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

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

Narrowing down the single homoeologous FaPFRU locus controlling flowering in cultivated octoploid strawberry using a selective mapping strategy

Extending the period of fruit production is a way to substantially increase crop yield in many fruit or ornamental species. In the cultivated octoploid strawberry (Fragaria × ananassa), the most consumed small fruit worldwide, fruit production season can be extended by selecting the perpetual flowering (PF) cultivars. This trait is of considerable interest to growers and to the food industry. Four homoeologous loci controlling a single trait can be expected in such a complex octoploid species. However, we recently showed that the PF trait is under the control of the single dominant FaPFRU locus (J. Exp. Bot., 2013, 64 , 1837), making it potentially amenable to marker‐assisted selection (MAS). Here, we report the successful use of a strategy, based on a selective mapping using a reduced sample of individuals, to identify nine markers in close linkage to the FaPFRU allelic variant. Thus, this strategy can be used to fine map the target homoeologous loci in other complex polyploid crop species. Recombinant analysis further enabled us to reduce the locus to a region flanked by two markers, Bx083_206 and Bx215_131, corresponding to a 1.1 Mb region in the diploid F. vesca reference genome. This region comprises 234 genes, including 15 flowering associated genes. Among these, the FLOWERING LOCUS T (FT) is known to be a key activator of flowering. The close association between the PF trait and the FaPFRU flanking markers was validated using an additional segregating population and genetic resources. This study lays the foundation for effective and rapid breeding of PF strawberry cultivars by MAS.     

INDUCER OF CBF EXPRESSION 1 integrates cold signals into FLOWERING LOCUS C‐mediated flowering pathways in Arabidopsis

Plants constantly monitor changes in photoperiod and temperature throughout the year to synchronize flowering with optimal environmental conditions. In the temperate zones, both photoperiod and temperature fluctuate in a somewhat predictable manner through the seasons, although a transient shift to low temperature is also encountered during changing seasons, such as early spring. Although low temperatures are known to delay flowering by inducing the floral repressor FLOWERING LOCUS C (FLC), it is not fully understood how temperature signals are coordinated with photoperiodic signals in the timing of seasonal flowering. Here, we show that the cold signaling activator INDUCER OF CBF EXPRESSION 1 (ICE1), FLC and the floral promoter SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) constitute an elaborate signaling network that integrates cold signals into flowering pathways. The cold‐activated ICE1 directly induces the gene encoding FLC, which represses SOC1 expression, resulting in delayed flowering. In contrast, under floral promotive conditions, SOC1 inhibits the binding of ICE1 to the promoters of the FLC gene, inducing flowering with a reduction of freezing tolerance. These observations indicate that the ICE1‐FLC‐SOC1 signaling network contributes to the fine‐tuning of flowering during changing seasons.     

Natural variation and CRISPR/Cas9‐mediated mutation in GmPRR37 affect photoperiodic flowering and contribute to regional adaptation of soybean

Flowering time is a critical determinant of the geographic distribution and regional adaptability of soybean (Glycine max) and is strongly regulated by photoperiod and temperature. In this study, quantitative trait locus (QTL) mapping and subsequent candidate gene analysis revealed that GmPRR37, encoding a pseudo‐response regulator protein, is responsible for the major QTL qFT12‐2, which was identified from a population of 308 recombinant inbred lines (RILs) derived from a cross between a very late‐flowering soybean cultivar, ‘Zigongdongdou (ZGDD)’, and an extremely early‐flowering cultivar, ‘Heihe27 (HH27)’, in multiple environments. Comparative analysis of parental sequencing data confirmed that HH27 contains a non‐sense mutation that causes the loss of the CCT domain in the GmPRR37 protein. CRISPR/Cas9‐induced Gmprr37‐ZGDD mutants in soybean exhibited early flowering under natural long‐day (NLD) conditions. Overexpression of GmPRR37 significantly delayed the flowering of transgenic soybean plants compared with wild‐type under long photoperiod conditions. In addition, both the knockout and overexpression of GmPRR37 in soybean showed no significant phenotypic alterations in flowering time under short‐day (SD) conditions. Furthermore, GmPRR37 down‐regulated the expression of the flowering‐promoting FT homologues GmFT2a and GmFT5a, and up‐regulated flowering‐inhibiting FT homologue GmFT1a expression under long‐day (LD) conditions. We analysed haplotypes of GmPRR37 among 180 cultivars collected across China and found natural Gmprr37 mutants flower earlier and enable soybean to be cultivated at higher latitudes. This study demonstrates that GmPRR37 controls soybean photoperiodic flowering and provides opportunities to breed optimized cultivars with adaptation to specific regions and farming systems.     

