HortJ

In Japan, over 95% of the acreage is covered with plastic to force June-bearing (seasonal flowering, SF) strawberry cultivars to produce fruit from late fall to early summer. During the late 1960s, a forcing technique was developed that advanced flower bud initiation to late summer and prevented the transplants from becoming dormant during winter. This new forcing technique involved nitrogen starvation of nursery plants to induce floral initiation. Until about 1980, strawberry growers in Japan used runner plants produced in waiting beds, but most transplants are now produced in plastic pots under rain shelters to avoid soil-borne diseases. Recently, the use of tray plants produced from hanging runner cuttings has become popular. To induce early floral initiation, the following artificial low temperature (LT) treatments have been established: (1) “Yarei”, a combination of a short day with solar radiation and LT under darkness in cooling facilities (Yarei-ko); (2) “Kaburei”, continuous dark-LT with refrigeration facilities including industrial warehouses; and (3) “Kanketsu-reizo”, intermittent LT storage. An overview of the technologies applied to plant propagation and the control of floral initiation of Japanese SF cultivars is provided in this review.

Strawberry flowering physiology has engaged the interest of researchers for almost a century after the initial reports demonstrating the photoperiodic control of flowering and vegetative reproduction through stolons called runners. Most strawberries possess a seasonal flowering habit with flower initiation occurring under short days in autumn and flowering during the following spring. Also perpetual flowering genotypes are known in diploid woodland strawberry (Fragaria vesca L.) and octoploid garden strawberry (F. × ananassa Duch.), and recent research have shown that this trait has evolved independently in different species. Studies in the perpetual flowering mutant of woodland strawberry led to the identification of TERMINAL FLOWER1 (FvTFL1) as a major floral repressor causing the seasonal flowering habit in this species and demonstrated that recessive mutation in this gene leads to perpetual flowering. This breakthrough opened an avenue for molecular understanding on the control of flowering by different environmental signals. Different loci control perpetual flowering in garden strawberry including one dominant major locus and additional environmentally regulated epistatic loci. The major gene is called Perpetual Flowering Runnering (PFRU) because it also reduces the number of runners. Growth regulator applications initially demonstrated the role of gibberellin in the control of runner formation, and molecular understanding on the role of gibberellin biosynthesis and signaling in this process has started to emerge. Here, we present current understanding and major open questions on the control of flowering and runnering in strawberries. In order to understand the control of flowering in the context of perennial growth cycle, we also discuss current knowledge on the control of dormancy.

Next generation sequencing (NGS) is one of the most impactful technologies to appear in the 21^st century, and has already brought important changes to agriculture, especially in the field of breeding. Construction of a reference genome is key to the advancement of genomic studies, and therefore, de novo whole genome assembly has been performed in various plants, including strawberry. Strawberry (Fragaria × ananassa) is an allo-octoploid species (2n = 8x = 56), which has four discriminable subgenomes. Because of its complex genome structure, de novo whole genome assembly in strawberry has been considered a difficult challenge. However, recent advances of NGS technologies have allowed the construction of chromosome-scale de novo whole genome assembly. In this manuscript, we review the recent advances in de novo whole genome sequencing in strawberry and other Fragaria species. The genome structure and domestication history in strawberry is one of the largest questions in genetic and genomic studies in strawberry. Therefore, the domestication history in strawberry is also be reviewed based on comparisons of genes and genome sequences across Fragaria species.

Japanese strawberries have quite high palatability, as well as softness, making them a valuable commodity for local consumption and export. To preserve these characteristics, several unique efforts have been made to improve the shelf-life of strawberry fruits in Japan. This review summarizes the specific breeding and technological developments currently in practice to distribute Japanese strawberries without losing palatability while prolonging the shelf-life. With respect to breeding, the amount and ratio of hydrochloric acid-soluble pectin have been improved, including the hydrochloric acid-soluble pectin to holocellulose ratio, which is related to fruit texture. In addition, although the optimal fruit shape is uncertain, the apex and equatorial parts can be bred to be more rounded in some new varieties. Inflorescence characteristics have also been improved to increase the uniformity of fruit shape and size, which can avoid the need for additional handling while sorting and packing the fruits. With respect to post-harvest techniques, pre-cooling methods, cold chain processes, and highly improved packages have been developed. In particular, the use of hammock-type and elastic film packages mean the strawberry fruit does not make contact with other fruits or trays. These packages also change the acceleration transmissibility under vibration, which can further prevent damage to the fruits during long-term transport. These emerging varieties, pre-cooling and cold-chain processing, and innovative packages are all compatible with sorting and packing robots, allowing for full integration of these processes. Therefore, these innovations and continuous improvements in breeding and packaging are expected to increase the export of Japanese strawberry fruits and provide guidance for achieving a balance between long shelf-life and high palatability in global strawberry production.

