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Anthocyanin accumulation is transcriptionally regulated by the MYB-bHLH-WD40 complex. Light is indispensable for anthocyanin accumulation, and light-inducible MYB and HY5 were considered to promote anthocyanin accumulation in many fruits. Whether and how light-inducible bHLH transcription factor and HY5 regulate anthocyanin synthesis in strawberry is unknown. In this study, we identified a bHLH transcription factor, FvbHLH9, which was induced by light as well as FvHY5, and found that, similar to FvHY5, the transient overexpression and interference FvbHLH9 in strawberry fruits can promote and decrease anthocyanin accumulation, respectively, indicating FvbHLH9 functions as a positive regulator of anthocyanin biosynthesis. Furthermore, we confirmed that both FvHY5 and FvbHLH9 specifically bind to the promoter region of some key enzyme genes, including FvDFR, and the expression of FvDFR was activated through the heterodimer formation between FvHY5 and FvbHLH9. Finally, we confirmed that FvbHLH9-promoted anthocyanin accumulation is dependent on HY5-bHLH heterodimerisation in Arabidopsis. Our findings provide insights into a mechanism involving the synergistic regulation of light-dependent coloration and anthocyanin biosynthesis via a HY5-bHLH heterodimer formed by the interaction of FvHY5 and FvbHLH9 in strawberry fruits., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Fruit crops, including apple, orange, grape, banana, strawberry, watermelon, kiwifruit and tomato, not only provide essential nutrients for human life but also contribute to the major agricultural output and economic growth of many countries and regions in the world. Recent advancements in genome editing provides an unprecedented opportunity for the genetic improvement of these agronomically important fruit crops. Here, we summarize recent reports of applying CRISPR/Cas9 to fruit crops, including efforts to reduce disease susceptibility, change plant architecture or flower morphology, improve fruit quality traits, and increase fruit yield. We discuss challenges facing fruit crops as well as new improvements and platforms that could be used to facilitate genome editing in fruit crops, including dCas9-base-editing to introduce desirable alleles and heat treatment to increase editing efficiency. In addition, we highlight what we see as potentially revolutionary development ranging from transgene-free genome editing to de novo domestication of wild relatives. Without doubt, we now see only the beginning of what will eventually be possible with the use of the CRISPR/Cas9 toolkit. Efforts to communicate with the public and an emphasis on the manipulation of consumer-friendly traits will be critical to facilitate public acceptance of genetically engineered fruits with this new technology., (© 2019 Institute of Botany, Chinese Academy of Sciences.)

The critical role of auxin in strawberry fruit set and receptacle enlargement was demonstrated previously. While fertilization is known to trigger auxin biosynthesis, the specific tissue source of fertilization-induced auxin is not well understood. Here, the auxin reporter DR5ver2::GUS was introduced into wild strawberry (Fragaria vesca) to reveal auxin distribution in the seed and fruit receptacle pre- and post-fertilization as well as in the root. In addition, the expression of TAR and YUCCA genes coding for enzymes catalysing the two-step auxin biosynthesis pathway was investigated using their respective promoters fused to the β-glucuronidase (GUS) reporter. Two FveTARs and four FveYUCs were shown to be expressed primarily in the endosperm and embryo inside the achenes as well as in root tips and lateral root primordia. Expression of these reporters in dissected tissues provided more detailed and precise spatial (cell and tissue) and temporal (pre- and post-fertilization) information on where auxin is synthesized and accumulates than previous studies in strawberry. Moreover, we generated CRISPR-mediated knock-out mutants of FveYUC10, the most abundant YUC in seeds; the mutants had a lower free auxin level in young fruit, but displayed no obvious morphological phenotypes. However, overexpression of FveYUC10 resulted in elongated hypocotyls in Arabidopsis caused by elevated auxin level. Overall, the study revealed auxin accumulation in the chalazal seed coat, embryo, receptacle vasculature, root tip, and lateral root primordia and highlighted the endosperm as the main auxin biosynthesis site for fruit set.

