such as branch count and flower count was assessed by genetic correlations. Simulated response to selection varied greatly, depending on the genotypic variances of the breeding population and traits. A varietal improvement of over 20% is possible for stem cutting count, root formation, branch count and flower count. In contrast, indirect selection of stem cutting count by branch count or flower count was found to be ineffective. The established phenotypic protocols and two-phase experimental designs are valuable tools for breeding of P. zonale.

Preharvest bagging is a simple, grower-friendly and safe physical protection technique commonly applied to many fruits, and the application of different fruit bags can have various effects. To explore the molecular mechanisms underlying the fruit quality effects of different bagging treatments, digital gene expression (DGE) profiling of bagged and unbagged ‘Chili’ (Pyrus bretschneideri Rehd.) pear pericarp during development was performed. Relative to unbagged fruit, a total of 3022 and 769 differentially expressed genes (DEGs) were detected in the polyethylene (PE)-bagged and non-woven fabric-bagged fruit, respectively. DEGs annotated as photosynthesis-antenna proteins and photosynthesis metabolism pathway were upregulated in non-woven fabric-bagged fruit but downregulated in the PE-bagged fruit. Non-woven fabric bagging inhibited lignin synthesis in ‘Chili’ pear pericarp by downregulating DEGs involved in phenylpropanoid biosynthesis; consequently, the fruit lenticels in non-woven fabric-bagged fruit were smaller than those in the other treatments. The results indicate that the non-woven fabric bagging method has a positive effect on the appearance of ‘Chili’ pear fruit but neither of the two bagging treatments is conducive to the accumulation of soluble sugar.

Crops of the Rosaceae family provide valuable contributions to rural economies and human health and enjoyment. Sustained solutions to production challenges and market demands can be met with genetically improved new cultivars. Traditional rosaceous crop breeding is expensive and time-consuming and would benefit from improvements in efficiency and accuracy. Use of DNA information is becoming conventional in rosaceous crop breeding, contributing to many decisions and operations, but only after past decades of solved challenges and generation of sufficient resources. Successes in deployment of DNA-based knowledge and tools have arisen when the ‘chasm’ between genomics discoveries and practical application is bridged systematically. Key steps are establishing breeder desire for use of DNA information, adapting tools to local breeding utility, identifying efficient application schemes, accessing effective services in DNA-based diagnostics and gaining experience in integrating DNA information into breeding operations and decisions. DNA-informed germplasm characterization for revealing identity and relatedness has benefitted many programs and provides a compelling entry point to reaping benefits of genomics research. DNA-informed germplasm evaluation for predicting trait performance has enabled effective reallocation of breeding resources when applied in pioneering programs. DNA-based diagnostics is now expanding from specific loci to genome-wide considerations. Realizing the full potential of this expansion will require improved accuracy of predictions, multi-trait DNA profiling capabilities, streamlined breeding information management systems, strategies that overcome plant-based features that limit breeding progress and widespread training of current and future breeding personnel and allied scientists.

Although chimeric repressors such as the Arabidopsis TCP3 repressor are known to have significant effects on flower morphology and color, their cellular-level effects on flower petals are not understood. The promoter sequences of the genes expressed in the flowers of cyclamen, a representative potted flower grown during the winter season, are also unknown. Here, we isolated eight promoters from cyclamen genes that are reportedly expressed in the petals. These promoters were then fused to four chimeric repressors and introduced into the model flower torenia to screen for effective combinations of promoters and repressors for flower breeding. As expected, some of the constructs altered flower phenotypes upon transformation. We further analyzed the effects of chimeric repressors at the cellular level. We observed that complicated petal and leaf serrations were accompanied by excessive vascular branching. Dichromatism in purple anthocyanin was inferred to result in bluish flowers, and imbalanced cell proliferation appeared to result in epinastic flowers. Thus, the genetic constructs and phenotypic changes described in this report will benefit the future breeding and characterization of ornamental flowers.

Apple dwarfing rootstocks cause earlier shoot termination and reduced root and shoot mass. To identify physiological factors responsible for rootstock-induced growth restriction, we compared vascular-enriched gene expression between two dwarfing rootstocks (‘M27’ and ‘M9’) and the vigorous rootstock ‘M793’ using RNA sequencing and quantitative reverse transcriptase PCR. Differentially expressed genes common to both dwarfing rootstocks belonged to five main biological processes: (1) primary metabolism, (2) cell wall synthesis and modification, (3) secondary metabolism, (4) hormone signalling and response and (5) redox homeostasis. Genes promoting the biosynthesis of amino acids, lipids and cell walls were downregulated in dwarfing rootstocks, whereas genes promoting the breakdown of these compounds were upregulated. The only exception to this trend was the upregulation of starch synthesis genes in dwarfing rootstocks. Non-structural carbohydrate analysis demonstrated that starch concentrations in ‘M9’ roots, stems and grafted ‘Royal Gala’ (‘RG’) scions were double that of equivalent tissues from ‘RG’ homo-grafted trees (‘RG’/‘RG’). Fructose and glucose concentrations were much lower in all three tissues of the ‘RG’/‘M9’ trees. Together, these data indicate that dwarfing rootstocks are in a state of sugar depletion and reduced cellular activity despite having large starch reserves. Another significant finding was the over-accumulation of flavonoids and the downregulation of auxin influx transporters MdAUX1 and MdLAX2 in dwarfing rootstocks. We propose that both factors reduce polar auxin transport. The results of this study contribute novel information about the physiological state of dwarfing rootstocks.