Strawberry plants are grown in hydroponics for higher quality and yield, as this system excludes soil-borne disease issues. Recycled hydroponics is practiced to make cultivation cost-effective, sustainable, and environmentally friendly. However, due to recycling of hydroponic nutrient solution, plant root exudates accumulate, leading to autotoxicity, a form of allelopathy that inhibits growth and development. In recent decades, commercial cultivation of strawberry under greenhouse and plant factory conditions following recycled hydroponics has been widely adopted globally. Subsequently, yield decline has also been reported due to development of autotoxicity from the accumulated root exudates. In recycled hydroponic systems, strawberry plant growth is inhibited by root exudates that contain mainly phenolic acids in the culture solution. In this regard, elimination of these accumulated root exudates or allelochemicals from the culture solution would restore inhibited plant growth and yield. A number of research studies have been conducted on autotoxicity in strawberry and possible mitigation methods. These studies suggested that addition of activated charcoal in the nutrient solution, supplementation of auxin on leaves, electro-degradation of root exudates in nutrient solution, and supplementation of amino acids and/or LEDs can effectively remove/degrade/mitigate autotoxicity in strawberry grown under recycling hydroponics. This review mainly discusses the autotoxicity phenomenon in strawberry under recycled hydroponics, the responsible allelochemicals and their mechanism of action, mitigation methods and future research endeavors in this field.

In Arabidopsis thaliana, the FLOWERING LOCUS T (FT) gene, acting as a floral promoter, is expressed and translated in leaves, and is then transported to the shoot apical meristem. In contrast, the expression pattern of the FaFT3 gene in the crown, which contains the shoot apical meristem, is coordinated with the initiation of the floral bud in the June-bearing type of cultivated strawberry (Fragaria × ananassa) ‘Nyoho’. However, whether the FaFT3 protein functions as a floral promoter and whether the expression pattern of FaFT3 in the crown observed in ‘Nyoho’ is conserved in other strawberry cultivars are not known. In this study, we investigated the floral inducer activity of the FaFT3 gene isolated from the cultivated strawberry ‘Tochiotome’ using FaFT3-overexpressing transgenic Arabidopsis lines and performed expression analysis on the FaFT3 gene in the crown tip of ‘Tochiotome’. Transgenic plants overexpressing the FaFT3 gene exhibited an early-flowering phenotype under both long-day and short-day conditions. Conversely, induction of FaFT3 expression at the crown tip specifically under floral induction conditions was not observed. However, RNA-seq analysis of laser microdissected meristem cells before and after floral bud initiation clearly revealed that the FaFT3 gene is specifically expressed in floral meristem cells. These results suggest that the FaFT3 gene acts as a common floral promoter in June-bearing Japanese cultivated strawberries.

Male sterility is defined as the loss of pollen fertility, and it represents a plant reproductive isolation symptom, along with self-incompatibility. It plays an important role in the efficient production of F₁-hybrid seeds, which results in affordable seed prices for farmers. Male sterile cultivated strawberry Fragaria × ananassa Duch. plants were found in an F₁ population and reciprocal backcrossed populations derived from a cross between ‘Fukuoka S6’ and ‘Kaorino’. Male sterile plants were clearly distinguished from male fertile plants in those populations based on the anther color. The pollen of the male sterile plants was a lighter yellow color and not maturely shaped compared with pollen of male fertile plants. Genotyping was performed using EST-SSR markers in the three populations. Quantitative trait locus analyses for pollen fertility were conducted independently using three kinds of populations, and this revealed that male sterility was controlled by three independent chromosomal regions in these populations, which corresponded to chromosome 4 in the wild strawberry (Fragaria vesca) genome. One region was derived from ‘Fukuoka S6’ and the other two regions from ‘Kaorino’. The segregation patterns of fertile and sterile plants in each population clearly supported the three gene theory of male sterility in cultivated strawberries. The accumulation of recessive alleles at the three regions led to male sterility, and the existence of a dominant allele in at least one region resulted in fertile pollen. Male sterile plants were also found in two self-pollenated populations derived from ‘Fukuoka S6’ and ‘Kaorino’, and the effects of the three regions were validated. The adaptability levels of the three genes with different genetic backgrounds were also evaluated using core collection cultivars and selected lines derived using recurrent selection. We also detected flanking DNA markers for the three regions associated with male sterility. The use of these markers, which are in the vicinity of quantitative trait loci and responsible for male-sterility, could increase the efficiency of producing seed-propagated strawberry F₁-hybrids.

Everbearing cultivars of the octoploid cultivated strawberry (Fragaria × ananassa) enable the expansion of production. Because the identification of everbearing individuals by phenotypic observation is time- and labor-consuming, marker-assisted selection is useful. In this study, we developed a sequence-tagged site marker, s2430859, which specifically amplifies a DNA fragment linked to the everbearing gene from homoeologous chromosomes for genotyping by agarose gel electrophoresis. In the analysis of eight F₁ populations produced from various crosses of everbearing by June-bearing cultivars, the concordance between the phenotype and the marker genotype ranged from 94.4% in the ‘Hecker’ × ‘Sagahonoka’ population to 100.0% in the ‘Miyazaki-natsuharuka’ × ‘Karenberry’ population. The average match was 96.8%. These results suggest that s2430859 is closely linked to the everbearing gene and is applicable to various breeding populations. Because s2430859 is located on the opposite side of the gene from already reported markers, it will contribute to the detection of recombinant individuals by use in combination with these markers.