The diploid strawberry, Fragaria vesca , is a developing model system for the economically important Rosaceae family. Strawberry fleshy fruit develops from the floral receptacle and its ripening is nonclimacteric. The external seed configuration of strawberry fruit facilitates the study of seed-to-fruit cross tissue communication, particularly phytohormone biosynthesis and transport. To investigate strawberry fruit development, we previously generated spatial and temporal transcriptome data profiling F. vesca flower and fruit development pre- and postfertilization. In this study, we combined 46 of our existing RNA-seq libraries to generate coexpression networks using the Weighted Gene Co-Expression Network Analysis package in R. We then applied a post-hoc consensus clustering approach and used bootstrapping to demonstrate consensus clustering's ability to produce robust and reproducible clusters. Further, we experimentally tested hypotheses based on the networks, including increased iron transport from the receptacle to the seed postfertilization and characterized a F. vesca floral mutant and its candidate gene. To increase their utility, the networks are presented in a web interface (www.fv.rosaceaefruits.org) for easy exploration and identification of coexpressed genes. Together, the work reported here illustrates ways to generate robust networks optimized for the mining of large transcriptome data sets, thereby providing a useful resource for hypothesis generation and experimental design in strawberry and related Rosaceae fruit crops., (© 2018 American Society of Plant Biologists. All rights reserved.)

Fruit set-the commitment of an angiosperm plant to develop fruit-is a key developmental process that normally occurs following successful fertilization. Parthenocarpy arises when fruit automatically develop in the absence of fertilization. This review uses parthenocarpic fruit development as a focal device through which to recapitulate and understand the molecular effectors that mediate and regulate fruit set. The review demonstrates that studies of parthenocarpy are providing vital insight into plant development, signaling and, potentially, high-value agricultural products.

Fragaria vesca is a species of diploid strawberry being developed as a model for the octoploid garden strawberry. This work sequenced and compared the genomes of three F. vesca accessions: 'Hawaii 4', 'Rügen', and 'Yellow Wonder'. Genome-scale analyses of shared and distinct SNPs among these three accessions have revealed that 'Rügen' and 'Yellow Wonder' are more similar to each other than they are to 'Hawaii 4'. Though all three accessions are inbred seven generations, each accession still possesses extensive heterozygosity, highlighting the inherent differences between individual plants even of the same accession. The identification of the impact of each SNP as well as the large number of Indel markers provides a foundation for locating candidate mutations underlying phenotypic variations among these F. vesca accessions and for mapping new mutations generated through forward genetics screens. Through systematic analysis of SNP variants affecting genes in anthocyanin biosynthesis and regulation, a candidate SNP in FveMYB10 was identified and then functionally confirmed to be responsible for the yellow color fruits made by many F. vesca accessions. As a whole, this study provides further resources for F. vesca and establishes a foundation for linking traits of economic importance to specific genes and variants.

Background: Long 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., Results: Here, 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., Conclusions: Our 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.

Fragaria vesca, a diploid woodland strawberry with a small and sequenced genome, is an excellent model for studying fruit development. The strawberry fruit is unique in that the edible flesh is actually enlarged receptacle tissue. The true fruit are the numerous dry achenes dotting the receptacle's surface. Auxin produced from the achene is essential for the receptacle fruit set, a paradigm for studying crosstalk between hormone signaling and development. To investigate the molecular mechanism underlying strawberry fruit set, next-generation sequencing was employed to profile early-stage fruit development with five fruit tissue types and five developmental stages from floral anthesis to enlarged fruits. This two-dimensional data set provides a systems-level view of molecular events with precise spatial and temporal resolution. The data suggest that the endosperm and seed coat may play a more prominent role than the embryo in auxin and gibberellin biosynthesis for fruit set. A model is proposed to illustrate how hormonal signals produced in the endosperm and seed coat coordinate seed, ovary wall, and receptacle fruit development. The comprehensive fruit transcriptome data set provides a wealth of genomic resources for the strawberry and Rosaceae communities as well as unprecedented molecular insight into fruit set and early stage fruit development.

The diploid woodland strawberry, Fragaria vesca, is being recognized as a model for the more complex octoploid commercial strawberry, Fragaria × ananassa. F. vesca exhibits a short seed to seed cycle, can be easily transformed by Agrobacteria, and a draft genome sequence has been published. These features, together with its similar flower structure, potentially make F. vesca a good model for studying the flower development of other members of the Rosaceae family, which contains many economically important fruit trees and ornamental plants. To propel F. vesca's role in genetic and genomic research and to facilitate the study of its reproductive development, we have investigated in detail F. vesca flower and early fruit development using a seventh generation inbred diploid line, Yellow Wonder 5AF7. We present here standardized developmental staging and detailed descriptions of morphological changes associated with flower and early fruit development based on images of hand dissected flowers, histological sections, and scanning electron microscopy. In situ hybridization with the F. vesca AGAMOUS homolog, FvAG, showed expression in young stamen and carpel primordia. This work lays the essential groundwork and standardization for future molecular, genetic, and genomic studies of F. vesca.

