Diploid Strawberry Fragaria Vesca Research Articles

The Short Inflorescence Mutation in Diploid Strawberry Fragaria vesca Affects Inflorescence Architecture and Runner Elongation

Mutants are useful for determining the genes that underlie a given trait. This information is highly useful for developing molecular markers for breeding and is the foundational knowledge required for future genomic crop improvements. The dessert strawberry, Fragaria ×ananassa, is a valuable crop with high potential for increased use in controlled environment agriculture. The genome of the woodland strawberry Fragaria vesca is the dominant genome of the four diploid strawberry subgenomes that contribute to the octoploid F. ×ananassa genome. F. vesca is therefore a useful reference system for determining gene function and should be a useful source of gene diversity for breeding of F. ×ananassa. Chemical mutagenesis of the inbred F. vesca line H4 F7-3 resulted in one M2 line with a smaller stature overall and which produces flowers on very short peduncles close to the crown. This line was named short inflorescence (sin). The sin phenotype results from a single gene recessive mutation that is pleiotropic in that the mutation also affects internode lengths of runners as well as petiole elongation of sin plants. Microscopic characterization revealed that sin peduncles are most likely short because of a failure of cells to elongate. Inflorescences, runners, and petioles of sin plants were found to elongate in response to exogenous gibberellin, indicating that sin could be a gibberellin biosynthesis or transport mutant. A brief characterization of sin plants is presented to facilitate collaborative studies of the line.

FvMYB44, a Strawberry R2R3-MYB Transcription Factor, Improved Salt and Cold Stress Tolerance in Transgenic Arabidopsis

MYB (v-MYB avian myeloblastosis viral oncogene homolog) transcription factor (TF) family has numerous members with complex and diverse functions, which perform an integral role in regulating the plant’s response to adversity. This study used cloning to obtain a novel MYB TF gene from the diploid strawberry Fragaria vesca, which was given the designation FvMYB44. Subcellular localization results showed that the protein of FvMYB44 was a nuclear localization protein. The resistance of Arabidopsis thaliana to salt and low temperature stresses was greatly enhanced by the overexpression of FvMYB44. When subjected to salt and temperature stress, transgenic plants showed higher proline and chlorophyll concentrations and higher superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities than wild-type (WT) and unloaded line (UL) of A. thaliana. In contrast, WT and UL lines had higher malondialdehyde (MDA) content and reactive oxygen species ROS (O2− and H2O2) content. These findings suggest that FvMYB44 may perform a role in controlling the response of A. thaliana to cold and salt stress.

Genome-Wide Identification and Characterization of Argonaute, Dicer-like and RNA-Dependent RNA Polymerase Gene Families and Their Expression Analyses in Fragaria spp.

In the growth and development of plants, some non-coding small RNAs (sRNAs) not only mediate RNA interference at the post-transcriptional level, but also play an important regulatory role in chromatin modification at the transcriptional level. In these processes, the protein factors Argonaute (AGO), Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) play very important roles in the synthesis of sRNAs respectively. Though they have been identified in many plants, the information about these gene families in strawberry was poorly understood. In this study, using a genome-wide analysis and a phylogenetic approach, 13 AGO, six DCL, and nine RDR genes were identified in diploid strawberry Fragaria vesca. We also identified 33 AGO, 18 DCL, and 28 RDR genes in octoploid strawberry Fragaria × ananassa, studied the expression patterns of these genes in various tissues and developmental stages of strawberry, and researched the response of these genes to some hormones, finding that almost all genes respond to the five hormone stresses. This study is the first report of a genome-wide analysis of AGO, DCL, and RDR gene families in Fragaria spp., in which we provide basic genomic information and expression patterns for these genes. Additionally, this study provides a basis for further research on the functions of these genes and some evidence for the evolution between diploid and octoploid strawberries.

Genetic modulation of RAP alters fruit coloration in both wild and cultivated strawberry.

