CRISPR-Cas9-mediated mutagenesis of the flowering repressor gene VcCENTRORADIALIS (VcCEN) induces early flowering in tetraploid highbush blueberry

CRISPR-Cas9-mediated mutagenesis of the flowering repressor gene VcCENTRORADIALIS (VcCEN) induces early flowering in tetraploid highbush blueberry | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CRISPR-Cas9-mediated mutagenesis of the flowering repressor gene VcCENTRORADIALIS ( VcCEN ) induces early flowering in tetraploid highbush blueberry Masafumi Omori, Hisayo Yamane, Keishi Osakabe, Yuriko Osakabe, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4642319/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Flowering marks the vegetative-to-reproductive growth transition and is the most important event in the plant life cycle. Unlike annual plants, perennial fruit trees flower and set fruits only after an extended juvenile phase (i.e., several years), which is an impediment to efficient breeding and gene function analyses. In this study, we generated an early flowering blueberry line via the CRISPR-Cas9-mediated mutagenesis of VcCENTRORADIALIS ( VcCEN ). The expression of VcCEN in the apical bud was negatively correlated with flower bud formation. Moreover, in the cultivar that flowers in both autumn and spring, the VcCEN expression level was lower and decreased earlier than in the normal cultivar that flowers in only spring. The expression data suggested that VcCEN functions as a flowering repressor. The CRISPR-Cas9 vector harboring a gRNA targeting VcCEN was introduced into the blueberry genome via Agrobacterium-mediated transformation. Mutations (e.g., 1–10 bp indels) were detected in the stable transformants, with all VcCEN alleles of the tetraploid genome mutated in some lines. Compared with the wild-type (WT), the cen mutants exhibited repressed vegetative growth. Additionally, in the mutants, first flowering occurred within 1 year after the Agrobacterium infection, which was approximately 1–2 years earlier than in WT. The mutants set a single terminal flower without entering dormancy, whereas WT produced an apical flower and multiple axillary flowers that bloomed after an exposure to chilling conditions and then warm temperatures. This early flowering trait is conducive to efficient breeding and gene functional analyses, especially in fruit crops with a long juvenile phase. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key Message We generated early flowering blueberry by mutagenizing VcCEN . The mutants flowered within 1 year after the Agrobacterium infection, making them useful for breeding and functional analyses. Introduction Blueberries are perennial plants classified in the section Cyanococcus of the genus Vaccinium . Most Vaccinium species originated in Central and South America and were subsequently dispersed to North America (Luby et al, 1991 ). There are several types of blueberries, including highbush blueberry ( V. corymbosum ), rabbiteye blueberry ( V. virgatum ), lowbush blueberry ( V. angustifolium ), and half-high blueberry (highbush and lowbush hybrid), all of which originated in North America. Blueberry is one of the most economically important woody plant species because its fruits contain substantial amounts of antioxidants with beneficial effects on human health (Prior et al, 1998 ; Ehlenfeldt and Prior 2001 ). Thus, there has been a continuous increase in the demand for blueberry and for novel elite cultivars. Recent genomic and transcriptomic analyses identified many candidate genomic regions responsible for important agronomic traits, including fruit firmness, fruit weight, pH, soluble solids content, epicuticular wax composition, and volatile organic compound content (Cappai et al. 2018 , 2020 ; Edger et al. 2022 ; Ferrão et al. 2018 , 2020 ; Qi et al. 2019 ). It takes several years to validate the functions of the candidate genes related to these fruit traits using stable transformants because of their long juvenile phase. Artificially manipulating the flowering of fruit trees with a long juvenile phase enables us to shorten the period required before the transformants can be phenotyped and to accelerate breeding by decreasing the generation time. Plants go through a series of phase transitions during their life cycle in response to internal and external factors. Flower initiation marks the switch from vegetative to reproductive growth and is the most important event in the plant life cycle. The differences in plant reproductive patterns reflect the diversity in adaptations to various ecological contexts. In annual herbaceous plants, flower initiation occurs only once, whereas flower initiation in perennial woody plants is an annual occurrence that ensures reproductive growth continues consistently over multiple years. The extended lifespan and polycarpic growth habits of perennial trees require a highly intricate system that coordinates responses to environmental signals and determines the appropriate flowering time (Albani and Coupland 2010 ). In fruit trees, there are perennial-specific mechanisms regulating flowering (e.g., phase transitions and bud dormancy) (Wang and Ding 2023 ). However, studies on the molecular mechanisms underlying flower development have mainly involved annual model plants. Accordingly, the corresponding mechanisms in perennial woody plants remain uncharacterized. Genetic research on flowering mechanisms has primarily focused on the phosphatidyl ethanolamine-binding protein (PEBP) gene family (Karlgren et al. 2011 ), which consists of FLOWERING LOCUS T ( FT ), TERMINAL FLOWER1 / CENTRORADIALIS ( TFL1 / CEN ), and MOTHER OF FT AND TFL1 ( MFT ). In angiosperms, PEBPs function as promoters and suppressors of flowering, while also controlling the plant architecture. Earlier research showed FT and its orthologs in other plant species promote flowering, whereas TFL1 / CEN and its orthologs encode a flowering repressor in many plant species (Karlgren et al. 2011 ). In Arabidopsis, FT is expressed mainly in leaves and the encoded protein is translocated to buds. Additionally, FT along with the transcription factor FLOWERING LOCUS D (FD) promotes the expression of downstream genes, including APETALA1 ( AP1 ), thereby inducing flower differentiation (Abe et al. 2005 ). Arabidopsis TFL1 / CEN is expressed in the shoot apical meristem and promotes vegetative growth along with FD (Huang et al. 2012 ). Loss-of-function mutations to TFL1 / CEN genes can lead to precocious and continuous flowering in perennial fruit crops, such as kiwifruit ( Actinidia chinensis ) (Varkonyi-Gasic et al. 2019 ), apple ( Malus domestica ) (Charrier et al. 2019 ), and pear ( Pyrus communis L.) (Charrier et al. 2019 ). Although researchers have attempted to knock out the tetraploid highbush blueberry CEN -like gene ( VcCEN ), a mutant with mutations to all VcCEN alleles was not obtained and promotion of flowering was not observed in the mutant (Omori et al. 2021 ). Therefore, VcCEN function has not been fully characterized in blueberry. Furthermore, expression patterns of VcCEN have yet to be thoroughly investigated. Compared with diploids, it is generally more difficult to confer phenotype mutation in polyploid crops because usually phenotype change could be achieved when all of the alleles are mutated in a specific gene and there are more target sequences. Furthermore, the accumulation of mutations during crossing is not feasible for fruit crops that have a long juvenile phase and a heterozygous genome. In previous genome editing studies involving highbush blueberry ( Vaccinium corymbosum ), the mutated allele frequency was 0–37.5% (Omori et al. 2021 ), 0–23.8% (Vaia et al. 2022 ), and 0–28.8% (Han et al. 2022 ); none of these studies generated an all-allelic mutant, reflecting the need for methods that can increase the genome editing efficiency in blueberry. In this study, we characterized the VcCEN expression profile and generated early flowering blueberry via the CRISPR-Cas9-mediated mutagenesis of VcCEN . This is the first report of obtaining all-allelic mutant in tetraploid blueberry. The phenotyping of the mutant lines provides new insights into the effects of VcCEN on vegetative growth and flowering in blueberry. Additionally, the mutants may be useful for shortening the generation time of fruit trees with a long juvenile phase, with potential implications for enhancing traditional breeding methods. Materials and Methods Plant materials For the gene expression analysis of highbush blueberry cultivars ‘Blue Muffin’ (‘BM’) and ‘Bluecrop’ (‘BC’), 3- to 5-year-old plants were grown in pots at the experimental farm of Kyoto University, Kyoto, Japan (34°N; 135°E). For genetic transformations, ‘BM’ was maintained and cultured under in vitro conditions. The in vitro shoots were propagated in shoot proliferation medium [MW basal medium (Tetsumura et al, 2008 ) supplemented with 20 g/L sucrose, 2.2 mg/L zeatin, and 6 g/L agar]. The MW medium consisted of equal volumes of MS (Murashige and Skoog 1962 ) and WPM (McCown and Lloyd 1981 ). Construction of a phylogenetic tree To identify the blueberry CEN -like gene in the blueberry genome, a phylogenetic tree was constructed using publicly available protein sequences from NCBI ( https://www.ncbi.nlm.nih.gov/ ) and MEGA X. Protein sequences encoded by PEBP gene family members were collected to identify the CEN clade. Analysis of VcCEN expression in blueberry Published transcriptome data for highbush blueberry ‘Draper’ (Colle et al. 2019 ) were analyzed using the Genome Database for Vaccinium (GDV; https://www.vaccinium.org/ ) to examine VcCEN expression in different organs. To thoroughly investigate the VcCEN expression pattern, a quantitative real-time PCR (qPCR) analysis was performed. Shoot tips were collected from ‘BC’ and ‘BM’ plants after the expanding leaves were removed. All samples were immediately frozen in liquid nitrogen and stored at − 80°C until used. To obtain sufficient amounts of RNA, 3–4 buds were pooled as one biological replicate