Understanding of genetic diversity and marker-trait relationships in pears (Pyrus spp.) forms an important part of gene conservation and cultivar breeding. Accessions of Asian and European pear species, and interspecific hybrids were planted in a common garden experiment. Genotyping-by-sequencing (GBS) was used to genotype 214 accessions, which were also phenotyped for fruit quality traits. A combination of selection scans and association analyses were used to identify signatures of selection. Patterns of genetic diversity, population structure and introgression were also investigated. About 15 000 high-quality SNP markers were identified from the GBS data, of which 25% and 11% harboured private alleles for European and Asian species, respectively. Bayesian clustering analysis suggested negligible gene flow, resulting in highly significant population differentiation (F_st = 0.45) between Asian and European pears. Interspecific hybrids displayed an average of 55% and 45% introgression from their Asian and European ancestors, respectively. Phenotypic (firmness, acidity, shape and so on) variation between accessions was significantly associated with genetic differentiation. Allele frequencies at large-effect SNP loci were significantly different between genetic groups, suggesting footprints of directional selection. Selection scan analyses identified over 20 outlier SNP loci with substantial statistical support, likely to be subject to directional selection or closely linked to loci under selection.

Globally, pea (Pisum sativum L.) is an important temperate legume crop for food, feed and fodder, and many breeding programs develop cultivars adapted to these end-uses. In order to assist pea development efforts, we assembled the USDA Pea Single Plant Plus Collection (PSPPC), which contains 431 P. sativum accessions with morphological, geographic and taxonomic diversity. The collection was characterized genetically in order to maximize its value for trait mapping and genomics-assisted breeding. To that end, we used genotyping-by-sequencing-a cost-effective method for de novo single-nucleotide polymorphism (SNP) marker discovery-to generate 66 591 high-quality SNPs. These data facilitated the identification of accessions divergent from mainstream breeding germplasm that could serve as sources of novel, favorable alleles. In particular, a group of accessions from Central Asia appear nearly as diverse as a sister species, P. fulvum, and subspecies, P. sativum subsp. elatius. PSPPC genotypes can be paired with new and existing phenotype data for trait mapping; as proof-of-concept, we localized Mendel’s A gene controlling flower color to its known position. We also used SNP data to define a smaller core collection of 108 accessions with similar levels of genetic diversity as the entire PSPPC, resulting in a smaller germplasm set for research screening and evaluation under limited resources. Taken together, the results presented in this study along with the release of a publicly available SNP data set comprise a valuable resource for supporting worldwide pea genetic improvement efforts.

Grapes are categorized as a non-climacteric type of fruit which its ripening is not associated to important rises in respiration and ethylene synthesis. The starch metabolism shares a certain role in the carbohydrate metabolic pathways during grape berry development, and is regarded as an important transient pool in the pathway of sugar accumulation. However, the comprehensive role of starch and its contribution to the quality and flavor of grape berry have not been explored thoroughly. In this study, the expression levels of genes enzyme activities and carbohydrate concentrations related to starch metabolism, were analyzed to understand the molecular mechanism of starch accumulation during grape berry development. The results indicated that starch granules in grape berry were located at the chloroplast in the sub-epidermal tissues, acting as the temporary reserves of photosynthetic products to meet the needs for berry development, and relatively high starch contents could be detected at véraison stage. Moreover, both ADP-glucose pyrophosphorylase (EC 2.7.7.27) and sucrose phosphate synthase (EC 2.3.1.14) involved in starch synthesis displayed elevated gene expression and enzymes activities in the sub-epidermal tissue, while α- and β-amylases involved in its degradation were highly transcribed and active in the central flesh, explaining the absence of starch in this last tissue. Change in the gene expression and activities of ADP-glucose pyrophosphorylase, β-amylase and sucrose phosphate synthase revealed that they were regulated by the circadian rhythms in the fruitlets compared with those in the leaves. Both the morphological, enzymological and transcriptional data in this study provide advanced understandings on the function of starch during berry development and ripening that are so important for berry quality. This study will further facilitate our understanding of the sugar metabolism in grape berry as well as in other plant species.

Flowering time is an important trait that affects survival, reproduction and yield in both wild and cultivated plants. Therefore, many studies have focused on the identification of flowering time quantitative trait locus (QTLs) in different crops, and molecular control of this trait has been extensively investigated in model species. Here we report the mapping of QTLs for flowering time and vegetative traits in a large woodland strawberry mapping population that was phenotyped both under field conditions and in a greenhouse after flower induction in the field. The greenhouse experiment revealed additive QTLs in three linkage groups (LG), two on both LG4 and LG7, and one on LG6 that explain about half of the flowering time variance in the population. Three of the QTLs were newly identified in this study, and one co-localized with the previously characterized FvTFL1 gene. An additional strong QTL corresponding to previously mapped PFRU was detected in both field and greenhouse experiments indicating that gene(s) in this locus can control the timing of flowering in different environments in addition to the duration of flowering and axillary bud differentiation to runners and branch crowns. Several putative flowering time genes were identified in these QTL regions that await functional validation. Our results indicate that a few major QTLs may control flowering time and axillary bud differentiation in strawberries. We suggest that the identification of causal genes in the diploid strawberry may enable fine tuning of flowering time and vegetative growth in the closely related octoploid cultivated strawberry.