SummaryFruit colour affects consumer preference and is an important trait for breeding in strawberry. Previously, we isolated the Reduced Anthocyanins in Petioles (RAP) gene encoding a glutathione S‐transferase (GST) that binds anthocyanins to facilitate their transport from cytosol to vacuole in the diploid strawberry Fragaria vesca. The parent of rap was the F. vesca variety ‘Yellow Wonder’ that develops white fruit due to a natural mutation in the FveMYB10 gene. Here, we investigated the application potential of RAP in modulating fruit colours by overexpression of RAP in F. vesca and knockout of RAP in the cultivated strawberry Fragaria × ananassa. Unexpectedly, the RAP overexpression in Yellow Wonder background caused formation of red fruit. In addition, the red coloration occurs precociously at floral stage 10 and continues in the receptacle during early fruit development. Transcriptome analysis revealed that the anthocyanin biosynthesis genes were not up‐regulated in RAP‐ox; rap myb10 flowers at anthesis and largely inhibited at the turning stage in fruit, suggesting a coloration mechanism independent of FveMYB10. Moreover, we used CRISPR/Cas9 to knockout RAP in cultivated strawberry which is octoploid. Six copies of RAP were simultaneously knocked out in the T0 generation leading to the green stem and white‐fruited phenotype. Several T1 progeny have segregated away the CRISPR/Cas9 transgene but maintain the green stem trait. Our results indicate that enhancing the anthocyanin transport could redirect the metabolic flux from proanthocyanidin to anthocyanin production at early developmental stages of fruit and that RAP is one promising candidate gene in fruit colour breeding of strawberry.

Updated annotation of the wild strawberry Fragaria vesca V4 genome

The diploid strawberry Fragaria vesca serves as an ideal model plant for cultivated strawberry (Fragaria × ananassa, 8x) and the Rosaceae family. The F. vesca genome was initially published in 2011 using older technologies. Recently, a new and greatly improved F. vesca genome, designated V4, was published. However, the number of annotated genes is remarkably reduced in V4 (28,588 genes) compared to the prior annotations (32,831 to 33,673 genes). Additionally, the annotation of V4 (v4.0.a1) implements a new nomenclature for gene IDs (FvH4_XgXXXXX), rather than the previous nomenclature (geneXXXXX). Hence, further improvement of the V4 genome annotation and assigning gene expression levels under the new gene IDs with existing transcriptome data are necessary to facilitate the utility of this high-quality F. vesca genome V4. Here, we built a new and improved annotation, v4.0.a2, for F. vesca genome V4. The new annotation has a total of 34,007 gene models with 98.1% complete Benchmarking Universal Single-Copy Orthologs (BUSCOs). In this v4.0.a2 annotation, gene models of 8,342 existing genes are modified, 9,029 new genes are added, and 10,176 genes possess alternatively spliced isoforms with an average of 1.90 transcripts per locus. Transcription factors/regulators and protein kinases are globally identified. Interestingly, the transcription factor family FAr-red-impaired Response 1 (FAR1) contains 82 genes in v4.0.a2 but only two members in v4.0.a1. Additionally, the expression levels of all genes in the new annotation across a total of 46 different tissues and stages are provided. Finally, miRNAs and their targets are reanalyzed and presented. Altogether, this work provides an updated genome annotation of the F. vesca V4 genome as well as a comprehensive gene expression atlas with the new gene ID nomenclature, which will greatly facilitate gene functional studies in strawberry and other evolutionarily related plant species.

An Easy-to-Follow Pipeline for Long Noncoding RNA Identification: A Case Study in Diploid Strawberry Fragaria vesca.

Long noncoding RNAs (lncRNAs), defined as transcripts longer than 200 nucleotides without coding potential, are a new class of regulatory molecules with roles in diverse biological processes. New lncRNAs can readily be identified by mining RNA-seq data from a wide range of plant species. However, challenges remain as to how one can distinguish functional lncRNAs from mRNAs coding for small peptides or products of pseudogenes without any function. In this chapter, stepwise instruction is provided using RNA-seq datasets of developing wild strawberry fruit to illustrate each step. The workflow can be divided into three parts. Part I concerns standard RNA-seq data processing and analysis; part II describes lncRNA identification; part III describes several approaches aimed at shedding lights on lncRNA function. The description is intended for beginners with easy-to-follow steps. Text boxes provide codes and explanations. While it is relatively easy to identify lncRNAs, it is difficult to infer their function in the absence of coding information. Multiple RNA-seq libraries across tissues and stages are useful resources for deducing possible function of lncRNAs based on their expression and co-regulation.