for the RNA extraction. Samples were collected monthly from May to October in 2017. Total RNA was isolated from the frozen samples using the PureLink™ Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA). A total of 1 µg RNA was used as the template for synthesizing full-length cDNA using ReverTra Ace reverse transcriptase (Toyobo, Osaka, Japan). The qPCR analysis was completed using the LightCycler 480 instrument (Roche Diagnostics, Mannheim, Germany) and the Thunderbird SYBR mix (Toyobo). The relative expression levels of VcCEN were calculated according to the 2 −∆∆Ct method, with the blueberry UBIQUITIN CONJUGATING ENZYME 28 ( VcUBC28 ) gene selected as the internal reference. Significant differences in gene expression among the months were determined on the basis of Tukey’s honest significant difference test (P < 0.05). Three biological replicates and two technical replicates were analyzed. CRISPR-Cas9 vector construction Two CRISPR-Cas9 vectors with different promoters were used. One of the vectors contains the Arabidopsis U6 ( AtU6 ) promoter for gRNA expression and the cauliflower mosaic virus (CaMV) 35S promoter for Cas9 expression, whereas the other vector contains blueberry gene promoters ( VcU6-7 and VcUBQ3b promoters for gRNA and Cas9 expression, respectively) that are useful for generating high gRNA and Cas9 expression levels in blueberry (Omori et al, 2023 ). The Focas software (Osakabe et al. 2016 ) was used to identify target sequences for the efficient and specific editing of VcCEN . Two target sites were selected and the gRNA sequences were inserted into the CRISPR-Cas9 vectors (Fig. 1 ) according to the Golden Gate Cloning method. The resulting plasmids were verified by Sanger sequencing using the 3730xl DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) and then inserted into Agrobacterium tumefaciens strain EHA105 cells via electroporation (MicroPulser Electroporator; Bio-Rad, Hercules, CA, USA). Genetic transformation of blueberry Leaf explants of ‘BM’ were co-cultivated with A. tumefaciens on co-cultivation medium for 6 days in darkness. The explants were placed on selection medium (MW supplemented with 20 g/L sucrose, 1.0 mg/L thidiazuron, 0.5 mg/L α-naphthaleneacetic acid, 15 mg/L kanamycin, 250 mg/L meropenem, and 6 g/L agar). After a 2-week incubation in darkness, the explants were transplanted to fresh selection medium. The transplanting was repeated every 3 weeks for 2 months to induce callus and adventitious bud formation. The transgenic shoots from separate explants were considered to have resulted from independent transgenic events. Regenerated shoots were transferred to the shoot proliferation medium (MW supplemented with 20 g/L sucrose, 2.2 mg/L zeatin, 30 mg/L kanamycin, 250 mg/L meropenem, and 6 g/L agar). For all media, the pH was adjusted to 5.2 before they were sterilized in an autoclave at 121°C for 20 min. All tissue-cultured plants were maintained at 25°C with a 16-h photoperiod (30 mE/m 2 /s from cool white fluorescent tubes). The regenerated non-infected explants served as the wild-type (WT) controls. Non-transgenic and transgenic shoots were immersed in 3 g/L indole-3-butyric acid to induce rooting. They were then transferred to the rooting medium, which comprised half-strength MW basal medium supplemented with 20 g/L sucrose and 2 g/L gellan gum, but no plant growth regulators (Tetsumura et al. 2008 ). All rooted shoots were transferred to a plant box containing peat moss and vermiculite (3:1, v/v). The plantlets were watered and fertilized every month with 0.1% Hyponex. Analysis of the CRISPR/Cas9-targeted sequence Genomic DNA was extracted from the regenerated kanamycin-resistant shoots using Genomic-tip (Qiagen, Venlo, The Netherlands). The genomic integration of transgenes was confirmed by PCR using a primer set designed for the Cas9 sequence. The VcCEN target sequences in the transgenic lines were amplified by PCR, purified, and analyzed by Sanger sequencing. Details regarding the primer sequences are provided in Supplementary Table 1. To estimate the mutated allele frequency in the pooled DNA samples, sequence chromatograms were analyzed using the online tool DECODR ( https://decodr.org/ ; Bloh et al. 2021 ). Some of the amplified fragments were inserted into the pGEM-T Easy vector (Promega, Madison, WI, USA). Sixteen clones were sequenced to estimate the mutated allele frequency. Phenotypic assessment of the CEN -mutated lines Six WT plants and three plants from two mutated ‘BM’ lines (12 plants in total) were included in the phenotypic analysis. At 2 months after potting, in August 2021, the potted plants were transferred to a greenhouse with no heating conditions. The plant height and the length of the longest shoot of each line were measured at 3, 6, and 9 months after potting. The date of first flowering was also recorded for each line. The significance of the differences between the WT and mutated lines was assessed on the basis of Student’s t -test (P < 0.05). A pollen germination test was conducted to determine pollen viability. Briefly, the collected pollen grains were placed on germination medium (15% sucrose, 1.5% agarose, and 0.01% boric acid) and incubated overnight at room temperature. Pollen germination was examined using a VH-S30 microscope (Keyence, Osaka, Japan). Results VcCEN expression in blueberry Three VcCEN alleles were identified in the highbush blueberry ‘Draper’ genome (Fig. 2 A). An in silico analysis revealed that VcCEN was expressed only in the shoot (Fig. 2 B). The analysis of seasonal changes in VcCEN expression in the ‘BC’ apical bud indicated that VcCEN expression peaked in June and gradually decreased until October (Fig. 2 C). Flower bud initiation in highbush blueberry in Japan starts in July to September depending on cultivars (Ogiwara et al. 2010), suggesting that VcCEN expression may be negatively correlated with flower bud formation. Most highbush blueberry cultivars, including ‘BC’, bloom in spring (typically at the end of March to April), whereas ‘BM’ blooms from July to December in addition to the spring bloom. The VcCEN expression level in ‘BM’ was lower than that in ‘BC’ and decreased in June, which was earlier than the decrease in ‘BC’ (Fig. 2 D). The comparison of these two cultivars suggested that the low VcCEN expression level in ‘BM’ may have resulted in unusual flowering. Overall, these findings imply VcCEN may encode a flowering repressor in blueberry, making it a potential candidate gene for manipulating blueberry flowering. Detection of CRISPR-Cas9-induced CEN mutations in genetic transformants Transgenic blueberry plants carrying the CRISPR-Cas9 vector targeting VcCEN were generated via Agrobacterium-mediated transformation. Mutations were detected in nine of the 11 transgenic lines (mutation rate of 82%) (Fig. 3 A). The highest mutated allele frequency was 100%, which means all alleles were edited. The mutated allele frequency was higher for gRNA2 than for gRNA1, indicative of the importance of selecting an appropriate gRNA for efficient genome editing. The mutation types in one of the edited plants are shown in Fig. 3 B–D. Specifically, DECODR and clone sequencing revealed 1- to 10-bp insertions/deletions a few bases upstream of the PAM sequence in the transgenic line (Fig. 3 C, D). Phenotypic observations of cen mutants At 4 months after rooting, approximately 90% of the rooted shoots were successfully acclimated to the ambient conditions. The average plant height data revealed that the three individuals from two selected mutant lines were significantly shorter than the non-transgenic control at 6 and 9 months after potting (Fig. 4 A). In addition, shoot lengths of cen mutants tended to be shorter than WT at 9 months after potting (Fig. 4 B). These results suggested that a mutation to VcCEN may induce suppressed vegetative growth in blueberry. First flowering of the cen mutants occurred earlier (8–11 months after potting) than the WT plants (20–28 months after potting) (Fig. 5 ). The mutant plants exhibited a compact growth habit and terminal flowering. The cen mutants produced a single or double flower at the top of the shoot, whereas the WT plants produced a florescence with several flowers (Supplementary Fig. 1). The terminal bud of the mutants did not enter dormancy and bloomed within 1 month after bud formation. In summer, the shoot apex of the mutant lines occasionally transformed directly into a flower without growth cessation and a typical bud set (Supplementary Fig. 2). The vegetative growth continued until just before the formation of the terminal flower. This flowering pattern in the mutants was reproduced under high temperature (28°C) and long photoperiod (16 h) conditions. In the cen mutant grown in the greenhouse without heating, flower buds formed throughout the year and they bloomed continuously from March to December without entering dormancy. In January and February, flower bud development was suppressed and no blooming was observed, probably because of the low temperatures during winter. When the mutants were grown in small pots and the longest shoot was shorter than 40 cm, they did not flower, similar to the WT control. The fact that flower buds only developed on shoots longer than approximately 60 cm for both the WT and mutant plants suggested there is a minimum shoot length threshold for plants to bear flower buds. There were no obvious morphological differences in the flowers and pollen germination between the WT and cen mutant plants (Supplementary Fig. 3). Discussion The TFL1 / CEN gene family plays a crucial role in the regulation of flowering and architecture in various plant species. Earlier research demonstrated that TFL1 / CEN genes encode flowering repressors in different plant species (Jin et al. 2021 ). The CEN gene was first identified as the gene responsible for controlling the inflorescence architecture in Antirrhinum (Bradley et al. 1996 ). The cen mutation changes inflorescence style from indeterminate to determinate single