The oomycete, Phytophthora capsici, infects cucumber (Cucumis sativus L.) fruit. An age-related resistance (ARR) to this pathogen was previously observed in fruit of cultivar ‘Vlaspik’ and shown to be associated with the peel. Young fruits are highly susceptible, but develop resistance at ~ 10–12 days post pollination (dpp). Peels from resistant (16 dpp) versus susceptible (8 dpp) age fruit are enriched with genes associated with defense, and methanolic extracts from resistant age peels inhibit pathogen growth. Here we compared developing fruits from ‘Vlaspik’ with those of ‘Gy14’, a line that does not exhibit ARR. Transcriptomic analysis of peels of the two lines at 8 and 16 dpp identified 80 genes that were developmentally upregulated in resistant ‘Vlaspik’ 16 dpp versus 8 dpp, but not in susceptible ‘Gy14’ at 16 dpp. A large number of these genes are annotated to be associated with defense and/or specialized metabolism, including four putative resistance (R) genes, and numerous genes involved in flavonoid and terpenoid synthesis and decoration. Untargeted metabolomic analysis was performed on extracts from 8 and 16 dpp ‘Vlaspik’ and ‘Gy14’ fruit peels using Ultra-Performance Liquid Chromatography and Quadrupole Time-of-Flight Mass Spectrometry. Multivariate analysis of the metabolomes identified 113 ions uniquely abundant in resistant ‘Vlaspik’ 16 dpp peel extracts. The most abundant compounds in this group had relative mass defects consistent with terpenoid glycosides. Two of the three most abundant ions were annotated as glycosylated nor-terpenoid esters. Together, these analyses reveal potential mechanisms by which ARR to P. capsici may be conferred.

The basic leucine zipper (bZIP) transcription factor HY5 plays a multifaceted role in plant growth and development. Here the apple MdHY5 gene was cloned based on its homology with Arabidopsis HY5. Expression analysis demonstrated that MdHY5 transcription was induced by light and abscisic acid treatments. Electrophoretic mobility shift assays and transient expression assays subsequently showed that MdHY5 positively regulated both its own transcription and that of MdMYB10 by binding to E-box and G-box motifs, respectively. Furthermore, we obtained transgenic apple calli that overexpressed the MdHY5 gene, and apple calli coloration assays showed that MdHY5 promoted anthocyanin accumulation by regulating expression of the MdMYB10 gene and downstream anthocyanin biosynthesis genes. In addition, the transcript levels of a series of nitrate reductase genes and nitrate uptake genes in both wild-type and transgenic apple calli were detected. In association with increased nitrate reductase activities and nitrate contents, the results indicated that MdHY5 might be an important regulator in nutrient assimilation. Taken together, these results indicate that MdHY5 plays a vital role in anthocyanin accumulation and nitrate assimilation in apple.

Willow (Salix) is one of the most important ornamental tree species in landscape plants. One species, Salix matsudana, is widely used as a shade tree and border tree because of its soft branches and plump crown. Some varieties of S. matsudana were salt tolerant and could grow normally in coastal regions. However, the molecular mechanisms of salt tolerance for S. matsudana have been less clear. Here, we addressed this issue by performing a mapping experiment containing 195 intraspecific F₁ progeny of S. matsudana, derived from salt-sensitive ‘yanjiang’ and salt-tolerant ‘9901’, grown by cuttings in a 100 mM NaCl solution. Growth performance of these progeny under salt stress was investigated, displaying marked genotypic variability with the coefficients of variance of 28.64–86.11% in shoot and root growth traits. We further mapped specific QTLs contributing to these differences to the Salix genome. Of the 204 QTLs identified, a few were detected to explain a remarkably larger portion of the phenotypic variation than many others. Many detected QTLs were found to reside in the region of candidate genes of known biological function. The discovery of growth QTLs expressed under salt stress provides important information for marker-assisted breeding of salt tolerant Salix varieties and founds the basis for the application of S. matsudana in coastal afforestation.

Wild strawberry Fragaria vesca is emerging as an important model system for the cultivated strawberry due to its diploid genome and availability of extensive transcriptome data and a range of molecular genetic tools. Being able to better utilize these tools, especially the transcriptome data, will greatly facilitate research progress in strawberry and other Rosaceae fruit crops. The electronic fluorescent pictograph (eFP) software is a useful and popular tool to display transcriptome data visually, and is widely used in other model organisms including Arabidopsis and mouse. Here we applied eFP to display wild strawberry RNA sequencing (RNA-seq) data from 42 different tissues and stages, including various flower and fruit developmental stages. In addition, we generated eight additional RNA-seq data sets to represent tissues from ripening-stage receptacle fruit from yellow-colored and red-colored wild strawberry varieties. Differential gene expression analysis between these eight data sets provides additional information for understanding fruit-quality traits. Together, this work greatly facilitates the utility of the extensive transcriptome data for investigating strawberry flower and fruit development as well as fruit-quality traits.

Non-coding RNAs (ncRNAs), that is, RNAs not translated into proteins, are crucial regulators of a variety of biological processes in plants. While protein-encoding genes have been relatively well-annotated in sequenced genomes, accounting for a small portion of the genome space in plants, the universe of plant ncRNAs is rapidly expanding. Recent advances in experimental and computational technologies have generated a great momentum for discovery and functional characterization of ncRNAs. Here we summarize the classification and known biological functions of plant ncRNAs, review the application of next-generation sequencing (NGS) technology and ribosome profiling technology to ncRNA discovery in horticultural plants and discuss the application of new technologies, especially the new genome-editing tool clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems, to functional characterization of plant ncRNAs.