Expression analysis suggests potential roles for PH-LIKE (PHL) genes in diploid strawberry Fragaria vesca L. seedling hormone response and fruit development

ABSTRACTAcidity is a critical determinant for fruit sweetness in fleshy fruits. A recent molecular genetic study in sweet melon has revealed that the PH gene, encoding a protein most closely related to the Arabidopsis auxin efflux carrier family (PILS), acts as a critical regulator of fruit acidity. To provide insights into the possible role of PH-LIKE (PHL) genes in strawberry fruit acidity control, we identified a total of six PHL genes, FvPHL1 to FvPHL6, in a diploid strawberry fruit model system, Fragaria vesca L. Yellow Wonder 5AF7. These FvPHL genes exhibit distinct organ expression patterns. FvPHL1, the melon PH ortholog, was expressed in roots, leaves, flowers and fruits, while FvPHL2/3/6 were preferentially expressed in fruits. In addition, FvPHL genes exhibit fruit development and ripening-related expression patterns. Interestingly, FvPHL1/3/5/6 were all induced by exogenous auxin treatments in seedlings. In addition, FvPHL5 was slightly inhibited by abscisic acid (ABA), while the other five PHL genes were up-regulated by ABA in seedlings. However, auxin and ABA did not seem to affect FvPHL expression in developing fruits. These results indicate that some of FvPHL genes may be involved in auxin and ABA response in seedling growth and act in fruit development including acidity control.

Cloning, expression and molecular modeling of the anthocyanidin reductase (FaANR) gene during strawberry fruit development

Introduction - Proanthocyanidins (PAs) are a group of polyphenolic secondary metabolites synthesized in plants via flavonoid pathway. Strawberry (Fragaria x ananassa Duch.) is a rich source of flavonoids and proanthocyanins, which are known to have multiple health benefits. The anthocyanidin reductase (ANR) is an interesting gene to study within the flavonoid biosynthesis pathway, since it diverts the anthocyanin pathway to flavonol synthesis. Materials and methods - The present study describes cloning, semi-quantitative expression analysis and molecular modeling of the strawberry (cv. Sweet Charlie) anthocyanidin reductase (FaANR) gene during the progressive stages of fruit development. Results and discussion - The FaANR gene was 1,020 bp long with an open reading frame encoding a protein of 308 amino acid residues. The estimated molecular mass and isoelectric point of the protein were 32.89 kD and 5.54, respectively. The expression of FaANR was only seen during early stages of fruit development indicating its early involvement in PA accumulation, well before ripening onset. Analysis of the FaANR sequence showed 98% similarity to ANR from diploid strawberry Fragaria vesca. The cladistic analysis indicated that the FaANR was phylogenetically similar to Pyrus and Prunus ANR genes. Protein modeling suggested protein-ligand interactions at active site with NADP binding as the plausible mechanism of action. Conclusion - This is the first report on cloning, expression study and in silico modeling of an anthocyanidin reductase of strawberry. The conditions of in vivo modulation of ANR expression open applied perspectives for commercial production of PAs in strawberry and other berries.

Bioinformatic and expression analyses on carotenoid dioxygenase genes in fruit development and abiotic stress responses in Fragaria vesca.

Carotenoid dioxygenases, including 9-cis-epoxycarotenoid dioxygenases (NCEDs) and carotenoid cleavage dioxygenases (CCDs), can selectively cleave carotenoids into various apocarotenoid products that play important roles in fleshy fruit development and abiotic stress response. In this study, we identified 12 carotenoid dioxygenase genes in diploid strawberry Fragaria vesca, and explored their evolution with orthologous genes from nine other species. Phylogenetic analyses suggested that the NCED and CCDL groups moderately expanded during their evolution, whereas gene numbers of the CCD1, CCD4, CCD7, and CCD8 groups maintained conserved. We characterized the expression profiles of FveNCED and FveCCD genes during flower and fruit development, and in response to several abiotic stresses. FveNCED1 expression positively responded to osmotic, cold, and heat stresses, whereas FveNCED2 was only induced under cold stress. In contrast, FveNCED2 was the unique gene highly and continuously increasing in receptacle during fruit ripening, which co-occurred with the increase in endogenous abscisic acid (ABA) content previously reported in octoploid strawberry. The differential expression patterns suggested that FveNCED1 and FveNCED2 were key genes for ABA biosynthesis in abiotic stress responses and fruit ripening, respectively. FveCCD1 exhibited the highest expression in most stages of flower and fruit development, while the other FveCCDs were expressed in a subset of stages and tissues. Our study suggests distinct functions of FveNCED and FveCCD genes in fruit development and stress responses and lays a foundation for future study to understand the roles of these genes and their metabolites, including ABA and other apocarotenoid products, in the growth and development of strawberry.

