Heterozygous frameshift mutation in FaMYB10 is responsible for the natural formation of red and white-fleshed strawberry (Fragaria x ananassa Duch).

During natural evolution and artificial selection, the fruit color of many species has been repeatedly gained or lost and is generally associated with mutations in genes encoding R2R3-MYB transcription factors, especially MYB10. In this study, we show that a heterozygous frameshift mutation (FaMYB10AG-insert/FaMYB10wild ) is responsible for the loss of anthocyanins in the flesh of cultivated strawberry. Comparative transcriptomic and metabolomic analyses of red- and white-fleshed strawberry indicated that the low expression level of FaUFGT (flavonol-O-glucosyltransferases) was responsible for the loss of anthocyanins and accumulation of proanthocyanidin in the white-fleshed strawberry and was the crucial gene that encodes enzymes of the anthocyanin biosynthesis pathway. Accordingly, overexpression and silencing of FaUFGT altered anthocyanin content and changed the flesh color of strawberry fruits. Furthermore, whole-genome resequencing analyses identified an AG insertion in the FaMYB10 coding region (FaMYB10AG-insert ) of white-fleshed strawberry. Y1H and EMSA assays showed that FaMYB10wild was able to bind to the promoter of the FaUFGT gene, while the FaMYB10AG-insert could not. The skin and flesh color were tightly linked to the number of fully functional FaMYB10 copies in the selfing progeny of white-fleshed strawberry. Our results suggested that heterozygous frameshift mutation of FaMYB10 resulted in the loss of the ability to activate the expression of the FaUFGT gene, was responsible for the natural formation of red and white-fleshed strawberry.

Combined transcriptomic and proteomic analysis reveals multiple pathways involved in self-pollen tube development and the potential roles of FviYABBY1 in self-incompatibility in Fragaria viridis.

Fragaria viridis exhibits S-RNase-based gametophytic self-incompatibility, in which S-RNase is the major factor inhibiting pollen tube growth. However, the pathways involved in and the immediate causes of the inhibition of pollen tube growth remain unknown. Here, interactive RNA sequencing and proteome analysis revealed changes in the transcriptomic and proteomic profiles of F. viridis styles harvested at 0 and 24 h after self-pollination. A total of 2,181 differentially expressed genes and 200 differentially abundant proteins were identified during the pollen development stage of self-pollination. Differentially expressed genes and differentially abundant proteins associated with self-incompatible pollination were further mined, and multiple pathways were found to be involved. Interestingly, the expression pattern of the transcription factor FviYABBY1, which is linked to polar growth, differed from those of other genes within the same family. Specifically, FviYABBY1 expression was extremely high in pollen, and its expression trend in self-pollinated styles was consistent with that of S-RNase. Furthermore, FviYABBY1 interacted with S-RNase in a non-S haplotype way. Therefore, FviYABBY1 affects the expression of polar growth-related genes in self-pollen tubes and is positively regulated by S-RNase.

Elevating Air Temperature May Enhance Future Epidemic Risk of the Plant Pathogen Phytophthora infestans.

Knowledge of pathogen adaptation to global warming is important for predicting future disease epidemics and food production in agricultural ecosystems; however, the patterns and mechanisms of such adaptation in many plant pathogens are poorly understood. Here, population genetics combined with physiological assays and common garden experiments were used to analyze the genetics, physiology, and thermal preference of pathogen aggressiveness in an evolutionary context using 140 Phytophthora infestans genotypes under five temperature regimes. Pathogens originating from warmer regions were more thermophilic and had a broader thermal niche than those from cooler regions. Phenotypic plasticity contributed ~10-fold more than heritability measured by genetic variance. Further, experimental temperatures altered the expression of genetic variation and the association of pathogen aggressiveness with the local temperature. Increasing experimental temperature enhanced the variation in aggressiveness. At low experimental temperatures, pathogens from warmer places produced less disease than those from cooler places; however, this pattern was reversed at higher experimental temperatures. These results suggest that geographic variation in the thermal preferences of pathogens should be included in modeling future disease epidemics in agricultural ecosystems in response to global warming, and greater attention should be paid to preventing the movement of pathogens from warmer to cooler places.

Complete chloroplast genome sequencing of ten wild Fragaria species in China provides evidence for phylogenetic evolution of Fragaria.