The shoot structure of cultivated grapevine Vitis vinifera L. typically exhibits a three-node modular repetitive pattern, two sequential leaf-opposed tendrils followed by a tendril-free node. In this study, we investigated the molecular basis of this pattern by characterizing differentially expressed genes in 10 bulk samples of young tendril tissue from two grapevine populations showing segregation of mutant or wild-type shoot/tendril phyllotaxy. One population was the selfed progeny and the other one, an outcrossed progeny of a Vitis hybrid, ‘Roger’s Red’. We analyzed 13 375 expressed genes and carried out in-depth analyses of 324 of them, which were differentially expressed with a minimum of 1.5-fold changes between the mutant and wild-type bulk samples in both selfed and cross populations. A significant portion of these genes were direct cis-binding targets of 14 transcription factor families that were themselves differentially expressed. Network-based dependency analysis further revealed that most of the significantly rewired connections among the 10 most connected hub genes involved at least one transcription factor. TCP3 and MYB12, which were known important for plant-form development, were among these transcription factors. More importantly, TCP3 and MYB12 were found in this study to be involved in regulating the lignin gene PRX52, which is important to plant-form development. A further support evidence for the roles of TCP3-MYB12-PRX52 in contributing to tendril phyllotaxy was the findings of two other lignin-related genes uniquely expressed in the mutant phyllotaxy background.

The downy mildew disease in grapevines is caused by Plasmopara viticola. This disease poses a serious threat wherever viticulture is practiced. Wild Vitis species showing resistance to P. viticola offer a promising pathway to develop new grapevine cultivars resistant to P. viticola which will allow reduced use of environmentally unfriendly fungicides. Here, transmission and scanning microscopy was used to compare the resistance responses to downy mildew of three resistant genotypes of V. davidii var. cyanocarpa, V. piasesezkii and V. pseudoreticulata and the suceptible V. vinifera cultivar ‘Pinot Noir’. Following inoculation with sporangia of P. viticola isolate ‘YL’ on V. vinifera cv. ‘Pinot Noir’, the infection was characterized by a rapid spread of intercellular hyphae, a high frequency of haustorium formation within the host’s mesophyll cells, the production of sporangia and by the absence of host-cell necrosis. In contrast zoospores were collapsed in the resistant V. pseudoreticulata ‘Baihe-35-1’, or secretions appeared arround stomata at the beginning of the infection period in V. davidii var. cyanocarpa ‘Langao-5’ and V. piasezkii ‘Liuba-8’. The main characteristics of the resistance responses were the rapid depositions of callose and the appearance of empty hyphae and the plasmolysis of penetrated tissue. Moreover, collapsed haustoria were observed in V. davidii var. cyanocarpa ‘Langao-5’ at 5 days post inoculation (dpi) and in V. piasezkii ‘Liuba-8’ at 7 dpi. Lastly, necrosis extended beyond the zone of restricted colonization in all three resistant genotypes. Sporangia were absent in V. piasezkii ‘Liuba-8’ and greatly decreased in V. davidii var. cyanocarpa ‘Langao-5’ and in V. pseudoreticulata ‘Baihe-35-1’ compared with in V. vinifera cv. ‘Pinot Noir’. Overall, these results provide insights into the cellular biological basis of the incompatible interactions between the pathogen and the host. They indicate a number of several resistant Chinese wild species that could be used in developing new cultivars having good levels of downy mildew resistance.

Grapes are one of the most economically and culturally important crops worldwide, and they have been bred for both winemaking and fresh consumption. Here we evaluate patterns of diversity across 33 phenotypes collected over a 17-year period from 580 table and wine grape accessions that belong to one of the world’s largest grape gene banks, the grape germplasm collection of the United States Department of Agriculture. We find that phenological events throughout the growing season are correlated, and quantify the marked difference in size between table and wine grapes. By pairing publicly available historical phenotype data with genome-wide polymorphism data, we identify large effect loci controlling traits that have been targeted during domestication and breeding, including hermaphroditism, lighter skin pigmentation and muscat aroma. Breeding for larger berries in table grapes was traditionally concentrated in geographic regions where Islam predominates and alcohol was prohibited, whereas wine grapes retained the ancestral smaller size that is more desirable for winemaking in predominantly Christian regions. We uncover a novel locus with a suggestive association with berry size that harbors a signature of positive selection for larger berries. Our results suggest that religious rules concerning alcohol consumption have had a marked impact on patterns of phenomic and genomic diversity in grapes.

Plant cell cultures represent important model systems to understand metabolism and its modulation by regulatory factors. Even in controlled conditions, cell metabolism is highly dynamic and can be fully characterized only by time course experiments. Here, we show that statistical analysis of this type of data gains power if it moves to approaches able to compare the ‘trends’ of the different metabolites. In particular, we show how generalized additive models can be used to model the time-dependent profile of anthocyanin synthesis in grapevine cell suspension cultures (Vitis vinifera cv. Gamay), following treatment with 100 μM methyl jasmonate. The sampling was performed daily for 20 days of culturing following elicitation at day 5. All samples were analyzed by UPLC-MS/MS for the identification and quantification of fifteen anthocyanin compounds. The models confirmed the separation in the anthocyanin biosynthetic pathway between delphinidin-based and cyanidin-based compounds, showing that methyl jasmonate modulates the anthocyanin concentration profiles. Our results clearly indicate that the combination of high-throughput metabolomics and state of the art statistical modeling is a powerful approach to study plant metabolism. This approach is expected to gain popularity due to the growing availability of low-cost high-throughput ‘omic’ assays.

Red pitaya (Hylocereus polyrhizus) fruit is a high-value, functional food, containing a high level of betalains. Several genes potentially related to betalain biosynthesis, such as cytochrome P450-like (CytP450-like), have been identified in pitaya fruit, while their transcriptional regulation remains unclear. In this work, the potential involvement of a WRKY transcription factor, HpWRKY44, in regulating CytP450-like1 expression in pitaya fruit was examined. HpWRKY44, a member of the Group 1 WRKY family, contains two conserved WRKY motifs and is localized in the nucleus. HpWRKY44 also exhibits trans-activation ability. Gene expression analysis showed that the expression of HpCytP450-like1 and HpWRKY44 increased steadily during pitaya fruit coloration, which corresponded with the production of elevated betalain levels in the fruit. HpWRKY44 was also demonstrated to directly bind to and activate the HpCytP450-like1 promoter via the recognition of the W-box element present in the promoter. Collectively, our findings indicate that HpWRKY44 transcriptionally activates HpCytP450-like1, which perhaps, at least in part, contributes to betalain biosynthesis in pitaya fruit. The information provided in the current study provides novel insights into the regulatory network associated with betalain biosynthesis during pitaya fruit coloration.