Microsatellite markers for Fragaria from ‘Strawberry Festival’ expressed sequence tags

We present 37 microsatellite primer pairs developed from a cDNA library of Fragaria ^xananassa Duch. cv. Strawberry Festival. Polymorphism was high and the number of presumptive alleles of 13 expressed sequence tag–simple sequence repeats (EST–SSRs) in 70 strawberry cultivars ranged from five to 32 per primer pairs, averaging 16.1. Cross‐species amplification was also high and ranged from 89% in Fragaria vesca L. to 100% in the progenitor species of octoploid strawberry, Fragaria chiloensis (L.) Duch. and Fragaria virginiana Duch.     

CRISPR/Cas9‐mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean

Flowering is an indication of the transition from vegetative growth to reproductive growth and has considerable effects on the life cycle of soya bean (Glycine max). In this study, we employed the CRISPR/Cas9 system to specifically induce targeted mutagenesis of GmFT2a, an integrator in the photoperiod flowering pathway in soya bean. The soya bean cultivar Jack was transformed with three sgRNA/Cas9 vectors targeting different sites of endogenous GmFT2a via Agrobacterium tumefaciens‐mediated transformation. Site‐directed mutations were observed at all targeted sites by DNA sequencing analysis. T1‐generation soya bean plants homozygous for null alleles of GmFT2a frameshift mutated by a 1‐bp insertion or short deletion exhibited late flowering under natural conditions (summer) in Beijing, China (N39°58′, E116°20′). We also found that the targeted mutagenesis was stably heritable in the following T2 generation, and the homozygous GmFT2a mutants exhibited late flowering under both long‐day and short‐day conditions. We identified some ‘transgene‐clean’ soya bean plants that were homozygous for null alleles of endogenous GmFT2a and without any transgenic element from the T1 and T2 generations. These ‘transgene‐clean’ mutants of GmFT2a may provide materials for more in‐depth research of GmFT2a functions and the molecular mechanism of photoperiod responses in soya bean. They will also contribute to soya bean breeding and regional introduction.     

Pollinator‐mediated assemblage processes in California wildflowers

Community assembly is the result of multiple ecological and evolutionary forces that influence species coexistence. For flowering plants, pollinators are often essential for plant reproduction and establishment, and pollinator‐mediated interactions may influence plant community composition. Here, we use null models and community phylogenetic analyses of co‐occurrence patterns to determine the role of pollinator‐mediated processes in structuring plant communities dominated by congeners. We surveyed three species‐rich genera (Limnanthes, Mimulus and Clarkia) with centres of diversity in the Sierra Nevada of California. Each genus contains species that co‐flower and share pollinators, and each has a robust phylogeny. Within each genus, we surveyed 44–48 communities at three spatial scales, measured floral and vegetative traits and tested for segregation or aggregation of: (i) species, (ii) floral traits (which are likely to be influenced by pollinators), and (iii) vegetative traits (which are likely affected by other environmental factors). We detected both aggregation and segregation of floral traits that were uncorrelated with vegetative trait patterns; we infer that pollinators have shaped the community assembly although the mechanisms may be varied (competition, facilitation, or filtering). We also found that mating system differences may play an important role in allowing species co‐occurrence. Together, it appears that pollinators influence community assemblage in these three clades.     