The everbearing strawberry cultivar ‘Natsuakari’ produces high-quality fruits and can be harvested in summer and autumn. However, flowering-defective individuals have been recently found at some farms in the Tohoku region. To solve this problem, we first investigated the behavior of flowering-defective individuals collected from three places in Aomori, Akita, and Miyagi prefectures and confirmed that the non-flowering phenomenon reproducibly occurred during summer and autumn. Next, we analyzed the genotypes of flowering-defective individuals using highly polymorphic simple sequence repeat (SSR) markers suitable for cultivar discrimination and SSR markers linked to the everbearing gene. As a result, flowering-defective individuals showed the same genotype as the original ‘Natsuakari’, and the fragment linked to the everbearing gene was not detected in flowering-defective individuals. It was suggested that cultivar contamination was not the cause of the non-flowering phenomenon, and that mutations may be present in flowering-defective individuals. To confirm the effects of previous history before transplanting on the flowering and runner development of normal flowering plants and the flowering-defective mutants, the influence of potting time of the daughter plant and overwintering conditions were investigated. The potting time had no effect on flowering or runner development. Inflorescences in the normal flowering plants continuously emerged, especially in summer and autumn under both heated and non-heated conditions during overwintering. The mutants hardly flowered in the summer and autumn under both conditions, although the inflorescences emerged in the early summer under the heated condition during overwintering. The number of runners observed in the mutants was higher than that in the normal plants throughout the study, especially in the summer, while in both heating during overwintering led to much less runner development in May through July. According to these results, although the mutants were suitable for plant propagation, defective flowering persisted in subsequent years. Therefore, to ensure stable production, it is important to observe flowering types in mother plants with adequate chilling and to select mother plants exhibiting flowering in summer and autumn.

In this study, the in vivo potential of lemon balm water extract on Fusarium wilt control in strawberry and the antifungal properties of secondary metabolites in the extract were investigated. Runner plants of strawberry (Fragaria × ananassa Duch., ‘Sachinoka’) were treated with water extracts (20%, w/v) of lemon balm (Melissa officinalis L.) and inoculated with Fusarium oxysporum f. sp. fragariae (Fof). Four weeks after Fof inoculation, lower disease incidence and indices in both shoots and roots were observed in lemon balm-treated plants. These effects could be attributed to reduced Fusarium populations due to the fungistasis and fungicidal effects induced by the extract in the rhizospheric soil. Consequently, dry weights of shoots and roots in the plants treated with lemon balm extracts were higher than those of the control. Based on the results of ultra performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) analyses, rosmarinic acid was the metabolite with the highest concentration and was also the most stable metabolite in the water extract. In addition, the antifungal effect of rosmarinic acid against Fof was confirmed by in vitro tests. Therefore, water extracts of lemon balm could suppress Fusarium wilt in strawberry plants and rosmarinic acid was one of the key metabolites with antifungal properties present in the water extract.

Fra a 1 is a strawberry allergen that causes oral allergic syndrome. Fra a 1.01 is a major isoform that accumulates abundantly in fruits during the winter season. Here, we tested the hypothesis that Fra a 1.01 responds to environmental factors, such as cold stress. We analyzed transcriptional and translational levels of Fra a 1.01 in strawberry calli and organs under various cold conditions. First, we incubated strawberry (Fragaria × ananassa ‘Akihime’) calli and post-harvested fruits at low temperatures from several hours to days. Fra a 1.01 did not show significant differences in either gene expression or protein accumulation levels, suggesting that short-term cold treatments did not affect Fra a 1.01 expression. Second, we exposed whole plants to low temperature conditions for ~28 days. Under conditions below 10°C, Fra a 1.01 transcripts were induced gradually throughout the cold treatment (crown and root), or from 2 days to the last day (leaf and fruit). The Fra a 1.01 protein remarkably accumulated in crowns and slightly in fruits after 28 days. Finally, the promoter region of Fra a 1.01e was analyzed to detect tissue-specific expression. The cloned and sequenced promoter included several cis-acting regulatory elements related to cold response. When the Fra a 1.01 promoter region was heterologously expressed in Arabidopsis, the promoter activities, as assessed by GUS staining, were observed mainly around the shoot apices and in roots. Thus, Fra a 1.01 was considered to be expressed in crowns and roots, and was additively induced by cold stress. These organ-specific expressions could be important in elucidating the mechanisms responsible for Fra a 1.01 protein’s accumulation in fruits during the winter season.