Candidatus Liberibacter asiaticus (Las) is a fastidious, phloem-restricted pathogen with a significantly reduced genome, and attacks all citrus species with no immune cultivars documented to date. Like other plant bacterial pathogens, Las deploys effector proteins into the organelles of plant cells, such as mitochondria and chloroplasts to manipulate host immunity and physiology. These organelles are responsible for the synthesis of adenosine triphosphate (ATP) and have a critical role in plant immune signaling during hydrogen peroxide (H₂O₂) production. In this study, we investigated H₂O₂ and ATP accumulation in relation to citrus huanglongbing (HLB) in addition to revealing the expression profiles of genes critical for the production and detoxification of H₂O₂ and ATP synthesis. We also found that as ATP and H₂O₂ concentrations increased in the leaf, so did the severity of the HLB symptoms, a trend that remained consistent among the four different citrus varieties tested. Furthermore, the upregulation of ATP synthase, a key enzyme for energy conversion, may contribute to the accumulation of ATP in infected tissues, whereas downregulation of the H₂O₂ detoxification system may cause oxidative damage to plant macromolecules and cell structures. This may explain the cause of some of the HLB symptoms such as chlorosis or leaf discoloration. The findings in this study highlight important molecular and physiological mechanisms involved in the host plants’ response to Las infection and provide new targets for interrupting the disease cycle.

Identification of genes with differential transcript abundance (GDTA) in seedless mutants may enhance understanding of seedless citrus development. Transcriptome analysis was conducted at three time points during early fruit development (Phase 1) of three seedy citrus genotypes: Fallglo (Bower citrus hybrid (Citrus reticulata × C. reticulata × C. paradisi) × Temple (C. reticulata × C. sinensis)), grapefruit (C. paradisi), Pineapple sweet orange (C. sinensis), and their seedless mutants. Seed abortion in seedless mutants was observed at 26 days post anthesis (Time point 2). Affymetrix transcriptomic analysis revealed 359 to 1077 probe sets with differential transcript abundance in the comparison of seedless versus seedy fruits for each citrus genotypes and time points. The GDTA identified by 18 microarray probe sets were validated by qPCR. Hierarchical clustering analysis revealed a range of GDTA associated with development, hormone and protein metabolism, all of which may reflect genes associated with seedless fruit development. There were 14, 9 and 12 genes found exhibiting similar abundance ratios in all three seedless versus seedy genotype comparisons at time point 1, 2 and 3, respectively. Among those genes were genes coding for an aspartic protease and a cysteine protease, which may play important roles in seedless fruit development. New insights into seedless citrus fruit development may contribute to biotech approaches to create seedless cultivars.

Raspberry (Rubus spp.) is an economically important crop with a restricted growing season and very limited fruit shelf-life due to its extreme tenderness. In order to prolong its shelf life, an aqueous composition containing hexanal as the key active ingredient (HC) was applied as a preharvest spray during fruit development. The effects of HC were assessed using physiological, biochemical and anatomical parameters on the treated fruits and compared with the effects of mock inoculation which lacked hexanal. Sugars and acidity did not show a significant change in response to HC treatment, while the pulling force (the tension required to detach the berry from the receptacle) significantly improved in the HC-treated fruits, compared to control. Scanning electron microscope (SEM) analysis revealed a high correlation between the presence of rigid epidermal hairs and a stronger degree of attachment between berries and their receptacle in the HC treated fruits. Further, electron micrographs also showed abnormal crystalline depositions on the epidermal drupelets of the treated berries. Energy Dispersive X-ray Spectroscopy (EDS) analysis showed those crystals to be largely composed of calcium. HC treatment also resulted in the reduction of transcript level of three phospholipase D genes, as well as altered expression pattern of five members of the annexin gene family, and four calmodulin-binding transcription activators. Quantification of PLD activity showed that hexanal inhibited PLD activity in treated berries. The potential crosstalk between hexanal, phospholipase D activity and calcium and this crosstalk’s role in delaying fruit softening and in prolonging storage life of fruits shelf life is discussed.

Exogenous application of a cytokinin-like compound forchlorfenuron (CPPU) can promote fruit growth, although often at the expense of dry matter (DM), an important indicator of fruit quality. Actinidia chinensis var. deliciosa ‘Hayward’ fruit are very responsive to CPPU treatments, but the mechanism underlying the significant fruit weight increase and associated decrease in DM is unclear. In this study, we hypothesised that CPPU-enhanced growth increases fruit carbohydrate demand, but limited carbohydrate supply resulted in decreased fruit DM. During fruit development, CPPU effects on physical parameters, metabolites, osmotic pressure and transcriptional changes were assessed under conditions of both standard and a high carbohydrate supply. We showed that CPPU increased fruit fresh weight but the dramatic DM decrease was not carbohydrate limited. Enhanced glucose and fructose concentrations contributed to an increase in soluble carbohydrate osmotic pressure, which was correlated with increased water accumulation in CPPU-treated fruit and up-regulation of water channel aquaporin gene PIP2.4 at 49 days after anthesis. Transcript analysis suggested that the molecular mechanism contributing to increased glucose and fructose concentrations was altered by carbohydrate supply. At standard carbohydrate supply, the early glucose increase in CPPU fruit was associated with reduced starch synthesis and increased starch degradation. When carbohydrate supply was high, the early glucose increase in CPPU fruit was associated with a general decrease in starch synthesis but up-regulation of vacuolar invertase and fructokinase genes. We conclude that CPPU affected fruit expansion by increasing the osmotically-driven water uptake and its effect was not carbohydrate supply-limited.