Pathway Analysis and Omics Data Visualization Using Pathway Genome Databases: FragariaCyc, a Case Study.

The species-specific plant Pathway Genome Databases (PGDBs) based on the BioCyc platform provide a conceptual model of the cellular metabolic network of an organism. Such frameworks allow analysis of the genome-scale expression data to understand changes in the overall metabolisms of an organism (or organs, tissues, and cells) in response to various extrinsic (e.g. developmental and differentiation) and/or extrinsic signals (e.g. pathogens and abiotic stresses) from the surrounding environment. Using FragariaCyc, a pathway database for the diploid strawberry Fragaria vesca, we show (1) the basic navigation across a PGDB; (2) a case study of pathway comparison across plant species; and (3) an example of RNA-Seq data analysis using Omics Viewer tool. The protocols described here generally apply to other Pathway Tools-based PGDBs.

Global identification and analysis of long non-coding RNAs in diploid strawberry Fragaria vesca during flower and fruit development.

BackgroundLong non-coding RNAs (lncRNAs) are a new class of regulatory molecules with roles in diverse biological processes. While much effort has been invested in the analysis of lncRNAs from established plant models Arabidopsis, maize, and rice, almost nothing is known about lncRNAs from fruit crops, including those in the Rosaceae family.ResultsHere, we present a genome-scale identification and characterization of lncRNAs from a diploid strawberry, Fragaria vesca, based on rich RNA-seq datasets from 35 different flower and fruit tissues. 5,884 Fve-lncRNAs derived from 3,862 loci were identified. These lncRNAs were carefully cataloged based on expression level and whether or not they contain repetitive sequences or generate small RNAs. About one fourth of them are termed high-confidence lncRNAs (hc-lncRNAs) because they are expressed at a level of FPKM higher than 2 and produce neither small RNAs nor contain repetitive sequence. To identify regulatory interactions between lncRNAs and their potential protein-coding (PC) gene targets, pairs of lncRNAs and PC genes with positively or negatively correlated expression trends were identified based on their expression; these pairs may be candidates of cis- or trans-acting lncRNAs and their targets. Finally, blast searches within plant species indicate that lncRNAs are not well conserved.ConclusionsOur study identifies a large number of tissue-specifically expressed lncRNAs in F. vesca, thereby highlighting their potential contributions to strawberry flower and fruit development and paving the way for future functional studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2014-2) contains supplementary material, which is available to authorized users.

The Arabidopsis NPR1 gene confers broad-spectrum disease resistance in strawberry.

Although strawberry is an economically important fruit crop worldwide, production of strawberry is limited by its susceptibility to a wide range of pathogens and the lack of major commercial cultivars with high levels of resistance to multiple pathogens. The objective of this study is to ectopically express the Arabidopsis thaliana NPR1 gene (AtNPR1) in the diploid strawberry Fragaria vesca L. and to test transgenic plants for disease resistance. AtNPR1 is a key positive regulator of the long-lasting broad-spectrum resistance known as systemic acquired resistance (SAR) and has been shown to confer resistance to a number of pathogens when overexpressed in Arabidopsis or ectopically expressed in several crop species. We show that ectopic expression of AtNPR1 in strawberry increases resistance to anthracnose, powdery mildew, and angular leaf spot, which are caused by different fungal or bacterial pathogens. The increased resistance is related to the relative expression levels of AtNPR1 in the transgenic plants. In contrast to Arabidopsis plants overexpressing AtNPR1, which grow normally and do not constitutively express defense genes, the strawberry transgenic plants are shorter than non-transformed controls, and most of them fail to produce runners and fruits. Consistently, most of the transgenic lines constitutively express the defense gene FvPR5, suggesting that the SAR activation mechanisms in strawberry and Arabidopsis are different. Nevertheless, our results indicate that overexpression of AtNPR1 holds the potential for generation of broad-spectrum disease resistance in strawberry.