Complete chloroplast genomes of ten wild Fragaria species native to China were sequenced. Phylogenetic analysis clustered Fragaria species into two clades: The south clade (F. iinumae, F. chinensis, F. pentaphylla, F. nilgerrensis, F. daltoniana, F. corymbosa, F. moupinensis, F. tibetica, F. nipponica, F. gracilis, and F. nubicola and north clade (F. viridis, F. orientalis, F. moschata, F. mandshurica, F. vesca, F. chiloensis, F. virginiana, and F. × ananassa), while F. iinumae is the oldest extant species. Molecular clock analysis suggested present Fragaria species share a common ancestor 3.57 million years ago (Ma), F. moschata and octoploid species evolve 0.89 and 0.97 Ma, respectively, but F. moschata be not directly involved in current octoploid species formation. Drastic global temperature change since the Palaeocene-Eocene, approx. 55 Ma, especially during uplifting of the Qinghai-Tibet plateau and quaternary glaciation may have driven the formation of Fragaria, separation of two groups and polyploidization.

Tracing the Diploid Ancestry of the Cultivated Octoploid Strawberry.

The commercial strawberry, Fragaria × ananassa, is a recent allo-octoploid that is cultivated worldwide. However, other than Fragaria vesca, which is universally accepted one of its diploid ancestors, its other early diploid progenitors remain unclear. Here, we performed comparative analyses of the genomes of five diploid strawberries, F. iinumae, F. vesca, F. nilgerrensis, F. nubicola, and F. viridis, of which the latter three are newly sequenced. We found that the genomes of these species share highly conserved gene content and gene order. Using an alignment-based approach, we show that F. iinumae and F. vesca are the diploid progenitors to the octoploid F. × ananassa, whereas the other three diploids that we analyzed in this study are not parental species. We generated a fully resolved, dated phylogeny of Fragaria, and determined that the genus arose ∼6.37 Ma. Our results effectively resolve conflicting hypotheses regarding the putative diploid progenitors of the cultivated strawberry, establish a reliable backbone phylogeny for the genus, and provide genetic resources for molecular breeding.

Metabolomic and Transcriptomic Analyses Reveal that Blue Light Promotes Chlorogenic Acid Synthesis in Strawberry.

Light-emitting diodes (LEDs) have been widely used in plant factories and agricultural facilities. Different LEDs can be designed in accordance with the light quality and intensity requirements of different plants, allowing the regulation of plant growth and development, as well as metabolic processes. Blue and red lights have significant effects on anthocyanin metabolism in strawberry fruit, but their effects on other metabolites are unknown. Here, we studied the effects of blue and red lights on the metabolism and gene expression of strawberry using metabolomics combined with transcriptomics. A total of 33 differentially expressed metabolites (DEMs) and 501 differentially expressed genes (DEGs) were isolated and identified. Among these DEMs, chlorogenic acid synthesis was upregulated by the blue light compared with the red light. Co-expression network analysis of DEMs and DEGs revealed that the expression of hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (FvHCT), the main gene in the chlorogenic acid synthetic pathway, was induced by blue light. Using multi-omics-based approach, our results suggest that different LED lights have multiple effects on strawberry fruit, with blue light able to co-upregulate chlorogenic acid synthesis and FvHCT gene expression.

Phenotypic analysis combined with tandem mass tags (TMT) labeling reveal the heterogeneity of strawberry stolon buds.

BACKGROUND: Ramet propagation in strawberry (Fragaria × ananassa) is the most effective way in production. However, the lack of systematically phenotypic observations and high-throughput methods limits our ability to analyze the key factors regulating the heterogeneity in strawberry stolon buds. RESULTS: From observation, we found that the axillary bud located in the first node quickly stepped into dormancy (DSB), after several bract and leaf buds were differentiated. The stolon apical meristem (SAM) degenerated as the new ramet leaf buds (RLB), and the new active axillary stolon buds (ASB) differentiated continually after the differentiation of the first leaf. Using the tandem mass tags (TMT) labeling method, a total of 7271 strawberry proteins were identified. Between ASB and DSB, the spliceosome DEPs, such as Ser/Arg-rich (SR) and heterogeneous nuclear ribonucleoprotein particle (hnRNP), showed the highest enrichment and high PPI connectivity. This indicated that the differences in DEPs (e.g., SF-3A and PK) at the transcriptional level may be causing the differences between the physiological statuses of ASB and DSB. As expected, the photosynthetic pre-form RLB mainly differentiated from ASB and DSB judging by the DEP enrichment of photosynthesis. However, there are still other specialized features of DEPs between RLB and DSB and between ASB and DSB. The DEPs relative to DNA duplication [e.g., minichromosome maintenance protein (MCM 2, 3, 4, 7)], provide a strong hint of functional gene duplication leading the bud heterogeneity between RLB and DSB. In addition, the top fold change DEP of LSH 10-like might be involved in the degeneration of SAM into RLBs, based on its significant function in modulating the plant shoot initiation. As for RLB/ASB, the phenylpropanoid biosynthesis pathway probably regulates the ramet axillary bud specialization, and further promotes the differentiation of xylem when ASB develops into a new stolon [e.g., cinnamyl alcohol dehydrogenase 1 (CAD1) and phenylalanine ammonia-lyase 1 (PAL1)]. CONCLUSIONS: By using phenotypic observation combined with proteomic networks with different types of strawberry stolon buds, the definite dormancy phase of DSB was identified, and the biological pathways and gene networks that might be responsible for heterogeneity among different stolon buds in strawberry were also revealed.