SUMMARY: Auxin plays an essential role in plant growth and development, particularly in fruit development. The YUCCA (YUC) genes encode flavin monooxygenases that catalyze a rate‐limiting step in auxin biosynthesis. Mutations that disrupt YUC gene function provide useful tools for dissecting general and specific functions of auxin during plant development. In woodland strawberry (Fragaria vesca), two ethyl methanesulfonate mutants, Y422 and Y1011, have been identified that exhibit severe defects in leaves and flowers. In particular, the width of the leaf blade is greatly reduced, and each leaflet in the mutants has fewer and deeper serrations. In addition, the number and shape of the floral organs are altered, resulting in smaller fruits. Mapping by sequencing revealed that both mutations reside in the FveYUC4 gene, and were therefore renamed as yuc4‐1 and yuc4‐2. Consistent with a role for FveYUC4 in auxin synthesis, free auxin and its metabolites are significantly reduced in the yuc4 leaves and flowers. This role of FveYUC4 in leaf and flower development is supported by its high and specific expression in young leaves and flower buds using GUS reporters. Furthermore, germline transformation of pYUC4::YUC4, which resulted in elevated expression of FveYUC4 in yuc4 mutants, not only rescued the leaf and flower defects but also produced parthenocarpic fruits. Taken together, our data demonstrate that FveYUC4 is essential for leaf and flower morphogenesis in woodland strawberry by providing auxin hormone at the proper time and in the right tissues. Significance Statement: Mutants of the auxin biosynthesis gene FveYUC4 have been identified in woodland strawberry, demonstrating the importance of local auxin biosynthesis in regulating leaf shape and floral organ formation. Germline transformation of pYUC4::YUC4 can not only rescue the leaf and flower defects of yuc4, but also produces pollination‐independent fruits due to increased expression of FveYUC4, a candidate gene for the introduction of parthenocarpy in strawberry. [ABSTRACT FROM AUTHOR]

The R2R3-MYB transcription factor FveMYB10 is a major regulator of anthocyanin pigmentation in the red fruits of strawberry. fvemyb10 loss-of-function mutants form yellow fruits but still accumulate purple-colored anthocyanins in the petioles, suggesting that anthocyanin biosynthesis is under distinct regulation in fruits and petioles. From chemical mutagenesis in the diploid wild strawberry Fragaria vesca , we identified a green petioles (gp) -1 mutant that lacks anthocyanins in petioles. Using mapping-by-sequencing and transient functional assays, we confirmed that the causative mutation resides in a FveMYB10-Like (FveMYB10L) gene and that FveMYB10 and FveMYB10L function independently in the fruit and petiole, respectively. In addition to their tissue-specific regulation, FveMYB10 and FveMYB10L respond differently to changes in light quality, produce distinct anthocyanin compositions, and preferentially activate different downstream anthocyanin biosynthesis genes in their respective tissues. This work identifies a new regulator of anthocyanin synthesis and demonstrates that two paralogous MYB genes with specialized functions enable tissue-specific regulation of anthocyanin biosynthesis in fruit and petiole tissues. [ABSTRACT FROM AUTHOR]

SUMMARY: Fruits represent key evolutionary innovations in angiosperms and exhibit diverse types adapted for seed dissemination. However, the mechanisms that underlie fruit type diversity are not understood. The Rosaceae family comprises many different fruit types, including 'pome' and 'drupe' fruits, and hence is an excellent family for investigating the genetic basis of fruit type specification. Using comparative transcriptomics, we investigated the molecular events that correlate with pome (apple) and drupe (peach) fleshy fruit development, focusing on the earliest stages of fruit initiation. We identified PI and TM6, MADS box genes whose expression negatively correlates with fruit flesh‐forming tissues irrespective of fruit type. In addition, the MADS box gene FBP9 is expressed in fruit‐forming tissues in both species, and was lost multiple times in the genomes of dry‐fruit‐forming eudicots including Arabidopsis. Network analysis reveals co‐expression between FBP9 and photosynthesis genes in both apple and peach, suggesting that FBP9 and photosynthesis may both promote fleshy fruit development. The large transcriptomic datasets at the earliest stages of pome and drupe fruit development provide rich resources for comparative studies, and the work provides important insights into fruit‐type specification. Significance Statement: Rose family of plants exhibit a diverse array of fruit types when fruit flesh develops from different floral parts. Through comparative transcriptomic analyses of carefully dissected and staged fruit tissues from apple (a pome) and peach (a drupe), candidate regulators of fleshy fruit identity are identified and a step‐wise specification of fruit type via MADS box genes and phytohormones is proposed. [ABSTRACT FROM AUTHOR]