Molecular cloning and expression analyses of FaFT, FaTFL, and FaAP1 genes in cultivated strawberry: their correlation to flower bud formation

In this study, we cloned flowering-related genes FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) from domesticated octaploid strawberries (Fragaria × ananassa) and analyzed their expression patterns in cultivars Tochiotome and Akihime. The floral meristem generation was induced under the short day and low temperature (SDLT), but not under the long day and high temperature (LDHT). We found that FaFT1, which is an orthologue of the Arabidopsis floral activator FT, was highly expressed in leaves under LDHT but not expressed in leaves under SDLT. On the other hand, the expression of FaTFL2, which belongs to the TFL1 family of flowering repressing genes, decreased in crowns (stem tissue including meristem) under SDLT. These results suggest that FaTFL2, as opposed to FvTFL1 in wild diploid strawberry Fragaria vesca, is related to flowering of the cultivated strawberry. Moreover, the FaTFL2 expression might be regulated by temperature rather than by photoperiod. We demonstrated that a reduction of the FaTFL2 expression is a key signal for flowering in domesticated strawberries.

An inbred line of the diploid strawberry Fragaria vesca f. semperflorens for genomic and molecular genetic studies in the Rosaceae.

BackgroundThe diploid woodland strawberry (Fragaria vesca) is an attractive system for functional genomics studies. Its small stature, fast regeneration time, efficient transformability and small genome size, together with substantial EST and genomic sequence resources make it an ideal reference plant for Fragaria and other herbaceous perennials. Most importantly, this species shares gene sequence similarity and genomic microcolinearity with other members of the Rosaceae family, including large-statured tree crops (such as apple, peach and cherry), and brambles and roses as well as with the cultivated octoploid strawberry, F. ×ananassa. F. vesca may be used to quickly address questions of gene function relevant to these valuable crop species. Although some F. vesca lines have been shown to be substantially homozygous, in our hands plants in purportedly homozygous populations exhibited a range of morphological and physiological variation, confounding phenotypic analyses. We also found the genotype of a named variety, thought to be well-characterized and even sold commercially, to be in question. An easy to grow, standardized, inbred diploid Fragaria line with documented genotype that is available to all members of the research community will facilitate comparison of results among laboratories and provide the research community with a necessary tool for functionally testing the large amount of sequence data that will soon be available for peach, apple, and strawberry.ResultsA highly inbred line, YW5AF7, of a diploid strawberry Fragaria vesca f. semperflorens line called "Yellow Wonder" (Y2) was developed and examined. Botanical descriptors were assessed for morphological characterization of this genotype. The plant line was found to be rapidly transformable using established techniques and media formulations.ConclusionThe development of the documented YW5AF7 line provides an important tool for Rosaceae functional genomic analyses. These day-neutral plants have a small genome, a seed to seed cycle of 3.0 - 3.5 months, and produce fruit in 7.5 cm pots in a growth chamber. YW5AF7 is runnerless and therefore easy to maintain in the greenhouse, forms abundant branch crowns for vegetative propagation, and produces highly aromatic yellow fruit throughout the year in the greenhouse. F. vesca can be transformed with Agrobacterium tumefaciens, making these plants suitable for insertional mutagenesis, RNAi and overexpression studies that can be compared against a stable baseline of phenotypic descriptors and can be readily genetically substantiated.