flower in Antirrhinum . Although extensive research has been conducted to characterize the function of CEN in model annual plants, the importance of its role in perennial plants has not been thoroughly investigated. In fruit trees, flowering comprises of perennial-specific several complex processes such as vegetative to reproductive phase transitions and bud dormancy (Wang et al. 2022). In the current study, the results of our analyses of VcCEN expression and function collectively provide new insights into the molecular mechanism underlying blueberry flowering. More specifically, our findings indicate VcCEN functions as a flowering repressor in blueberry. Additionally, the pleiotropic effects of VcCEN were revealed; the mutation to VcCEN resulted in changes to vegetative growth, photoperiod sensitivity, juvenile phase duration, flowering architecture, and flower bud dormancy. Moreover, VcCEN was expressed at high levels in actively growing shoots in the summer, but the cen mutants were shorter than the WT control, indicative of the positive effects of VcCEN on vegetative growth. In blueberry, flower initiation is induced by short-day conditions, with a critical day length threshold of approximately 12 h (Hall 1963; Phatak and Austin 1990). Increasing the day length (e.g., 16-h photoperiod) reportedly leads to the lack of flower bud formation in three highbush blueberry cultivars (Bañados and Strik, 2006). In the present study, the WT plants did not bloom under long-day conditions, whereas flowers were detected on the mutants, suggesting a mutation to VcCEN can alter photoperiod sensitivity. Under high temperature conditions, the shoot apex of the mutant lines occasionally transformed directly into a flower (Supplementary Fig. 2). Moreover, in the mutant, there was a spontaneous transition from vegetative growth to reproductive growth, resulting in the rapid formation of floral organs. In a recent study, a similar phenomenon was often observed in rabbiteye blueberry plants grown in a warm subtropical climate (Omori et al. 2022 ). The growing shoots of some rabbiteye blueberry cultivars directly turn into inflorescences, with decreases in VcCEN expression as the floral meristem forms (Omori et al. 2022 ). These two phenomena may be mediated by a similar mechanism; Due to the concurrent occurrence of vegetative growth stimulated by high temperatures and reproductive growth triggered by the mutation or decreased expression of VcCEN , elongating shoots abruptly differentiate into floral organs. A mutated VcCEN also substantially changed the inflorescence architecture in blueberry. This phenotype is a typical consequence of mutations to TFL1 / CEN genes in various plant species (Bradley et al. 1996 ; Charrier et al. 2019 ; Varkonyi-Gasic et al. 2019 ; Shannon et al. 1991). Similar to other studies, the mutagenesis of VcCEN limited the development of the normally indeterminate inflorescence and led to the production of a terminal flower. Thus, this study and reported studies collectively suggested that mutating VcCEN is useful for promoting flowering and overcoming several traits typical of woody plants that restrict flowering, including a long juvenile phase and dormancy. The annualization of fruit trees may revolutionize the breeding of fruit tree crops with a long juvenile phase. Most perennial plants have a long juvenile phase and lack flowers for a certain period. This characteristic is detrimental to efficient breeding and genetic analyses. For example, introducing disease resistance genes via backcrossing involving closely related wild species and pyramiding useful genes are time-consuming processes. ‘Fast-track breeding’ has been developed as a novel strategy for efficiently breeding stress-resistant fruit trees. This approach is based on the overexpression of genes that promote flowering, including FT orthologs. It has been used to introduce fire blight and apple scab resistance genes from wild apple into an elite cultivar, with five generations produced within 7 years (Schlathölter et al. 2018 ). Similarly, the citrus tristeza virus resistance gene from trifoliate orange ( Poncirus trifoliata ; Endo et al. 2020 ) was introduced into other citrus germplasm. A similar approach may be applied to incorporate some important traits from wild Vaccinium plants into blueberry cultivars. Some wild Vaccinium species that may be used for the cross-pollination of blueberry have valuable traits, including vigor, unique fruit volatile compositions, fruit firmness, waxy foliage, low chilling requirements, cold hardiness, and drought tolerance (Edger et al. 2022 ). The early flowering trait of the cen mutant may also be useful for validating agronomically important genes in blueberry. Many recent genetic studies have identified candidate genes associated with agronomically important traits of blueberry (Edger et al. 2022 ). The precise characterization of the functions and practical utility of genes requires experiments involving stable transformations or mutagenesis. However, even though many candidate genes have been identified, very few have been functionally validated, especially in fruit tree crops. Creating and phenotyping transgenic plants require a lot of time, area, and labor if the individual plants are large and have a long generation time. Shortening the juvenile phase can accelerate gene validation studies. For example, “rapid flowering” kiwifruit ( A. chinensis ) was previously used to functionally validate Friendly boy ( FrBy ), which is a Y chromosome-encoded sex-determinant gene (Akagi et al. 2019 ). In another study, an early flowering grape mutant ‘microvine’ that flowers continuously and has a shortened generation time was used to generate a segregating population and validate the contribution of the candidate gene VviPLATZ1 to female flower morphology (Iocco-Corena et al. 2021 ). In the current study, the cen mutant had a continuous flowering phenotype with no dormancy period, suggesting that flower bud development was enhanced. The continuous flowering trait may be exploited for off-season fruit production. The shelf life of blueberry is shorter than that of most fruits. Thus, a year-round production system is desirable. Genome editing technology has been utilized to improve crop yield, nutrient, stress tolerance (Atia et al. 2024 ) and many countries adapted guidelines that permit the cultivation of non-transgenic gene-edited lines similar to conventionally bred lines (Buchholzer and Frommer. 2023). The continuous flowering cen mutant generated in this study may also enable blueberry growers to extend the harvest period. However, the quality of the cen mutant fruit will need to be assessed for this purpose. Declarations Acknowledgments This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI [No. 19KK0156] to H.Y. and R.T. and Grant-in-Aid for JSPS Fellows for [No. 21J22977] to M.O. We thank Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. Contributions H.Y. and M.O. designed project and M.O. performed the experiments and wrote the manuscript. Y. O. and K. O. provided the CRISPR-Cas9 vector. 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(in Japanese) JATAFF journal 4:41–46 Omori M, Yamane H, Osakabe K , Osakabe Y, Tao R (2021) Targeted mutagenesis of CENTRORADIALIS using CRISPR/Cas9 system through the improvement of genetic transformation efficiency of tetraploid highbush blueberry. J Hortic Sci Biotechnol 96:153–161 Omori M, Cheng CC, Hsu FC, Chen SJ, Yamane H, Tao R, Li KT (2022) Off-season flowering and expression of flowering-related genes during floral bud differentiation of rabbiteye blueberry in a subtropical climate. Sci Hortic 306: 111458 Omori M, Yamane H, Osakabe K, Osakabe Y, Tao R (2023). The evaluation of CRISPR-Cas9-mediated editing efficiency using endogenous promoters in tetraploid blueberry. Acta Hortic 1362, 49–56. https://doi.org/10.17660/ActaHortic.2023.1362.8 Osakabe Y, Osakabe K (2015) Genome editing with engineered nucleases in plants. Plant Cell Physiol 56:389–400. https://doi.org/10.1093/pcp/pcu170 Osakabe Y, Watanabe T, Sugano SS, Ueta R, Ishihara R, Shinozaki K, Osakabe (2016) Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants. Sci Rep 6:26685. https://doi.org/10.1038/srep26685 Prior RL, Cao G, Martin A , Sofic E, McEwen J, O'Brien C, Lischner N, Ehlenfeldt M, Kalt W, Krewer G (1998) Antioxidant capacity is influenced by total phenolic and anthocyanin content, maturity, and variety of Vaccinium species. J Agric Food Chem 46:2686–2693. https://doi.org/10.1021/jf980145d Qi X, Ogden EL, Die JV, Ehlenfeldt MK, Polashock JJ, Darwish O, Alkharouf N, Rowland LJ (2019) Transcriptome analysis identifies genes related to the waxy coating on blueberry fruit in two northern-adapted rabbiteye breeding populations. BMC Plant Biol 19: 460. 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Bioinformatics 30:1180–1182 https://doi.org/10.1093/bioinformatics/btt764 Supplementary Files SupplementaryTableFigure.