Rosaceae, a large plant family of more than 3,000 species, consists of many economically important fruit and ornamental crops, including peach, apple, strawberry, raspberry, cherry, and rose. These horticultural crops are not only important economic drivers in many regions of the world, but also major sources of human nutrition. Additionally, due to the diversity of fruit types in Rosaceae, this plant family offers excellent opportunities for investigations into fleshy fruit diversity, evolution, and development. With the development of high-throughput sequencing technologies and computational tools, an increasing number of high-quality genomes and transcriptomes of Rosaceae species have become available and will greatly facilitate Rosaceae research and breeding. This review summarizes major genomic resources and genome research progress in Rosaceae, highlights important databases, and suggests areas for further improvement. The availability of these big data resources will greatly accelerate research progress and enhance the agricultural productivity of Rosaceae. [ABSTRACT FROM AUTHOR]

Summary: Fruit 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. [ABSTRACT FROM AUTHOR]

Hawthorn (Crataegus spp.) is an important pome with a long history as a fruit, an ornamental, and a source of medicine. Fruits of hawthorn are marked by hard stony endocarps, but a hawthorn germplasm with soft and thin endocarp was found in Liaoning province of China. To elucidate the molecular mechanism underlying the soft endocarp of hawthorn, we conducted a de novo assembly of the fruit transcriptome of Crataegus pinnatifida and compared gene expression profiles between the soft-endocarp and the hard-endocarp hawthorn varieties. De novo assembly yielded 52,673 putative unigenes, 20.4% of which are longer than 1,000 bp. Among the high-quality unique sequences, 35,979 (68.3%) had at least one significant match to an existing gene model. A total of 1,218 genes, represented 2.31% total putative unigenes, were differentially expressed between the soft-endocarp hawthorn and the hard-endocarp hawthorn. Among these differentially expressed genes, a number of lignin biosynthetic pathway genes were down-regulated while almost all the flavonoid biosynthetic pathway genes were strongly up-regulated, concomitant with the formation of soft endocarp. In addition, we have identified some MYB and NAC transcription factors that could potentially control lignin and flavonoid biosynthesis. The altered expression levels of the genes encoding lignin biosynthetic enzymes, MYB and NAC transcription factors were confirmed by quantitative RT-PCR. This is the first transcriptome analysis of Crataegus genus. The high quality ESTs generated in this study will aid future gene cloning from hawthorn. Our study provides important insights into the molecular mechanisms underlying soft endocarp formation in hawthorn. [ABSTRACT FROM AUTHOR]

The accumulation of anthocyanins can be found in both the fruit and petioles of strawberries, but the fruit appears red while the petioles appear purple-red. Additionally, in the white-fruited diploid strawberries, the petioles can accumulate anthocyanins normally, suggesting a different synthesis pattern between the petioles and fruits. We screened the EMS mutagenized population of a red-fruited diploid strawberry 'Ruegen' and discovered a mutant which showed no anthocyanin accumulation in the petioles but normal accumulation in the fruit. Through BSA sequencing and allelic test, it was found that a mutation in FvDFR2 was responsible for this phenotype. Furthermore, the complex formed by the interaction between the petiole-specific FvMYB10L and FvTT8 only binds the promoter of FvDFR2 but not FvDFR1 , resulting in the expression of only FvDFR2 in the petiole. FvDFR2 can catalyze the conversion of DHQ and eventually the formation of cyanidin and peonidin, giving the petiole a purplish-red color. In the fruit, however, both FvDFR1 and FvDFR2 can be expressed, which can mediate the synthesis of cyanidin and pelargonidin. Our study clearly reveals different regulation of FvDFR1 and FvDFR2 in mediating anthocyanin synthesis in petioles and fruits. • Mutations in FvDFR2 result in a phenotype of green petiole in strawberry. • In petioles, only FvDFR2 expressed while both FvDFR1 and FvDFR2 expressed in fruits. • FvMYB10L and FvTT8 selectively binds to the FvDFR2 promoter instead of FvDFR1. • FvDFR1 compensates for the loss of FvDFR2 function in forming cyanidin in fruits. [ABSTRACT FROM AUTHOR]