Auxin regulates adventitious root formation in tomato cuttings.

BACKGROUND: Adventitious root (AR) formation is a critical developmental process in cutting propagation for the horticultural industry. While auxin has been shown to regulate this process, the exact mechanism and details preceding AR formation remain unclear. Even though AR and lateral root (LR) formation share common developmental processes, there are exist some differences that need to be closely examined at the cytological level. Tomato stem cuttings, which readily form adventitious roots, represent the perfect system to study the influence of auxin on AR formation and to compare AR and LR organogenesis. RESULTS: Here we show the progression by which AR form from founder cells in the basal pericycle cell layers in tomato stem cuttings. The first disordered clumps of cells assumed a dome shape that later differentiated into functional AR cell layers. Further growth resulted in emergence of mature AR through the epidermis following programmed cell death of epidermal cells. Auxin and ethylene levels increased in the basal stem cutting within 1 h. Tomato lines expressing the auxin response element DR5pro:YFP showed an increase in auxin distribution during the AR initiation phase, and was mainly concentrated in the meristematic cells of the developing AR. Treatment of stem cuttings with auxin, increased the number of AR primordia and the length of AR, while stem cuttings treated with the pre-emergent herbicide/auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) occasionally developed thick, agravitropic AR. Hormone profile analyses showed that auxin positively regulated AR formation, whereas perturbations to zeatin, salicylic acid, and abscisic acid homeostasis suggested minor roles during tomato stem rooting. The gene expression of specific auxin transporters increased during specific developmental phases of AR formation. CONCLUSION: These data show that AR formation in tomato stems is a complex process. Upon perception of a wounding stimulus, expression of auxin transporter genes and accumulation of auxin at founder cell initiation sites in pericycle cell layers and later in the meristematic cells of the AR primordia were observed. A clear understanding and documentation of these events in tomato is critical to resolve AR formation in recalcitrant species like hardwoods and improve stem cutting propagation efficiency and effectiveness.

The complexity of the Fragaria x ananassa (octoploid) transcriptome by single-molecule long-read sequencing.

Strawberry (Fragaria x ananassa) is an allopolyploid species with diverse and complex transcripts. The regulatory mechanisms of fruit development and maturation have been extensively studied; however, little is known about the signaling mechanisms that direct this process in octoploid strawberry (Fragaria x ananassa). Here, we used long-read sequencing (LRS) technology and RNA-seq analysis to investigate the diversity and complexity of the polyploid transcriptome and differentially expressed transcripts along four successive fruit developmental stages of cultivated strawberry. We obtained a reference transcriptome with 119,897 unique full-length isoforms, including 2017 new isoforms and 2510 long noncoding RNAs. Based on the genome of the plausible progenitor (Fragaria vesca), 20,229 alternative splicing (AS) events were identified. Using this transcriptome, we found 17,485 differentially expressed transcripts during strawberry fruit development, including 527 transcription factors (TFs) belonging to 41 families. The expression profiles of all members of the auxin, ABA pathway, and anthocyanin biosynthesis gene families were also examined, and many of them were highly expressed at the ripe fruit stage, strongly indicating that the role of those genes is in the regulation of fruit ripening. We produce a high-quality reference transcriptome for octoploid strawberry, including much of the full-length transcript diversity, to help understand the regulatory mechanisms of fruit development and maturation of polyploid species, particularly via elucidation of the biochemical pathways involved in auxin, ABA, and anthocyanin biosynthesis.

Erratum: Author Correction: The complexity of the Fragaria x ananassa(octoploid) transcriptome by single-molecule long-read sequencing.

[This corrects the article DOI: 10.1038/s41438-019-0126-6.].

[Genome-wide identification and expression analysis of auxin-related gene families in grape].

The auxin response gene family adjusts the auxin balance and the growth hormone signaling pathways in plants. Using bioinformatics methods, the auxin-response genes from the grape genome database are identified and their chromosomal location, gene collinearity and phylogenetic analysis are performed. Probable genes include 25 AUX_IAA, 19 ARF, 9 GH3 and 42 LBD genes, which are unevenly distributed on all 19 chromosomes and some of them formed distinct tandem duplicate gene clusters. The available grape microarray databases show that all of the auxin-response genes are expressed in fruit and leaf buds, and significant overexpressed during fruit color-changing, bud break and bud dormancy periods. This paper provides a resource for functional studies of auxin-response genes in grape leaf and fruit development.