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4642319","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":326784048,"identity":"b8d7ab60-7bde-46c9-ba9c-ac3f320d7e6b","order_by":0,"name":"Masafumi Omori","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABM0lEQVRIie2QMUvDQBTHX4iky2E2uRhpv8KVA0GM+FUSAucSUHBxs1O6BLIGHPoV4uIcOEgWP0AkDpVC53QpGRy8xBJL0hZHkfvBPR53/HjvfwASyV/FbqoyByAwhIG//YYOKSqpFQooba/2K9AqAM4Es22lj37C03IOL6PRlGun+R1Q8rZkeFVZQBL1o4Sz965iPDE3sqEYx6+uZnoii7hJjchmQtEoBrTsKqTwqNiiUGK4TWuF6uaNbyKbP8YJnIssfJ9yPQsXzRTHN7JGEVMG60OKM8m/F3NCrKUbBe2cUmcBmxRunC/US49gagTMvYgYA4Oje2z3s+imy5Xqobiaha5SeJ/WkGTpOC8tC46z6XO5Cno/tlmv7XBdjpoKqjhOkOxWOqjlT1/9TpFIJJL/zBcjZmbb0SGWsAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0009-6859-7927","institution":"Kyoto University Graduate School of Agriculture Faculty of Agriculture: Kyoto Daigaku Nogaku Kenkyuka Nogakubu","correspondingAuthor":true,"prefix":"","firstName":"Masafumi","middleName":"","lastName":"Omori","suffix":""},{"id":326784049,"identity":"5bdc3085-bdfa-4f53-9814-649426735f2f","order_by":1,"name":"Hisayo Yamane","email":"","orcid":"https://orcid.org/0000-0002-9044-7863","institution":"Kyoto University Graduate School of Agriculture Faculty of Agriculture: Kyoto Daigaku Nogaku Kenkyuka Nogakubu","correspondingAuthor":false,"prefix":"","firstName":"Hisayo","middleName":"","lastName":"Yamane","suffix":""},{"id":326784050,"identity":"0cc557e5-9c5a-4a10-aef7-297a1e4d3fb6","order_by":2,"name":"Keishi Osakabe","email":"","orcid":"","institution":"University of Tokushima: Tokushima Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Keishi","middleName":"","lastName":"Osakabe","suffix":""},{"id":326784051,"identity":"acb06b70-a71c-4d94-b3fb-64a4953196e7","order_by":3,"name":"Yuriko Osakabe","email":"","orcid":"","institution":"Tokyo Institute of Technology: Tokyo Kogyo Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Yuriko","middleName":"","lastName":"Osakabe","suffix":""},{"id":326784052,"identity":"466a36df-b59d-485b-8b6b-cae84b3eb7c8","order_by":4,"name":"Ryutaro Tao","email":"","orcid":"","institution":"Kyoto University Graduate School of Agriculture Faculty of Agriculture: Kyoto Daigaku Nogaku Kenkyuka Nogakubu","correspondingAuthor":false,"prefix":"","firstName":"Ryutaro","middleName":"","lastName":"Tao","suffix":""}],"badges":[],"createdAt":"2024-06-26 11:20:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4642319/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4642319/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61930391,"identity":"c80f2701-4d64-4295-b908-2593a138e973","added_by":"auto","created_at":"2024-08-07 08:06:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":693115,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eVcCEN\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e sequences targeted by gRNA and schematic diagrams of the CRISPR-Cas9 vectors used in this study\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) \u003cem\u003eVcCEN \u003c/em\u003egene structure and gRNA sequences. Two \u003cem\u003eVcCEN \u003c/em\u003etarget sequences are presented and red boxes indicate the PAM sequence. The \u003cem\u003eVcCEN \u003c/em\u003egene has four exons (white box) and three introns (green line).\u003c/p\u003e\n\u003cp\u003e(B) Schematic diagrams of the CRISPR-Cas9 vectors with different promoters. AtU6: Arabidopsis \u003cem\u003eU6 \u003c/em\u003epromoter; VcU6-7: blueberry \u003cem\u003eU6\u003c/em\u003e promoter; 2×35SΩ: 2× CaMV 35S promoter with the omega enhancer sequence; VcUBQ3b: blueberry ubiquitin gene promoter; gRNA: location of the gRNA insertion; AtCas9: \u003cem\u003eArabidopsis thaliana\u003c/em\u003e codon-optimized SpCas9; 2A: 2A self-cleaving peptide; Km: kanamycin resistance marker expression cassette; RB: T-DNA right border; LB: T-DNA left border.\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-4642319/v1/9581391fd29de8c83caf1ed7.png"},{"id":61930396,"identity":"9e48411e-1011-42a7-8e88-bd769b4d4960","added_by":"auto","created_at":"2024-08-07 08:06:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":898265,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic and expression analyses of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVcCEN\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Phylogenetic tree of flowering-related PEBP family proteins constructed using the neighbor-joining method and the MEGA X program. The scale indicates the average substitutions per site. VaccDscaff: gene ID from GDV; AcCEN: \u003cem\u003eActinidia chinensis \u003c/em\u003eCENTRORADIALIS (JX417424.1); AcCEN4: \u003cem\u003eActinidia chinensis \u003c/em\u003eCENTRORADIALIS4 (ARE72519.1); MdCEN: \u003cem\u003eMalus domestica \u003c/em\u003eCENTRORADIALIS (BAG31958.1); AtCEN: \u003cem\u003eArabidopsis thaliana \u003c/em\u003eCENTRORADIALIS homolog (AT2G27550.1); PnTFL1: \u003cem\u003ePopulus nigra \u003c/em\u003eTERMINAL FLOWER 1 (BAG12897.1); PtCEN1: \u003cem\u003ePopulus trichocarpa \u003c/em\u003eCENTRORADIALIS (AAQ88444.1); AtTFL1: \u003cem\u003eArabidopsis thaliana \u003c/em\u003eTERMINAL FLOWER 1 (AAM27956.1); AcCEN3: \u003cem\u003eActinidia chinensis \u003c/em\u003eCENTRORADIALIS3 (ARE72518.1); AcCEN2: \u003cem\u003eActinidia chinensis \u003c/em\u003eCENTRORADIALIS2 (ARE72517.1); AcCEN1: \u003cem\u003eActinidia chinensis \u003c/em\u003eCENTRORADIALIS1\u003cem\u003e \u003c/em\u003e(ARE72516.1); MiTFL1: \u003cem\u003eMangifera indica \u003c/em\u003eTERMINAL FLOWER 1 (AGA19027.1); LcTFL1: \u003cem\u003eLitchi chinensis \u003c/em\u003eTERMINAL FLOWER 1 (QNH85212.1); CsTFL1: \u003cem\u003eCitrus sinensis \u003c/em\u003eTERMINAL FLOWER 1 (KAH9738276.1); MdTFL1: \u003cem\u003eMalus domestica \u003c/em\u003eTERMINAL FLOWER 1 (NP_001280887.1); ClBFT: \u003cem\u003eCitrus limon \u003c/em\u003eBROTHER of FT and TFL1 (AWW25017.1); FaBFT: \u003cem\u003eFragaria \u003c/em\u003e×\u003cem\u003e ananassa\u003c/em\u003e BROTHER of FT and TFL1 (AMR34798.1); AtBFT: \u003cem\u003eArabidopsis thaliana \u003c/em\u003eBROTHER of FT and TFL1 (Q9FIT4.1); AtFT: \u003cem\u003eArabidopsis thaliana\u003c/em\u003e FLOWERING LOCUS T (AAF03936.1); AcFT: \u003cem\u003eActinidia chinensis \u003c/em\u003eFLOWERING LOCUS T (AGK89939.1).\u003c/p\u003e\n\u003cp\u003e(B) Expression levels of \u003cem\u003eVcCEN \u003c/em\u003ein different organs. (C) Seasonal changes in \u003cem\u003eVcCEN\u003c/em\u003e expression in the ‘BC’ apical bud. (D) Seasonal changes in \u003cem\u003eVcCEN \u003c/em\u003eexpression in the ‘BM’ apical bud.\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-4642319/v1/b52a61fdcea54181ade55408.png"},{"id":61930392,"identity":"630309a3-d078-4cd4-9bb0-e8d666c0fc0e","added_by":"auto","created_at":"2024-08-07 08:06:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1909807,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCRISPR-Cas9-induced \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVcCEN \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emutations.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Mutated allele frequency of the transgenic lines transformed with different CRISPR-Cas9 vectors. The conventional vector (\u003cem\u003eAtU6\u003c/em\u003e and 35S promoters) is indicated in red.\u003cstrong\u003e \u003c/strong\u003eThe vector containing blueberry promoters (\u003cem\u003eVcU6-7\u003c/em\u003eand \u003cem\u003eVcUBQ3b\u003c/em\u003e promoters) is indicated in blue. To estimate the mutated allele frequency of each transformant, sequence chromatograms were analyzed using the online tool DECODR. (B) Direct sequencing of WT and the mutant. The PAM and 20 bp target sequences are indicated in red and green, respectively. (C) Sequence chromatograms were used to predict mutations. In this transgenic line, three kinds of mutations (−1, +1, and −10) were detected, which resulted in a frame shift. (D) Clone sequencing results. WT (upper) and mutated (lower) sequences were aligned. An intact WT sequence was not detected in the transgenic line. The PAM sequence is highlighted in red. The gRNA target sequences are indicated by a green underline. The mutation types and mutated allele ratios of each line are presented to the right of the sequences. *(mutation type, number of clones with the mutation/total number of clones).\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-4642319/v1/0ab00292f0270201e9ada869.png"},{"id":61930397,"identity":"5ebd455d-7af3-4526-8d2a-22fb655ad81f","added_by":"auto","created_at":"2024-08-07 08:06:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":409707,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the vegetative growth between the WT control (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCEN\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) and the mutated lines (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecen\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Plant height of six individuals from two selected lines. A significant difference was detected between WT and \u003cem\u003ecen#1\u003c/em\u003e at 9 months. (B) Length of the longest shoot of the six individuals. Data are presented as the mean ± standard error. Significant differences (P \u0026lt; 0.05) were determined according to Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-4642319/v1/26a6c67989e5ab31886b8e5e.png"},{"id":61930394,"identity":"1107e095-ba52-47a5-879a-ea4532c5a3db","added_by":"auto","created_at":"2024-08-07 08:06:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":550981,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEarly flowering in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecen \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emutant.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Flowering was observed in the \u003cem\u003ecen \u003c/em\u003emutant at 8 months after potting. (B) Number of individuals for specific first flowering dates (i.e., months after potting). At 2 months after potting, in August 2021, the potted plants were transferred to a greenhouse with no heating conditions.\u003c/p\u003e","description":"","filename":"Picture5.png","url":"https://assets-eu.researchsquare.com/files/rs-4642319/v1/fbf9088b9b17ede8164c1a88.png"},{"id":63082783,"identity":"75fee38e-e32c-4a00-b484-b7b67e88f516","added_by":"auto","created_at":"2024-08-23 01:55:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5860447,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4642319/v1/dc3357c7-fa1d-4b11-b9af-b220d0ab087f.pdf"},{"id":61930926,"identity":"56b353ea-e85e-4ba0-8da6-8a7aa95cf7da","added_by":"auto","created_at":"2024-08-07 08:14:03","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1207828,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-4642319/v1/f28eda045f20695a5bb584e7.