The trxG family histone methyltransferase SET DOMAIN GROUP 26 promotes flowering via a distinctive genetic pathway

Histone methylation is a major component in numerous processes such as determination of flowering time, which is fine‐tuned by multiple genetic pathways that integrate both endogenous and environmental signals. Previous studies identified SET DOMAIN GROUP 26 (SDG26) as a histone methyltransferase involved in the activation of flowering, as loss of function of SDG26 caused a late‐flowering phenotype in Arabidopsis thaliana. However, the SDG26 function and the underlying molecular mechanism remain largely unknown. In this study, we undertook a genetic analysis by combining the sdg26 mutant with mutants of other histone methylation enzymes, including the methyltransferase mutants Arabidopsis trithorax1 (atx1), sdg25 and curly leaf (clf), as well as the demethylase double mutant lsd1‐like1 lsd1‐like2 (ldl1 ldl2). We found that the early‐flowering mutants sdg25, atx1 and clf interact antagonistically with the late‐flowering mutant sdg26, whereas the late‐flowering mutant ldl1 ldl2 interacts synergistically with sdg26. Based on microarray analysis, we observed weak overlaps in the genes that were differentially expressed between sdg26 and the other mutants. Our analyses of the chromatin of flowering genes revealed that the SDG26 protein binds at the key flowering integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1/AGAMOUS‐LIKE 20 (SOC1/AGL20), and is required for histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 36 trimethylation (H3K36me3) at this locus. Together, our results indicate that SDG26 promotes flowering time through a distinctive genetic pathway, and that loss of function of SDG26 causes a decrease in H3K4me3 and H3K36me3 at its target gene SOC1, leading to repression of this gene and the late‐flowering phenotype.     

Bee visit rates vary with floral morphology among highbush blueberry cultivars (Vaccinium corymbosum L.)

As the human population has increased, so too has the demand for biotically pollinated crops. Bees (Apoidea) are essential for pollen transfer and fruit production in many crops, and their visit patterns can be influenced by floral morphology. Here, we considered the role of floral morphology on visit rates and behaviour of managed honey bees (Apis mellifera) and wild bumble bees (genus Bombus), for four highbush blueberry cultivars (Vaccinium corymbosum L.). We measured five floral traits for each cultivar, finding significant variation among cultivars. Corolla throat diameter may be the main morphological determinant of visit rates of honey bees, which is significantly higher on the wider flowers of cv. ‘Duke’ than on ‘Bluecrop’ or ‘Draper’. Honey bees also visited cv. ‘Duke’ legitimately but were frequent nectar robbers on the long, narrow flowers of cv. ‘Bluecrop’. Bumble bees were infrequent (and absent on cv. ‘Draper’) but all observed visits were legitimate. Crop yield was highest for the cultivar with the highest combined (honey bee + bumble bee) visit rate, suggesting that aspects of floral morphology that affect pollinator visit patterns should be considered in crop breeding initiatives.     

The control of red colour by a family of MYB transcription factors in octoploid strawberry (Fragaria × ananassa) fruits

Octoploid strawberry (Fragaria × ananassa Duch.) is a model plant for research and one of the most important non‐climacteric fruit crops throughout the world. The associations between regulatory networks and metabolite composition were explored for one of the most critical agricultural properties in octoploid strawberry, fruit colour. Differences in the levels of flavonoids are due to the differences in the expression of structural and regulatory genes involved in flavonoid biosynthesis. The molecular mechanisms underlying differences in fruit colour were compared between red and white octoploid strawberry varieties. FaMYB genes had combinatorial effects in determining the red colour of fruit through the regulation of flavonoid biosynthesis in response to the increase in endogenous ABA at the final stage of fruit development. Analysis of alleles of FaMYB10 and FaMYB1 in red and white strawberry varieties led to the discovery of a white‐specific variant allele of FaMYB10, FaMYB10‐2. Its coding sequence possessed an ACTTATAC insertion in the genomic region encoding the C‐terminus of the protein. This insertion introduced a predicted premature termination codon, which suggested the loss of intact FaMYB10 protein playing a critical role in the loss of red colour in white octoploid strawberry.     

A photo‐responsive F‐box protein FOF2 regulates floral initiation by promoting FLC expression in Arabidopsis

Floral initiation is regulated by various genetic pathways in response to light, temperature, hormones and developmental status; however, the molecular mechanisms underlying the interactions between different genetic pathways are not fully understood. Here, we show that the photoresponsive gene FOF2 (F‐box of flowering 2) negatively regulates flowering. FOF2 encodes a putative F‐box protein that interacts specifically with ASK14, and its overexpression results in later flowering under both long‐day and short‐day photoperiods. Conversely, transgenic plants expressing the F‐box domain deletion mutant of FOF2 (FOF2ΔF), or double loss of function mutant of FOF2 and FOL1 (FOF2‐LIKE 1) present early flowering phenotypes. The late flowering phenotype of the FOF2 overexpression lines is suppressed by the flc‐3 loss‐of‐function mutation. Furthermore, FOF2 mRNA expression is regulated by autonomous pathway gene FCA, and the repressive effect of FOF2 in flowering can be overcome by vernalization. Interestingly, FOF2 expression is regulated by light. The protein level of FOF2 accumulates in response to light, whereas it is degraded under dark conditions via the 26S proteasome pathway. Our findings suggest a possible mechanistic link between light conditions and the autonomous floral promotion pathway in Arabidopsis.     