Characterization of a Calmodulin-binding Transcription Factor from Strawberry (Fragaria × ananassa).

Calmodulin-binding transcription activator (CAMTA) is a calmodulin-binding transcription factor that has a broad range of functions from sensory mechanisms to regulating many growth and developmental processes. In this study, we isolated four strawberry CAMTA (FaCAMTA) genes using HMMER and BLAST analysis. The chromosome scaffold locations of these CAMTA genes in the strawberry genome were determined and the protein domain and motif organization [CG-1, transcription factor immunoglobulin, ankyrin (ANK) repeats, calmodulin-binding IQ motif) of FaCAMTAs were also assessed. All FaCAMTAs were predicted to be Ca- and calmodulin-binding proteins. The expression profiles of FaCAMTA genes were measured in different tissues and revealed distinct FaCAMTA gene expression patterns under heat, cold, and salt stress. These data not only contribute to a better understanding of the complex regulation of the FaCAMTA gene family but also provide evidence supporting the role of CAMTAs in multiple signaling pathways involved in stress responses. This investigation can provide useful information for further study.

Genome-wide identification and analysis of FK506-binding protein family gene family in strawberry (Fragaria × ananassa).

The FK506 binding proteins (FKBPs) are abundant and ubiquitous proteins belonging to the large peptidyl-prolylcis-trans isomerase superfamily. FKBPs are known to be involved in many biological processes including hormone signaling, plant growth, and stress responses through a chaperone or an isomerization of proline residues during protein folding. The availability of complete strawberry genome sequences allowed the identification of 23 FKBP genes by HMMER and blast analysis. Chromosome scaffold locations of these FKBP genes in the strawberry genome were determined and the protein domain and motif organization of FaFKBPs analyzed. The phylogenetic relationships between strawberry FKBPs were also assessed. The expression profiles of FaFKBPs genes results revealed that most FaFKBPs were expressed in all tissues, while a few FaFKBPs were specifically expressed in some of the tissues. These data not only contribute to some better understanding of the complex regulation of the strawberry FKBP gene family, but also provide valuable information for further research in strawberry functional genomics.

Computational identification of microRNAs in the strawberry (Fragaria x ananassa) genome sequence and validation of their precise sequences by miR-RACE.

In plants, microRNAs (miRNAs) play significant roles in post-transcriptional gene regulation and have been found to control many genes involved in different biological and metabolic processes. Extensive studies were carried out to discover miRNAs and analyze their functions in model plant species, such as in Arabidopsis and rice that have been reported. In this research, we used bioinformatics to predict microRNAs in an important strawberry rootstock cultivar to discover and validate precise sequences of microRNAs in strawberry. By adopting a range of filtering criteria, we obtained 59 potential miRNAs belonging to 40 miRNA families from the Fragaria vesca genome. Using two specific 5' and 3' miRNA RACE PCR reactions and a sequence-directed cloning method, we accurately determined 34 precise sequences of candidate miRNAs, while six other sequences exhibited some minor divergence in their termini nucleotides, and 19 miRNAs that could not be cloned owing to expression abundance may be too low or these mirRNAs predicted could not be existing in strawberry. Potential target genes were further predicted for the miRNAs above. The expression of the 16 miRNAs unreported and having exact sequences and their targets by experiment could be detected in different tissues of strawberry ranging from roots, stems, leaves, flowers and fruits by qRT-PCR and some of them showed differential expression in various tissues. The functional analysis of 16 miRNAs and their targets was carried out. Finally, we conclude that there are 34 mirRNAs in strawberry and their targets play vital roles not only in growth and development, but also in diverse physiological processes. These results show that regulatory miRNAs exist in agronomically important strawberry and might have an important function in strawberry growth and development.

Computational identification of MicroRNAs in strawberry expressed sequence tags and validation of their precise sequences by miR-RACE.

MicroRNAs (miRNAs) are small, endogenously expressed, nonprotein-coding RNAs that regulate gene expression at the post-transcriptional level in both animals and plants through repressing translation or inducing mRNA degradation. A comprehensive strategy to identify new miRNA homologs by mining the repository of available strawberry expressed sequence tags (ESTs) was developed. By adopting a range of filtering criteria, we identified 11 potential miRNAs belonging to 5 miRNA families from 47 890 Fragaria vesca EST sequences. Using 2 specific 5' and 3' miRNA RACE PCR reactions and a sequence-directed cloning method, we accurately determined both end sequences of 5 candidate miRNAs. Meanwhile, qRT-PCR was used to detect the expression of these 5 miRNAs in different strawberry organs and tissues at several growing stages. These newly identified F. vesca miRNAs (fve-miRNAs) and their expression information can improve our understanding of possible roles of fve-miRNAs in regulating the growth and development of F. vesca.