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003e\u003cstrong\u003eCRISPR-Cas9-mediated mutagenesis of the flowering repressor gene \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVcCENTRORADIALIS \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVcCEN\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) induces early flowering in tetraploid highbush blueberry\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Key Message","content":"\u003cp\u003eWe generated early flowering blueberry by mutagenizing \u003cem\u003eVcCEN\u003c/em\u003e. The\u003cem\u003e\u0026nbsp;\u003c/em\u003emutants flowered within 1 year after the Agrobacterium infection, making them useful for breeding and functional analyses.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eBlueberries are perennial plants classified in the section \u003cem\u003eCyanococcus\u003c/em\u003e of the genus \u003cem\u003eVaccinium\u003c/em\u003e. Most \u003cem\u003eVaccinium\u003c/em\u003e species originated in Central and South America and were subsequently dispersed to North America (Luby et al, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). There are several types of blueberries, including highbush blueberry (\u003cem\u003eV. corymbosum\u003c/em\u003e), rabbiteye blueberry (\u003cem\u003eV. virgatum\u003c/em\u003e), lowbush blueberry (\u003cem\u003eV. angustifolium\u003c/em\u003e), and half-high blueberry (highbush and lowbush hybrid), all of which originated in North America. Blueberry is one of the most economically important woody plant species because its fruits contain substantial amounts of antioxidants with beneficial effects on human health (Prior et al, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Ehlenfeldt and Prior \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Thus, there has been a continuous increase in the demand for blueberry and for novel elite cultivars. Recent genomic and transcriptomic analyses identified many candidate genomic regions responsible for important agronomic traits, including fruit firmness, fruit weight, pH, soluble solids content, epicuticular wax composition, and volatile organic compound content (Cappai et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Edger et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ferr\u0026atilde;o et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Qi et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It takes several years to validate the functions of the candidate genes related to these fruit traits using stable transformants because of their long juvenile phase. Artificially manipulating the flowering of fruit trees with a long juvenile phase enables us to shorten the period required before the transformants can be phenotyped and to accelerate breeding by decreasing the generation time.\u003c/p\u003e \u003cp\u003ePlants go through a series of phase transitions during their life cycle in response to internal and external factors. Flower initiation marks the switch from vegetative to reproductive growth and is the most important event in the plant life cycle. The differences in plant reproductive patterns reflect the diversity in adaptations to various ecological contexts. In annual herbaceous plants, flower initiation occurs only once, whereas flower initiation in perennial woody plants is an annual occurrence that ensures reproductive growth continues consistently over multiple years. The extended lifespan and polycarpic growth habits of perennial trees require a highly intricate system that coordinates responses to environmental signals and determines the appropriate flowering time (Albani and Coupland \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In fruit trees, there are perennial-specific mechanisms regulating flowering (e.g., phase transitions and bud dormancy) (Wang and Ding \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, studies on the molecular mechanisms underlying flower development have mainly involved annual model plants. Accordingly, the corresponding mechanisms in perennial woody plants remain uncharacterized.\u003c/p\u003e \u003cp\u003eGenetic research on flowering mechanisms has primarily focused on the phosphatidyl ethanolamine-binding protein (PEBP) gene family (Karlgren et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), which consists of \u003cem\u003eFLOWERING LOCUS T\u003c/em\u003e (\u003cem\u003eFT\u003c/em\u003e), \u003cem\u003eTERMINAL FLOWER1\u003c/em\u003e/\u003cem\u003eCENTRORADIALIS\u003c/em\u003e (\u003cem\u003eTFL1\u003c/em\u003e/\u003cem\u003eCEN\u003c/em\u003e), and \u003cem\u003eMOTHER OF FT AND TFL1\u003c/em\u003e (\u003cem\u003eMFT\u003c/em\u003e). In angiosperms, PEBPs function as promoters and suppressors of flowering, while also controlling the plant architecture. Earlier research showed \u003cem\u003eFT\u003c/em\u003e and its orthologs in other plant species promote flowering, whereas \u003cem\u003eTFL1\u003c/em\u003e/\u003cem\u003eCEN\u003c/em\u003e and its orthologs encode a flowering repressor in many plant species (Karlgren et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In Arabidopsis, \u003cem\u003eFT\u003c/em\u003e is expressed mainly in leaves and the encoded protein is translocated to buds. Additionally, FT along with the transcription factor FLOWERING LOCUS D (FD) promotes the expression of downstream genes, including \u003cem\u003eAPETALA1\u003c/em\u003e (\u003cem\u003eAP1\u003c/em\u003e), thereby inducing flower differentiation (Abe et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Arabidopsis \u003cem\u003eTFL1\u003c/em\u003e/\u003cem\u003eCEN\u003c/em\u003e is expressed in the shoot apical meristem and promotes vegetative growth along with \u003cem\u003eFD\u003c/em\u003e (Huang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Loss-of-function mutations to \u003cem\u003eTFL1\u003c/em\u003e/\u003cem\u003eCEN\u003c/em\u003e genes can lead to precocious and continuous flowering in perennial fruit crops, such as kiwifruit (\u003cem\u003eActinidia chinensis\u003c/em\u003e) (Varkonyi-Gasic et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), apple (\u003cem\u003eMalus domestica\u003c/em\u003e) (Charrier et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and pear (\u003cem\u003ePyrus communis\u003c/em\u003e L.) (Charrier et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Although researchers have attempted to knock out the tetraploid highbush blueberry \u003cem\u003eCEN\u003c/em\u003e-like gene (\u003cem\u003eVcCEN\u003c/em\u003e), a mutant with mutations to all \u003cem\u003eVcCEN\u003c/em\u003e alleles was not obtained and promotion of flowering was not observed in the mutant (Omori et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, VcCEN function has not been fully characterized in blueberry. Furthermore, expression patterns of \u003cem\u003eVcCEN\u003c/em\u003e have yet to be thoroughly investigated. Compared with diploids, it is generally more difficult to confer phenotype mutation in polyploid crops because usually phenotype change could be achieved when all of the alleles are mutated in a specific gene and there are more target sequences. Furthermore, the accumulation of mutations during crossing is not feasible for fruit crops that have a long juvenile phase and a heterozygous genome. In previous genome editing studies involving highbush blueberry (\u003cem\u003eVaccinium corymbosum\u003c/em\u003e), the mutated allele frequency was 0\u0026ndash;37.5% (Omori et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), 0\u0026ndash;23.8% (Vaia et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and 0\u0026ndash;28.8% (Han et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e); none of these studies generated an all-allelic mutant, reflecting the need for methods that can increase the genome editing efficiency in blueberry.\u003c/p\u003e \u003cp\u003eIn this study, we characterized the \u003cem\u003eVcCEN\u003c/em\u003e expression profile and generated early flowering blueberry via the CRISPR-Cas9-mediated mutagenesis of \u003cem\u003eVcCEN\u003c/em\u003e. This is the first report of obtaining all-allelic mutant in tetraploid blueberry. The phenotyping of the mutant lines provides new insights into the effects of \u003cem\u003eVcCEN\u003c/em\u003e on vegetative growth and flowering in blueberry. Additionally, the mutants may be useful for shortening the generation time of fruit trees with a long juvenile phase, with potential implications for enhancing traditional breeding methods.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials\u003c/h2\u003e \u003cp\u003eFor the gene expression analysis of highbush blueberry cultivars \u0026lsquo;Blue Muffin\u0026rsquo; (\u0026lsquo;BM\u0026rsquo;) and \u0026lsquo;Bluecrop\u0026rsquo; (\u0026lsquo;BC\u0026rsquo;), 3- to 5-year-old plants were grown in pots at the experimental farm of Kyoto University, Kyoto, Japan (34\u0026deg;N; 135\u0026deg;E).\u003c/p\u003e \u003cp\u003eFor genetic transformations, \u0026lsquo;BM\u0026rsquo; was maintained and cultured under \u003cem\u003ein vitro\u003c/em\u003e conditions. The \u003cem\u003ein vitro\u003c/em\u003e shoots were propagated in shoot proliferation medium [MW basal medium (Tetsumura et al, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) supplemented with 20 g/L sucrose, 2.2 mg/L zeatin, and 6 g/L agar]. The MW medium consisted of equal volumes of MS (Murashige and Skoog \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) and WPM (McCown and Lloyd \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1981\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of a phylogenetic tree\u003c/h2\u003e \u003cp\u003eTo identify the blueberry \u003cem\u003eCEN\u003c/em\u003e-like gene in the blueberry genome, a phylogenetic tree was constructed using publicly available protein sequences from NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and MEGA X. Protein sequences encoded by PEBP gene family members were collected to identify the \u003cem\u003eCEN\u003c/em\u003e clade.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAnalysis of\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eVcCEN\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eexpression in blueberry\u003c/span\u003e\u003c/p\u003e \u003cp\u003ePublished transcriptome data for highbush blueberry \u0026lsquo;Draper\u0026rsquo; (Colle et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) were analyzed using the Genome Database for Vaccinium (GDV; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.vaccinium.org/\u003c/span\u003e\u003cspan address=\"https://www.vaccinium.