Water lilies are not only highly favored aquatic ornamental plants with cultural and economic importance but they also occupy a critical evolutionary space that is crucial for understanding the origin and early evolutionary trajectory of flowering plants. The birth and rapid radiation of flowering plants has interested many scientists and was considered ‘an abominable mystery’ by Charles Darwin. In searching for the angiosperm evolutionary origin and its underlying mechanisms, the genome of Amborella has shed some light on the molecular features of one of the basal angiosperm lineages; however, little is known regarding the genetics and genomics of another basal angiosperm lineage, namely, the water lily. In this study, we reviewed current molecular research and note that water lily research has entered the genomic era. We propose that the genome of the water lily is critical for studying the contentious relationship of basal angiosperms and Darwin’s ‘abominable mystery’. Four pantropical water lilies, especially the recently sequenced Nymphaea colorata, have characteristics such as small size, rapid growth rate and numerous seeds and can act as the best model for understanding the origin of angiosperms. The water lily genome is also valuable for revealing the genetics of ornamental traits and will largely accelerate the molecular breeding of water lilies.

Pears with red skin are attractive to consumers and provide additional health benefits. Identification of the gene(s) responsible for skin coloration can benefit cultivar selection and breeding. The use of QTL-seq, a bulked segregant analysis method, can be problematic when heterozygous parents are involved. The present study modified the QTL-seq method by introducing a |Δ(SNP- index)| parameter to improve the accuracy of mapping the red skin trait in a group of highly heterozygous Asian pears. The analyses were based on mixed DNA pools composed of 28 red-skinned and 27 green-skinned pear lines derived from a cross between the ‘Mantianhong’ and ‘Hongxiangsu’ red-skinned cultivars. The ‘Dangshansuli’ cultivar genome was used as reference for sequence alignment. An average single-nucleotide polymorphism (SNP) index was calculated using a sliding window approach (200-kb windows, 20-kb increments). Nine scaffolds within the candidate QTL interval were in the fifth linkage group from 111.9 to 177.1 cM. There was a significant linkage between the insertions/deletions and simple sequence repeat markers designed from the candidate intervals and the red/green skin (R/G) locus, which was in a 582.5-kb candidate interval that contained 81 predicted protein-coding gene models and was composed of two subintervals at the bottom of the fifth chromosome. The ZFRI 130-16, In2130-12 and In2130-16 markers located near the R/G locus could potentially be used to identify the red skin trait in Asian pear populations. This study provides new insights into the genetics controlling the red skin phenotype in this fruit.

Huanglongbing (HLB), a systemic and destructive disease of citrus, is associated with ‘ Candidatus Liberibacter asiaticus’ (Las) in the United States. Our earlier work has shown that Las bacteria were significantly reduced or eliminated when potted HLB-affected citrus were continuously exposed to high temperatures of 40 to 42 °C for a minimum of 48 h. To determine the feasibility and effectiveness of solar thermotherapy in the field, portable plastic enclosures were placed over commercial and residential citrus, exposing trees to high temperatures through solarization. Within 3–6 weeks after treatment, most trees responded with vigorous new growth. Las titer in new growth was greatly reduced for 18–36 months after treatment. Unlike with potted trees, exposure to high heat did not eradicate the Las population under field conditions. This may be attributed to reduced temperatures at night in the field compared to continuous high temperature exposure that can be maintained in growth chambers, and the failure to achieve therapeutic temperatures in the root zone. Despite the presence of Las in heat-treated commercial citrus, many trees produced abundant flush and grew vigorously for 2 to 3 years after treatment. Transcriptome analysis comparing healthy trees to HLB-affected citrus both before and after heat treatment demonstrated that post-treatment transcriptional expression patterns more closely resembled the expression patterns of healthy controls for most differentially expressed genes and that genes involved with plant-bacterium interactions are upregulated after heat treatment. Overall, these results indicate that solar thermotherapy can be an effective component of an integrated control strategy for citrus HLB.

Vitis species, including grapevine, produce a class of secondary metabolites called stilbenes that are important for plant disease resistance and can have positive effects on human health. Mitogen-activated protein kinase (MAPK) signaling cascades not only play key roles in plant defense responses but also contribute to stilbene biosynthesis in grapevine. MAPKKKs function at the upper level of the MAPK network and initiate signaling through this pathway. In this study, a Raf-like MAPKKK gene, VqMAPKKK38, was identified and functionally characterized from the Chinese wild grapevine V. quinquangularis accession ‘Danfeng-2’. We observed that VqMAPKKK38 transcript levels were elevated by powdery mildew infection, high salinity conditions and chilling stresses, as well as in response to treatments by the hormones salicylic acid (SA), methyl jasmonate (MeJA), ethylene (Eth) and abscisic acid (ABA). In addition, based on both transient overexpression and gene suppression of VqMAPKKK38 in grapevine leaves, we found that VqMAPKKK38 positively regulates stilbene synthase transcription and stilbene accumulation probably by mediating the activation of the transcription factor MYB14. In addition, both hydrogen peroxide (H₂O₂) and calcium influx activated VqMAPKKK38 expression and stilbene biosynthesis, which suggests that VqMAPKKK38 may be involved in the calcium signaling and ROS signaling pathways.