The circadian clock‐associated gene gigantea1 affects maize developmental transitions

The circadian clock is an internal timing mechanism that allows plants to make developmental decisions in accordance with environmental conditions. In model plants, circadian clock‐associated gigantea (gi) genes are directly involved in control of growth and developmental transitions. The maize gigantea1 (gi1) gene is the more highly expressed of the two gi homeologs, and its function is uncharacterized. To understand the role of gi1 in the regulatory networks of the maize circadian clock system, gi1 mutants were evaluated for changes in flowering time, phase change and growth control. When grown in long‐day (LD) photoperiods, gi1 mutants flowered earlier than non‐mutant plants, but this difference was not apparent in short‐day (SD) photoperiods. Therefore, gi1 participates in a pathway that suppresses flowering in LD photoperiods, but not in SD. Part of the underlying cause of early flowering was up‐regulated expression of the FT‐like floral activator gene zea mays centroradialis8 (zcn8) and the CONSTANS‐like flowering regulatory gene constans of zea mays1 (conz1). gi1 mutants also underwent vegetative phase change earlier and grew taller than non‐mutant plants. These findings indicate gi1 has a repressive function in multiple regulatory pathways that govern maize growth and development.     

Assessing cultivar resistance to Sitodiplosis mosellana (Géhin) (Diptera: Cecidomyiidae) using a phenotyping method under semi‐field conditions

The orange wheat blossom midge, Sitodiplosis mosellana (Géhin), can significantly reduce wheat yield. Growing resistant wheat cultivars is an effective way of managing this pest. The assessment of cultivar resistance in field trials is difficult because of unequal pressure of S. mosellana caused by differences in cultivar heading dates relative to the flight period of S. mosellana adult females and huge variations of egg laying conditions from 1 day to another. To overcome these hurdles and to expose all cultivars homogeneously to the pest, an assessment method of cultivar resistance was developed under semi‐field conditions. In 2015, the resistance of 64 winter wheat cultivars to S. mosellana was assessed. Few or no larvae developed in the ears of resistant cultivars, but in susceptible cultivars, large numbers of larvae developed. Seventeen cultivars proved to be resistant, whereas 47 were susceptible. The identification of new resistant cultivars offers more opportunities to manage S. mosellana. The phenotyping method is easy, cheap, efficient and reliable. It can be used to guide the breeding of new resistant wheat cultivars. Using specific midge populations, this method could also be used in research on new resistance mechanisms in winter wheat or in other cereal species.     

The Fragaria vesca Homolog of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 Represses Flowering and Promotes Vegetative Growth

In the annual long-day plant Arabidopsis thaliana, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) integrates endogenous and environmental signals to promote flowering. We analyzed the function and regulation of the SOC1 homolog (Fragaria vesca [Fv] SOC1) in the perennial short-day plant woodland strawberry (Fragaria vesca). We found that Fv SOC1 overexpression represses flower initiation under inductive short days, whereas its silencing causes continuous flowering in both short days and noninductive long days, similar to mutants in the floral repressor Fv TERMINAL FLOWER1 (Fv TFL1). Molecular analysis of these transgenic lines revealed that Fv SOC1 activates Fv TFL1 in the shoot apex, leading to the repression of flowering in strawberry. In parallel, Fv SOC1 regulates the differentiation of axillary buds to runners or axillary leaf rosettes, probably through the activation of gibberellin biosynthetic genes. We also demonstrated that Fv SOC1 is regulated by photoperiod and Fv FLOWERING LOCUS T1, suggesting that it plays a central role in the photoperiodic control of both generative and vegetative growth in strawberry. In conclusion, we propose that Fv SOC1 is a signaling hub that regulates yearly cycles of vegetative and generative development through separate genetic pathways.