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to examine \u003cem\u003eVcCEN\u003c/em\u003e expression in different organs.\u003c/p\u003e \u003cp\u003eTo thoroughly investigate the \u003cem\u003eVcCEN\u003c/em\u003e expression pattern, a quantitative real-time PCR (qPCR) analysis was performed. Shoot tips were collected from \u0026lsquo;BC\u0026rsquo; and \u0026lsquo;BM\u0026rsquo; plants after the expanding leaves were removed. All samples were immediately frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until used. To obtain sufficient amounts of RNA, 3\u0026ndash;4 buds were pooled as one biological replicate for the RNA extraction. Samples were collected monthly from May to October in 2017. Total RNA was isolated from the frozen samples using the PureLink\u0026trade; Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA). A total of 1 \u0026micro;g RNA was used as the template for synthesizing full-length cDNA using ReverTra Ace reverse transcriptase (Toyobo, Osaka, Japan). The qPCR analysis was completed using the LightCycler 480 instrument (Roche Diagnostics, Mannheim, Germany) and the Thunderbird SYBR mix (Toyobo). The relative expression levels of \u003cem\u003eVcCEN\u003c/em\u003e were calculated according to the 2\u003csup\u003e\u0026minus;∆∆Ct\u003c/sup\u003e method, with the blueberry \u003cem\u003eUBIQUITIN CONJUGATING ENZYME 28\u003c/em\u003e (\u003cem\u003eVcUBC28\u003c/em\u003e) gene selected as the internal reference. Significant differences in gene expression among the months were determined on the basis of Tukey\u0026rsquo;s honest significant difference test (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Three biological replicates and two technical replicates were analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCRISPR-Cas9 vector construction\u003c/h2\u003e \u003cp\u003eTwo CRISPR-Cas9 vectors with different promoters were used. One of the vectors contains the Arabidopsis \u003cem\u003eU6\u003c/em\u003e (\u003cem\u003eAtU6\u003c/em\u003e) promoter for gRNA expression and the cauliflower mosaic virus (CaMV) 35S promoter for \u003cem\u003eCas9\u003c/em\u003e expression, whereas the other vector contains blueberry gene promoters (\u003cem\u003eVcU6-7\u003c/em\u003e and \u003cem\u003eVcUBQ3b\u003c/em\u003e promoters for gRNA and \u003cem\u003eCas9\u003c/em\u003e expression, respectively) that are useful for generating high gRNA and \u003cem\u003eCas9\u003c/em\u003e expression levels in blueberry (Omori et al, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The Focas software (Osakabe et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) was used to identify target sequences for the efficient and specific editing of \u003cem\u003eVcCEN\u003c/em\u003e. Two target sites were selected and the gRNA sequences were inserted into the CRISPR-Cas9 vectors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) according to the Golden Gate Cloning method. The resulting plasmids were verified by Sanger sequencing using the 3730xl DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) and then inserted into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain EHA105 cells via electroporation (MicroPulser Electroporator; Bio-Rad, Hercules, CA, USA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eGenetic transformation of blueberry\u003c/h2\u003e \u003cp\u003eLeaf explants of \u0026lsquo;BM\u0026rsquo; were co-cultivated with \u003cem\u003eA. tumefaciens\u003c/em\u003e on co-cultivation medium for 6 days in darkness. The explants were placed on selection medium (MW supplemented with 20 g/L sucrose, 1.0 mg/L thidiazuron, 0.5 mg/L α-naphthaleneacetic acid, 15 mg/L kanamycin, 250 mg/L meropenem, and 6 g/L agar). After a 2-week incubation in darkness, the explants were transplanted to fresh selection medium. The transplanting was repeated every 3 weeks for 2 months to induce callus and adventitious bud formation. The transgenic shoots from separate explants were considered to have resulted from independent transgenic events. Regenerated shoots were transferred to the shoot proliferation medium (MW supplemented with 20 g/L sucrose, 2.2 mg/L zeatin, 30 mg/L kanamycin, 250 mg/L meropenem, and 6 g/L agar). For all media, the pH was adjusted to 5.2 before they were sterilized in an autoclave at 121\u0026deg;C for 20 min. All tissue-cultured plants were maintained at 25\u0026deg;C with a 16-h photoperiod (30 mE/m\u003csup\u003e2\u003c/sup\u003e/s from cool white fluorescent tubes).\u003c/p\u003e \u003cp\u003eThe regenerated non-infected explants served as the wild-type (WT) controls. Non-transgenic and transgenic shoots were immersed in 3 g/L indole-3-butyric acid to induce rooting. They were then transferred to the rooting medium, which comprised half-strength MW basal medium supplemented with 20 g/L sucrose and 2 g/L gellan gum, but no plant growth regulators (Tetsumura et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). All rooted shoots were transferred to a plant box containing peat moss and vermiculite (3:1, v/v). The plantlets were watered and fertilized every month with 0.1% Hyponex.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of the CRISPR/Cas9-targeted sequence\u003c/h2\u003e \u003cp\u003eGenomic DNA was extracted from the regenerated kanamycin-resistant shoots using Genomic-tip (Qiagen, Venlo, The Netherlands). The genomic integration of transgenes was confirmed by PCR using a primer set designed for the \u003cem\u003eCas9\u003c/em\u003e sequence. The \u003cem\u003eVcCEN\u003c/em\u003e target sequences in the transgenic lines were amplified by PCR, purified, and analyzed by Sanger sequencing. Details regarding the primer sequences are provided in Supplementary Table\u0026nbsp;1. To estimate the mutated allele frequency in the pooled DNA samples, sequence chromatograms were analyzed using the online tool DECODR (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://decodr.org/\u003c/span\u003e\u003cspan address=\"https://decodr.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; Bloh et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Some of the amplified fragments were inserted into the pGEM-T Easy vector (Promega, Madison, WI, USA). Sixteen clones were sequenced to estimate the mutated allele frequency.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePhenotypic assessment of the\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eCEN\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e-mutated lines\u003c/span\u003e\u003c/p\u003e \u003cp\u003eSix WT plants and three plants from two mutated \u0026lsquo;BM\u0026rsquo; lines (12 plants in total) were included in the phenotypic analysis. At 2 months after potting, in August 2021, the potted plants were transferred to a greenhouse with no heating conditions. The plant height and the length of the longest shoot of each line were measured at 3, 6, and 9 months after potting. The date of first flowering was also recorded for each line. The significance of the differences between the WT and mutated lines was assessed on the basis of Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A pollen germination test was conducted to determine pollen viability. Briefly, the collected pollen grains were placed on germination medium (15% sucrose, 1.5% agarose, and 0.01% boric acid) and incubated overnight at room temperature. Pollen germination was examined using a VH-S30 microscope (Keyence, Osaka, Japan).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eVcCEN\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eexpression in blueberry\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThree \u003cem\u003eVcCEN\u003c/em\u003e alleles were identified in the highbush blueberry \u0026lsquo;Draper\u0026rsquo; genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). An \u003cem\u003ein silico\u003c/em\u003e analysis revealed that \u003cem\u003eVcCEN\u003c/em\u003e was expressed only in the shoot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The analysis of seasonal changes in \u003cem\u003eVcCEN\u003c/em\u003e expression in the \u0026lsquo;BC\u0026rsquo; apical bud indicated that \u003cem\u003eVcCEN\u003c/em\u003e expression peaked in June and gradually decreased until October (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Flower bud initiation in highbush blueberry in Japan starts in July to September depending on cultivars (Ogiwara et al. 2010), suggesting that \u003cem\u003eVcCEN\u003c/em\u003e expression may be negatively correlated with flower bud formation. Most highbush blueberry cultivars, including \u0026lsquo;BC\u0026rsquo;, bloom in spring (typically at the end of March to April), whereas \u0026lsquo;BM\u0026rsquo; blooms from July to December in addition to the spring bloom. The \u003cem\u003eVcCEN\u003c/em\u003e expression level in \u0026lsquo;BM\u0026rsquo; was lower than that in \u0026lsquo;BC\u0026rsquo; and decreased in June, which was earlier than the decrease in \u0026lsquo;BC\u0026rsquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The comparison of these two cultivars suggested that the low \u003cem\u003eVcCEN\u003c/em\u003e expression level in \u0026lsquo;BM\u0026rsquo; may have resulted in unusual flowering. Overall, these findings imply \u003cem\u003eVcCEN\u003c/em\u003e may encode a flowering repressor in blueberry, making it a potential candidate gene for manipulating blueberry flowering.