The cultivated strawberry (Fragaria × ananassa) is consumed worldwide for its flavor and nutritional benefits. Genetic analysis of commercially important traits in strawberry are important for the development of breeding methods and tools for this species. Although several quantitative trait loci (QTL) have been previously detected for fruit quality and flowering traits using low-density genetic maps, clarity on the sub-genomic locations of these QTLs was missing. Recent discoveries in allo-octoploid strawberry genomics led to the development of the IStraw90 single-nucleotide polymorphism (SNP) array, enabling high-density genetic maps and finer resolution QTL analysis. In this study, breeder-specified traits were evaluated in the Eastern (Michigan) and Western (Oregon) United States for a common set of breeding populations during 2 years. Several QTLs were validated for soluble solids content (SSC), fruit weight (FWT), pH and titratable acidity (TA) using a pedigree-based QTL analysis approach. For fruit quality, a QTL for SSC on linkage group (LG) 6A, a QTL for FWT on LG 2BII, a QTL for pH on LG 4CII and two QTLs for TA on LGs 2A and 5B were detected. In addition, a large-effect QTL for flowering was detected at the distal end of LG 4A, coinciding with the FaPFRU locus. Marker haplotype analysis in the FaPFRU region indicated that the homozygous recessive genotype was highly predictive of seasonal flowering. SNP probes in the FaPFRU region may help facilitate marker-assisted selection for this trait.

Huanglongbing (HLB) in citrus infected by Candidatus Liberibacter asiaticus (CLas) has caused tremendous losses to the citrus industry. No resistant genotypes have been identified in citrus species or close relatives. Among citrus varieties, rough lemon (Citrus jambhiri) has been considered tolerant due to its ability to produce a healthy flush of new growth after infection. The difference between tolerance and susceptibility is often defined by the speed and intensity of a plant’s response to a pathogen, especially early defense responses. RNA-seq data were collected from three biological replicates of CLas- and mock-inoculated rough lemon and sweet orange at week 0 and 7 following infection. Functional analysis of the differentially expressed genes (DEGs) indicated that genes involved in the mitogen activated protein kinase (MAPK) signaling pathway were highly upregulated in rough lemon. MAPK induces the transcription of WRKY and other transcription factors which potentially turn on multiple defense-related genes. A Subnetwork Enrichment Analysis further revealed different patterns of regulation of several functional categories, suggesting DEGs with different functions were subjected to reprogramming. In general, the amplitude of the expression of defense-related genes is much greater in rough lemon than in sweet orange. A quantitative disease resistance response may contribute to the durable tolerance level to HLB observed in rough lemon.

Huanglongbing (HLB) is the most destructive bacterial disease of citrus worldwide. While most citrus varieties are susceptible to HLB, Poncirus trifoliata, a close relative of Citrus, and some of its hybrids with Citrus are tolerant to HLB. No specific HLB tolerance genes have been identified in P. trifoliata but recent studies have shown that constitutive disease resistance (CDR) genes were expressed at much higher levels in HLB-tolerant Poncirus hybrids and the expression of CDR genes was modulated by Candidatus Liberibacter asiaticus (CLas), the pathogen of HLB. The current study was undertaken to mine and characterize the CDR gene family in Citrus and Poncirus and to understand its association with HLB tolerance in Poncirus. We identified 17 CDR genes in two citrus genomes, deduced their structures, and investigated their phylogenetic relationships. We revealed that the expansion of the CDR family in Citrus seems to be due to segmental and tandem duplication events. Through genome resequencing and transcriptome sequencing, we identified eight CDR genes in the Poncirus genome (PtCDR1-PtCDR8). The number of SNPs was the highest in PtCDR2 and the lowest in PtCDR7. Most of the deletion and insertion events were observed in the UTR regions of Citrus and Poncirus CDR genes. PtCDR2 and PtCDR8 were in abundance in the leaf transcriptomes of two HLB-tolerant Poncirus genotypes and were also upregulated in HLB-tolerant, Poncirus hybrids as revealed by real-time PCR analysis. These two CDR genes seem to be good candidate genes for future studies of their role in citrus-CLas interactions.

The new plant breeding technologies (NPBTs) have recently emerged as powerful tools in the context of ‘green’ biotechnologies. They have wide potential compared to classical genetic engineering and they are attracting the interest of politicians, stakeholders and citizens due to the revolutionary impact they may have on agriculture. Cisgenesis and genome editing potentially allow to obtain pathogen-resistant plants or plants with enhanced qualitative traits by introducing or disrupting specific genes in shorter times compared to traditional breeding programs and by means of minimal modifications in the plant genome. Grapevine, the most important fruit crop in the world from an economical point of view, is a peculiar case for NPBTs because of the load of cultural aspects, varietal traditions and consumer demands, which hinder the use of classical breeding techniques and, furthermore, the application of genetic engineering to wine grape cultivars. Here we explore the technical challenges which may hamper the application of cisgenesis and genome editing to this perennial plant, in particular focusing on the bottlenecks of the Agrobacterium-mediated gene transfer. In addition, strategies to eliminate undesired sequences from the genome and to choose proper target sites are discussed in light of peculiar features of this species. Furthermore is reported an update of the international legislative frameworks regulating NPBT products which shows conflicting positions and, in the case of the European Union, a prolonged lack of regulation.