Light as a Growth Regulator: Controlling Plant Biology with Narrow-bandwidth Solid-state Lighting Systems

In the mission of plant husbandry, light is a critical yet passive entity. The potential to actively implement dynamic lighting strategies to control plant growth and development holds great promise in the future of plant cultivation. In other words, rather than simply using a single stable light condition to maintain photosynthesis, might it be possible to continually adjust fluence rate, wavelength combinations, and photoperiods to actively manipulate plant morphology and production? Research over the past 100 years suggests that it is so, and today's solid-state, narrow bandwidth lighting platforms offer a unique opportunity to test this hypothesis. The goal of this report is to describe the potential use of light as a growth regulator. Here light-emitting diode technology is well suited for the application, because light quantity, quality, photoperiod, and combinations thereof can be controlled with great precision. Specific light combinations may be adjusted throughout the life of a plant to potentially optimize traits of interest such as synchronization of flowering, maintenance of vegetative growth programs, control of plant stature, or acceleration of juvenility. This report describes the plant photosensory networks and how they sense and respond to light. The connection between light and internal hormone stasis is explored and then extended to questions of designing specific regimes to control plant growth and development. The concept of static signaling states is presented as a means to tightly control plant habits, in essence, using light to stabilize plant signal transduction pathways and their associated outcomes. Finally, the concepts presented are applied to the diploid strawberry Fragaria vesca to demonstrate the usefulness of the approach. These experiments provide proof-of-concept and lay a foundation for further studies.

Long-day rather than autonomous control of flowering in the diploid everbearing strawberry Fragaria vesca ssp. semperflorens

SummaryThe environmental control of flowering in the perpetual-flowering (everbearing) diploid strawberry Fragaria vesca ssp. semperflorens cultivars ‘Rügen’ and ‘Baron Solemacher’ has been studied in controlled environments. Seedpropagated plants were exposed to 10-h short-day (SD) and 24-h long-day (LD) conditions at temperatures ranging from 9° – 27°C. The results revealed a quantitative LD response of flowering that increased in strength with increasing temperature, to become almost obligatory at 27°C. Occasional runner formation was observed in SD at high temperature, conditions which were inhibitory to flowering, demonstrating that runnering ability is not completely lost in these genotypes. A comparison with the perpetual-flowering octoploid F. ananassa ‘Elan’, in one experiment, demonstrated an identical LD temperature interaction in the two species. The results are discussed in relation to available information on the genetics of flowering habits in the two species. Since seasonal flowering types of F. vesca and F. ananassa have also been shown to share a principally identical flowering response, controlled by SD and low temperature, it is concluded that a remarkably similar flowering control system is present in the diploid F. vesca and the octoploid F. ananassa. Despite the large genetic differences between the two species, and regardless of the origin of the cultivars, the seasonal flowering types are all SD plants, while their perpetual-flowering counterparts all appear to be LD plants. In both cases, there is a pronounced interaction with temperature; the photoperiodic responses increasing with increasing temperature, in both cases. This raises the question whether a common genetic flowering control system is present in both species.

(265) HSP101 in the Model Strawberry Fragaria vesca

Our lab has initiated a project to determine if specific proteins expressed by strawberry function as part of the thermotolerance system. We have developed tools for investigating the role of heat shock proteins (HSPs) and other gene products in strawberry thermotolerance. These tools include an inbred diploid testing system, EST sequence data, and molecular markers for heat tolerance. We developed an inbred line, 5AF7, of the diploid strawberry Fragariavesca for testing gene function because the diploid genome is small (164Mbp), the life cycle of the plant is short (about 4 months), the plant size is small (a plant will produce fruit in a 4-inch pot), some genetic work is already done, F. vesca is transformable with Agrobacteriumtumefaciens, and results should be transferable to the commercial octoploid varieties. A cDNA library was constructed in the pCMVsport 6.1 vector using combined RNA from batches of aseptically grown F. vesca seedlings treated to various elevated temperature regimes. Over 1500 EST sequences from the library have been deposited in GenBank and are available annotated at the ESTAP Fragaria database (http://staff.vbi.vt.edu/estap/index.shtml). HSP101 affects thermotolerance in Arabidopsis(Queitsch et al., 2000, Plant Cell 12:479). A PCR fragment for HSP101 was generated from F. vesca with degenerate primers, and used to obtain a full-length cDNA clone from the library. Primers spanning an intron were designed for RTPCR from the sequence of the cDNA. Semi-quantitative RTPCR indicates that HSP101 is expressed constitutively in young leaves at 25 °C and is not induced at moderately higher temperatures (32 °C) even after 5 hours. Induction occurs within 1 hour at 37 °C.