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eDetection of CRISPR-Cas9-induced\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eCEN\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003emutations in genetic transformants\u003c/span\u003e\u003c/p\u003e \u003cp\u003eTransgenic blueberry plants carrying the CRISPR-Cas9 vector targeting \u003cem\u003eVcCEN\u003c/em\u003e were generated via Agrobacterium-mediated transformation. Mutations were detected in nine of the 11 transgenic lines (mutation rate of 82%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The highest mutated allele frequency was 100%, which means all alleles were edited. The mutated allele frequency was higher for gRNA2 than for gRNA1, indicative of the importance of selecting an appropriate gRNA for efficient genome editing. The mutation types in one of the edited plants are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026ndash;D. Specifically, DECODR and clone sequencing revealed 1- to 10-bp insertions/deletions a few bases upstream of the PAM sequence in the transgenic line (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePhenotypic observations of\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003ecen\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003emutants\u003c/span\u003e\u003c/p\u003e \u003cp\u003eAt 4 months after rooting, approximately 90% of the rooted shoots were successfully acclimated to the ambient conditions. The average plant height data revealed that the three individuals from two selected mutant lines were significantly shorter than the non-transgenic control at 6 and 9 months after potting (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In addition, shoot lengths of \u003cem\u003ecen\u003c/em\u003e mutants tended to be shorter than WT at 9 months after potting (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These results suggested that a mutation to \u003cem\u003eVcCEN\u003c/em\u003e may induce suppressed vegetative growth in blueberry.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFirst flowering of the \u003cem\u003ecen\u003c/em\u003e mutants occurred earlier (8\u0026ndash;11 months after potting) than the WT plants (20\u0026ndash;28 months after potting) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The mutant plants exhibited a compact growth habit and terminal flowering. The \u003cem\u003ecen\u003c/em\u003e mutants produced a single or double flower at the top of the shoot, whereas the WT plants produced a florescence with several flowers (Supplementary Fig.\u0026nbsp;1). The terminal bud of the mutants did not enter dormancy and bloomed within 1 month after bud formation. In summer, the shoot apex of the mutant lines occasionally transformed directly into a flower without growth cessation and a typical bud set (Supplementary Fig.\u0026nbsp;2). The vegetative growth continued until just before the formation of the terminal flower. This flowering pattern in the mutants was reproduced under high temperature (28\u0026deg;C) and long photoperiod (16 h) conditions. In the \u003cem\u003ecen\u003c/em\u003e mutant grown in the greenhouse without heating, flower buds formed throughout the year and they bloomed continuously from March to December without entering dormancy. In January and February, flower bud development was suppressed and no blooming was observed, probably because of the low temperatures during winter. When the mutants were grown in small pots and the longest shoot was shorter than 40 cm, they did not flower, similar to the WT control. The fact that flower buds only developed on shoots longer than approximately 60 cm for both the WT and mutant plants suggested there is a minimum shoot length threshold for plants to bear flower buds. There were no obvious morphological differences in the flowers and pollen germination between the WT and \u003cem\u003ecen\u003c/em\u003e mutant plants (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe \u003cem\u003eTFL1\u003c/em\u003e/\u003cem\u003eCEN\u003c/em\u003e gene family plays a crucial role in the regulation of flowering and architecture in various plant species. Earlier research demonstrated that \u003cem\u003eTFL1\u003c/em\u003e/\u003cem\u003eCEN\u003c/em\u003e genes encode flowering repressors in different plant species (Jin et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The \u003cem\u003eCEN\u003c/em\u003e gene was first identified as the gene responsible for controlling the inflorescence architecture in \u003cem\u003eAntirrhinum\u003c/em\u003e (Bradley et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). The \u003cem\u003ecen\u003c/em\u003e mutation changes inflorescence style from indeterminate to determinate single flower in \u003cem\u003eAntirrhinum\u003c/em\u003e. Although extensive research has been conducted to characterize the function of \u003cem\u003eCEN\u003c/em\u003e in model annual plants, the importance of its role in perennial plants has not been thoroughly investigated. In fruit trees, flowering comprises of perennial-specific several complex processes such as vegetative to reproductive phase transitions and bud dormancy (Wang et al. 2022). In the current study, the results of our analyses of \u003cem\u003eVcCEN\u003c/em\u003e expression and function collectively provide new insights into the molecular mechanism underlying blueberry flowering. More specifically, our findings indicate \u003cem\u003eVcCEN\u003c/em\u003e functions as a flowering repressor in blueberry. Additionally, the pleiotropic effects of \u003cem\u003eVcCEN\u003c/em\u003e were revealed; the mutation to \u003cem\u003eVcCEN\u003c/em\u003e resulted in changes to vegetative growth, photoperiod sensitivity, juvenile phase duration, flowering architecture, and flower bud dormancy. Moreover, \u003cem\u003eVcCEN\u003c/em\u003e was expressed at high levels in actively growing shoots in the summer, but the \u003cem\u003ecen\u003c/em\u003e mutants were shorter than the WT control, indicative of the positive effects of \u003cem\u003eVcCEN\u003c/em\u003e on vegetative growth. In blueberry, flower initiation is induced by short-day conditions, with a critical day length threshold of approximately 12 h (Hall 1963; Phatak and Austin 1990). Increasing the day length (e.g., 16-h photoperiod) reportedly leads to the lack of flower bud formation in three highbush blueberry cultivars (Ba\u0026ntilde;ados and Strik, 2006). In the present study, the WT plants did not bloom under long-day conditions, whereas flowers were detected on the mutants, suggesting a mutation to \u003cem\u003eVcCEN\u003c/em\u003e can alter photoperiod sensitivity. Under high temperature conditions, the shoot apex of the mutant lines occasionally transformed directly into a flower (Supplementary Fig.\u0026nbsp;2). Moreover, in the mutant, there was a spontaneous transition from vegetative growth to reproductive growth, resulting in the rapid formation of floral organs. In a recent study, a similar phenomenon was often observed in rabbiteye blueberry plants grown in a warm subtropical climate (Omori et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The growing shoots of some rabbiteye blueberry cultivars directly turn into inflorescences, with decreases in \u003cem\u003eVcCEN\u003c/em\u003e expression as the floral meristem forms (Omori et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These two phenomena may be mediated by a similar mechanism; Due to the concurrent occurrence of vegetative growth stimulated by high temperatures and reproductive growth triggered by the mutation or decreased expression of \u003cem\u003eVcCEN\u003c/em\u003e, elongating shoots abruptly differentiate into floral organs. A mutated \u003cem\u003eVcCEN\u003c/em\u003e also substantially changed the inflorescence architecture in blueberry. This phenotype is a typical consequence of mutations to \u003cem\u003eTFL1\u003c/em\u003e/\u003cem\u003eCEN\u003c/em\u003e genes in various plant species (Bradley et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Charrier et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Varkonyi-Gasic et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Shannon et al. 1991). Similar to other studies, the mutagenesis of \u003cem\u003eVcCEN\u003c/em\u003e limited the development of the normally indeterminate inflorescence and led to the production of a terminal flower. Thus, this study and reported studies collectively suggested that mutating \u003cem\u003eVcCEN\u003c/em\u003e is useful for promoting flowering and overcoming several traits typical of woody plants that restrict flowering, including a long juvenile phase and dormancy.\u003c/p\u003e \u003cp\u003eThe annualization of fruit trees may revolutionize the breeding of fruit tree crops with a long juvenile phase. Most perennial plants have a long juvenile phase and lack flowers for a certain period. This characteristic is detrimental to efficient breeding and genetic analyses. For example, introducing disease resistance genes via backcrossing involving closely related wild species and pyramiding useful genes are time-consuming processes. \u0026lsquo;Fast-track breeding\u0026rsquo; has been developed as a novel strategy for efficiently breeding stress-resistant fruit trees. This approach is based on the overexpression of genes that promote flowering, including \u003cem\u003eFT\u003c/em\u003e orthologs. It has been used to introduce fire blight and apple scab resistance genes from wild apple into an elite cultivar, with five generations produced within 7 years (Schlath\u0026ouml;lter et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Similarly, the citrus tristeza virus resistance gene from trifoliate orange (\u003cem\u003ePoncirus trifoliata\u003c/em\u003e; Endo et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) was introduced into other citrus germplasm. A similar approach may be applied to incorporate some important traits from wild \u003cem\u003eVaccinium\u003c/em\u003e plants into blueberry cultivars. Some wild \u003cem\u003eVaccinium\u003c/em\u003e species that may be used for the cross-pollination of blueberry have valuable traits, including vigor, unique fruit volatile compositions, fruit firmness, waxy foliage, low chilling requirements, cold hardiness, and drought tolerance (Edger et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe early flowering trait of the \u003cem\u003ecen\u003c/em\u003e mutant may also be