Transcriptional analyses identified molecular mechanisms associated with the response of leaf and root potato tissues to ‘Candidatus. Liberibacter solanacearum’ (Lso) infection, presumptive causal agent of zebra chip disease (ZC). Putative Lso infection affected several host processes including defense response-, regulation-, starch metabolism- and energy production-related processes. Interestingly, while proteinase inhibitors were strongly upregulated in leaf tissues, a concomitant downregulation was observed in root tissues. Quantitative polymerase chain reaction (qPCR) analysis suggests that alternative splicing might play a role. Furthermore, the transcriptional expression of redox homeostasis-related genes, including superoxide dismutase, showed the most inconsistent response to Lso in leaf and root tissues, highlighting potential targets of Lso susceptibility. Additionally, a net increase in gene expression in ZC-affected tissues despite the concomitant downregulation of photosynthesis-related processes, suggests a putative Lso-mediated low resource-use-efficiency. Subsequent nutritional analyses revealed a hypothesized Lso-mediated increase in nutrient accumulation, particularly a 210 and 108% increases in the potassium concentration of ZC-affected leaf and root tissues, respectively, suggesting an important role for potassium in ZC pathophysiology. This study highlights insights of above and below ground tissues in molecular and physiological aspects associated with potato response to ZC.

Anthocyanins are secondary metabolites in land plants that contribute to the colors of leaves and flowers, and are nutritionally valuable components of the human diet. The DFR gene plays an important role in the anthocyanin biosynthetic pathway. In this study, we investigated the regulation of DFR expression and in different Malus crabapple cultivars that show distinct patterns of leaf coloration, and how it influences leaf anthocyanin accumulation and coloration. Specifically, we studied the ever-red leaved cultivar ‘Royalty’, the ever-green leaved cultivar ‘Flame’ and the spring-red leaved cultivar ‘Radiant’. RT-PCR analysis showed that the expression of McDFR1 correlated with the expression of a MYB transcription factor, McMYB10, and with anthocyanin accumulation. We isolated five McDFR1 promoter fragments from the three cultivars and identified four different fragments (F1–4) that were present either in several cultivars, or only in one. Yeast one-hybrid and electrophoretic mobility shift assay analyses showed that McMYB10 could bind to all the McDFR1 promoters, except McDFR1-Ra2. The F1, F2 and F3 fragments did not affect McMYB10 binding to the McDFR1 promoters; however, we found evidence that the F4 fragment suppressed binding, and that the MYBGAHV amino-acid sequence maybe an important cis-element for McMYB10 protein binding. This information has potential value for strategies to modify plant color through genetic transformation.

Endogenous auxin is an important regulator of in vivo organ development, but its role in in vitro organogenesis is unclear. It has been observed that the basal end of epicotyl cuttings of juvenile citrus seedlings produces fewer shoots than the apical end. Here, we report that elevated endogenous auxin levels in the basal end of citrus epicotyl cuttings are inhibitory for in vitro shoot organogenesis. Using transgenic citrus plants expressing an auxin-inducible GUS reporter gene, we have observed elevated levels of auxin at the basal end of stem cuttings that are mediated by polar auxin transport. Depleting endogenous auxin or blocking polar auxin transport enhances shoot organogenesis. An auxin transport inhibitor, N-1-naphthylphthalamic acid (NPA), can also enhance shoot organogenesis independent of its action on polar auxin transport. Finally, we demonstrate that the promotional effects of depleting endogenous auxin or blocking polar auxin transport on shoot organogenesis are cytokinin-dependent. Our study thus provides meaningful insights into possible roles of endogenous auxin and polar auxin transport, as well as auxin–cytokinin interactions, in in vitro shoot organogenesis. Meanwhile, our results may also provide practical strategies for improving in vitro shoot organogenesis for citrus and many other plant species.

Mitogen-activated protein kinase (MAPK) cascade signal transduction modules play crucial roles in regulating many biological processes in plants. These cascades are composed of three classes of hierarchically organized protein kinases, MAPKKKs, MAPKKs and MAPKs. Here, we analyzed gene retention, phylogenetic, evolution and expression patterns of MAPK cascade genes in Brassica rapa. We further found that the MAPK branches, classes III and IV, appeared after the split of bryophytes and green algae after analyzing the MAPK cascade genes in 8 species, and their rapid expansion led to the great size of the families of MAPKs. In contrast, the ancestral class I subfamily of MAPKK gene families have been highly conserved from algae to angiosperms. For the MAPKKK family, the MEKK and Raf subfamily share a common evolutionary origin, and Raf plays a major role in the expansion of the MAPKKK gene family. The cis-elements and interaction network analyses showed the important function of MAPK cascade genes in development and stress responses in B. rapa. This study provides a solid foundation for molecular evolution analyses of MAPK cascade genes.

Jujube witches’ broom (JWB) is caused by infection with a phytoplasma. A multi-omics approach was taken during graft infection of jujube by JWB-infected scion through the analysis of the plant transcriptome, proteome and phytohormone levels. A high number of differentially expressed genes (DEGs) were identified 37 weeks after grafting (WAG), followed by observation of typical symptoms of JWB at 48 WAG. At 37 WAG, the majority of the upregulated DEGs and differentially expressed proteins (DEPs) were related to flavonoid biosynthesis, phenylalanine metabolism and phenylpropanoid biosynthesis. Two of the four upregulated proteins were similar to jasmonate-induced protein-like. Among the downregulated genes, the two most populated GO terms were plant–pathogen interaction and plant hormone signal transduction (mainly for tryptophan metabolism). Moreover, phytoplasma infection resulted in reduced auxin content and increased jasmonate content, indicating that auxin and jasmonic acid have important roles in regulating jujube responses during the first and second stages of phytoplasma infection. At 48 WAG, the two largest groups of upregulated genes were involved in phenylpropanoid biosynthesis and flavonoid biosynthesis. Both genes and proteins involved in carbon metabolism and carbon fixation in photosynthetic organisms were downregulated, indicating that photosynthesis was affected by the third stage of phytoplasma infection.