useful for validating agronomically important genes in blueberry. Many recent genetic studies have identified candidate genes associated with agronomically important traits of blueberry (Edger et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The precise characterization of the functions and practical utility of genes requires experiments involving stable transformations or mutagenesis. However, even though many candidate genes have been identified, very few have been functionally validated, especially in fruit tree crops. Creating and phenotyping transgenic plants require a lot of time, area, and labor if the individual plants are large and have a long generation time. Shortening the juvenile phase can accelerate gene validation studies. For example, \u0026ldquo;rapid flowering\u0026rdquo; kiwifruit (\u003cem\u003eA. chinensis\u003c/em\u003e) was previously used to functionally validate \u003cem\u003eFriendly boy\u003c/em\u003e (\u003cem\u003eFrBy\u003c/em\u003e), which is a Y chromosome-encoded sex-determinant gene (Akagi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In another study, an early flowering grape mutant \u0026lsquo;microvine\u0026rsquo; that flowers continuously and has a shortened generation time was used to generate a segregating population and validate the contribution of the candidate gene \u003cem\u003eVviPLATZ1\u003c/em\u003e to female flower morphology (Iocco-Corena et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the current study, the \u003cem\u003ecen\u003c/em\u003e mutant had a continuous flowering phenotype with no dormancy period, suggesting that flower bud development was enhanced. The continuous flowering trait may be exploited for off-season fruit production. The shelf life of blueberry is shorter than that of most fruits. Thus, a year-round production system is desirable. Genome editing technology has been utilized to improve crop yield, nutrient, stress tolerance (Atia et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and many countries adapted guidelines that permit the cultivation of non-transgenic gene-edited lines similar to conventionally bred lines (Buchholzer and Frommer. 2023). The continuous flowering \u003cem\u003ecen\u003c/em\u003e mutant generated in this study may also enable blueberry growers to extend the harvest period. However, the quality of the \u003cem\u003ecen\u003c/em\u003e mutant fruit will need to be assessed for this purpose.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI [No. 19KK0156] to H.Y. and R.T. and Grant-in-Aid for JSPS Fellows for [No. 21J22977] to M.O.\u003c/p\u003e\n\u003cp\u003eWe thank Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.Y. and M.O. designed project and M.O. performed the experiments and wrote the manuscript. Y. O. and K. O. provided the CRISPR-Cas9 vector. H.Y and R.T. supervised all the work and revised the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbe M, Kobayashi Y, Yamamoto S, Daimon Y, Yamaguchi A, Ikeda Y, Ichinoki H, Notaguchi M, Goto K, Araki T (2005) FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex. Science 309:1052\u0026ndash;1056. DOI: 10.1126/science.11159\u003c/li\u003e\n\u003cli\u003eAkagi T, Pilkington SM, Varkonyi-Gasic E, Henry IM, Sugano SS, Sonoda M, Firl A, McNeilage MA, Douglas MJ, Wang T, Rebstock R, Voogd C, Datson P, Allan AC, Beppu K, Kataoka I, Tao R (2019) Two Y-chromosome-encoded genes determine sex in kiwifruit. 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New Phytol 226:1725\u0026ndash;1737\u003c/li\u003e\n\u003cli\u003eHall IV, Craig DL, Aalders LE (1963) The effect of photoperiod on the growth and flowering of the highbush blueberry (\u003cem\u003eVaccinium corymbosum\u003c/em\u003e L.). J Amer Soc Hortic Sci 82, 260\u0026ndash;263\u003c/li\u003e\n\u003cli\u003eHan X, Yang Y, Han X, Ryner JT, Ahmed EAH, Qi Y, Zhong G, Song G (2022) CRISPR Cas9-and Cas12a-mediated gusA editing in transgenic blueberry. Plant Cell Tissue Organ Cult 1\u0026ndash;13\u003c/li\u003e\n\u003cli\u003eHuang NC, Jane WN, Chen J, Yu TS (2012) Arabidopsis thaliana CENTRORADIALIS homologue (ATC) acts systemically to inhibit floral initiation in Arabidopsis. Plant J 72:175\u0026ndash;184\u003c/li\u003e\n\u003cli\u003eIocco-Corena P, Cha\u0026iuml;b J, Torregrosa L, Mackenzie D, Thomas MR, Smith HM (2021) VviPLATZ1 is a major factor that controls female flower morphology determination in grapevine. Nat Commun 12:6995\u003c/li\u003e\n\u003cli\u003eJin S, Nasim Z, Susila H, Ahn JH (2021) Evolution and functional diversification of FLOWERING LOCUS T/TERMINAL FLOWER 1 family genes in plants. Seminars in cell \u0026amp; developmental biology 109:20\u0026ndash;30\u003c/li\u003e\n\u003cli\u003eKarlgren A, Gyllenstrand N, K\u0026auml;llman T Sundstr\u0026ouml;m JF, Moore D, Lascoux M, Lagercrantz U (2011) Evolution of the PEBP gene family in plants: functional diversification in seed plant evolution. Plant Physiol 156:1967\u0026ndash;1977\u003c/li\u003e\n\u003cli\u003eLuby JJ, Ballington JR, Draper AD , Pliszka K, Austin ME (1991) Blueberries and cranberries (\u003cem\u003eVaccinium\u003c/em\u003e). Acta Hortic 290:393\u0026ndash;458. https://doi.org/10.17660/ActaHortic.1991.290.9\u003c/li\u003e\n\u003cli\u003eMcCown BH, Lloyd G. Woody plant medium (WPM)-a mineral nutrient formulation for microculture of woody plant-species. HortScience. 1981;16:453\u003c/li\u003e\n\u003cli\u003eMurashige T, Skoog F. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant. 1962;15:473\u0026ndash;497\u003c/li\u003e\n\u003cli\u003eOgiwara I, Horiuchi N, Chitose A (2016) Experimental study on the production and shipping of off-season blueberry. (in Japanese) JATAFF journal 4:41\u0026ndash;46\u003c/li\u003e\n\u003cli\u003eOmori M, Yamane H, Osakabe K , Osakabe Y, Tao R (2021) Targeted mutagenesis of \u003cem\u003eCENTRORADIALIS\u003c/em\u003e using CRISPR/Cas9 system through the improvement of genetic transformation efficiency of tetraploid highbush blueberry. J Hortic Sci Biotechnol 96:153\u0026ndash;161\u003c/li\u003e\n\u003cli\u003eOmori M, Cheng CC, Hsu FC, Chen SJ, Yamane H, Tao R, Li KT (2022) Off-season flowering and expression of flowering-related genes during floral bud differentiation of rabbiteye blueberry in a subtropical climate. Sci Hortic 306: 111458\u003c/li\u003e\n\u003cli\u003eOmori M, Yamane H, Osakabe K, Osakabe Y, Tao R (2023). The evaluation of CRISPR-Cas9-mediated editing efficiency using endogenous promoters in tetraploid blueberry. Acta Hortic 1362, 49\u0026ndash;56. https://doi.org/10.17660/ActaHortic.2023.1362.8\u003c/li\u003e\n\u003cli\u003eOsakabe Y, Osakabe K (2015) Genome editing with engineered nucleases in plants. Plant Cell Physiol 56:389\u0026ndash;400. https://doi.org/10.1093/pcp/pcu170\u003c/li\u003e\n\u003cli\u003eOsakabe Y, Watanabe T, Sugano SS, Ueta R, Ishihara R, Shinozaki K, Osakabe (2016) Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants. 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Forestry Res 3:2\u003c/li\u003e\n\u003cli\u003eXiao A, Cheng Z, Kong L, Zhu Z, Lin S, Gao G, Zhang B (2014) . CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics 30:1180\u0026ndash;1182 https://doi.org/10.1093/bioinformatics/btt764\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4642319/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4642319/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFlowering marks the vegetative-to-reproductive growth transition and is the most important event in the plant life cycle. Unlike annual plants, perennial fruit trees flower and set fruits only after an extended juvenile phase (i.e., several years), which is an impediment to efficient breeding and gene function analyses. In this study, we generated an early flowering blueberry line via the CRISPR-Cas9-mediated mutagenesis of \u003cem\u003eVcCENTRORADIALIS\u003c/em\u003e (\u003cem\u003eVcCEN\u003c/em\u003e). The expression of \u003cem\u003eVcCEN\u003c/em\u003e in the apical bud was negatively correlated with flower bud formation. Moreover, in the cultivar that flowers in both autumn and spring, the \u003cem\u003eVcCEN \u003c/em\u003eexpression level was lower and decreased earlier than in the normal cultivar that flowers in only spring. The expression data suggested that \u003cem\u003eVcCEN \u003c/em\u003efunctions as a flowering repressor. The CRISPR-Cas9 vector harboring a gRNA targeting \u003cem\u003eVcCEN\u003c/em\u003ewas introduced into the blueberry genome via Agrobacterium-mediated transformation. Mutations (e.g., 1–10 bp indels) were detected in the stable transformants, with all \u003cem\u003eVcCEN\u003c/em\u003e alleles of the tetraploid genome mutated in some lines. Compared with the wild-type (WT), the \u003cem\u003ecen\u003c/em\u003e mutants exhibited repressed vegetative growth. Additionally, in the mutants, first flowering occurred within 1 year after the Agrobacterium infection, which was approximately 1–2 years earlier than in WT. The mutants set a single terminal flower without entering dormancy, whereas WT produced an apical flower and multiple axillary flowers that bloomed after an exposure to chilling conditions and then warm temperatures. This early flowering trait is conducive to efficient breeding and gene functional analyses, especially in fruit crops with a long juvenile phase.\u003c/p\u003e","manuscriptTitle":"CRISPR-Cas9-mediated mutagenesis of the flowering repressor gene VcCENTRORADIALIS (VcCEN) induces early flowering in tetraploid highbush blueberry","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-07 08:05:58","doi":"10.21203/rs.3.rs-4642319/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7bc33906-0574-477b-8012-9e1aa9f1abd0","owner":[],"postedDate":"August 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-10T09:32:28+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-07 08:05:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4642319","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4642319","identity":"rs-4642319","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}