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Deep neural network (DNN) techniques, as an advanced machine learning framework, have allowed various image diagnoses in plants, which often achieve better prediction performance than human experts in each specific field. Notwithstanding, in plant biology, the application of deep neural networks is still mostly limited to rapid and effective phenotyping. Recent development of explainable CNN frameworks has allowed visualization of the features in the prediction by convolutional neural network (CNN), which potentially contributes to the understanding of physiological mechanisms in objective phenotypes. In this study, we propose an integration of explainable CNN and transcriptomic approach to make a physiological interpretation of a fruit internal disorder in persimmon, rapid over-softening. We constructed CNN models to accurately predict the fate to be rapid softening in persimmon cv. Soshu, only with photo images. The explainable CNNs, such as Grad-CAM and Guided Grad-CAM, visualized specific featured regions relevant to the prediction of rapid-softening, which would correspond to the premonitory symptoms in a fruit. Transcriptomic analyses to compare the featured regions of predicted rapid-softening and control fruits suggested that rapid softening is triggered by precocious ethylene signal-dependent cell wall modification, despite exhibiting no direct phenotypic changes. Further transcriptomic comparison between the featured and non-featured regions in predicted rapid-softening fruit suggested that premonitory symptoms reflected hypoxia and the related stress signals finally to induce ethylene signals. These results would provide a good example for the collaboration of image analysis and omics approaches in plant physiology, which uncovered a novel aspect of fruit premonitory reactions in the rapid softening fate.

Frequent polyploidization events in plants have led to the establishment of many lineage-specific traits representing each species. Little is known about the genetic bases for these specific traits in polyploids, presumably due to plant genomic complexity and their difficulties of applying genetic approaches. Hexaploid Oriental persimmon (Diospyros kaki) has evolved specific fruit characters, including wide variations in fruit shapes and astringency. In this study, using whole-genome diploidized/quantitative genotypes from ddRAD-Seq data of 173 persimmon cultivars, we examined their population structures and potential correlations between their structural transitions and variations in nine fruit traits. The population structures of persimmon cultivars were highly randomized and not substantially correlated with the representative fruit traits focused on in this study, except for fruit astringency. With genome-wide association analytic tools considering polyploid alleles, we identified the loci associated with the nine fruit traits; we mainly focused on fruit-shape variations, which have been numerically characterized by principal component analysis of elliptic Fourier descriptors. The genomic regions that putatively underwent selective sweep exhibited no overlap with the loci associated with these persimmon-specific fruit traits. These insights will contribute to understanding of the genetic mechanisms by which fruit traits are independently established, possibly due to polyploidization events.

Sex chromosome evolution is thought to be tightly associated with the acquisition and maintenance of sexual dimorphisms. Plant sex chromosomes have evolved independently in many lineages, and can provide a powerful comparative framework to study this. We assembled and annotated genome sequences of three kiwifruit species (genus Actinidia) and uncovered recurrent sex chromosome turnovers in multiple lineages. Specifically, we observed structural evolution of the neo-Y chromosomes, which was driven via rapid bursts of transposable element insertions. Surprisingly, sexual dimorphisms were conserved in the different species studied, despite the fact that the partially sex-linked genes differ between them. Using gene-editing in kiwifruit, we demonstrated that one of the two Y chromosome-encoded sex determining genes, Shy Girl, shows pleiotropic effects that can explain the conserved sexual dimorphisms. These plant sex chromosomes therefore maintain sexual dimorphisms through the conservation of a single gene, without a process involving interactions between separate sex-determining genes and genes for sexually dimorphic traits.

Sex chromosomes have evolved independently in many plant lineages. They have often undergone rapid structural degeneration and extension of non-recombining regions, which is conventionally considered to be strongly associated with the expression of sexually dimorphic traits. In this study, we assembled a monoecious hexaploid persimmon (Diospyros kaki) in which the Y chromosome had lost its function in male determination. Comparative genomic analysis amongD. kakiand its dioecious relatives revealed that the non-functional Y chromosome (Ym) via silencing of the sex-determining gene,OGI, arose approximately two million years ago. Comparative analyses of the whole X and Ymchromosomes suggested that the non-functional male-specific region of the Y-chromosome (MSY), or post-MSY, retained certain conserved characteristics of the original functional MSY. Specifically, comparison of the functional MSY inD. lotusand the non-functional post-MSY inD. kakiindicated that the post-MSY had been rapidly rearranged mainly via ongoing transposable element bursts, as well as in the functional MSY. These results suggest a novel interpretation that the rapid evolution of the post-MSY (and possibly also MSYs in dioeciousDiospyrosspecies) might reflect the ancestral genomic properties of these regions, rather than the evolution of male-determining functions and/or sexually dimorphic traits.

Purpose Although the imaging of luminescence emitted in water during irradiation of protons and carbon ions is a useful method for range and dose estimations, the intensity of the images is relatively low due to the low photon production of the luminescence phenomenon. Therefore, a relatively long time is required for the imaging. Since a fluorescent dye, fluorescein, may increase the intensity of the optical signal, we measured the luminescence images of water with different concentrations of fluorescein during irradiation of protons and carbon ions and compared the results with those by measurements with water. Methods A cooled charge‐coupled device (CCD) camera was used for imaging a water phantom with different concentrations of fluorescein from 0.0063 to 0.025 mg/cm3, in addition to a water phantom without fluorescein during irradiation of 150‐MeV protons and 241.5‐MeV/n carbon ions. Results For both protons and carbon ions, the intensity of the luminescence images increased as the concentration of fluorescein increased. With a fluorescein concentration of 0.025 mg/cm3, the intensities increased to more than 10 times those of water for both protons and carbon ions. Although the shape of the depth profiles of luminescence images of water with fluorescein appeared similar to that of water for protons, those for carbon ions were different from those of water due to the increase in the Cherenkov light component at shallow depths by the decrease in the angular dependencies of the Cherenkov light. Conclusion We confirmed the increase in intensity of the luminescence of water by adding fluorescein for particle ions. With a small amount of Cherenkov light contamination in the images, such as protons, the relative distributions of the luminescence images with fluorescein were similar to that of water and will be used for range or dose determination in a short time.

This study aimed to evaluate the clinical beam commissioning results and lateral penumbra characteristics of our new pencil beam scanning (PBS) proton therapy using a multi-leaf collimator (MLC) calculated by use of a commercial Monte Carlo dose engine. Eighteen collimated uniform dose plans for cubic targets were optimized by the RayStation 9A treatment planning system (TPS), varying scan area, modulation widths, measurement depths, and collimator angles. To test the patient-specific measurements, we also created and verified five clinically realistic PBS plans with the MLC, such as the liver, prostate, base-of-skull, C-shape, and head-and-neck. The verification measurements consist of the depth dose (DD), lateral profile (LP), and absolute dose (AD). We compared the LPs and ADs between the calculation and measurements. For the cubic plans, the gamma index pass rates (gamma-passing) were on average 96.5% +/- 4.0% at 3%/3 mm for the DD and 95.2% +/- 7.6% at 2%/2 mm for the LP. In several LP measurements less than 75 mm depths, the gamma-passing deteriorated (increased the measured doses) by less than 90% with the scattering such as the MLC edge and range shifter. The deteriorated gamma-passing was satisfied by more than 90% at 2%/2 mm using uncollimated beams instead of collimated beams except for three planes. The AD differences and the lateral penumbra width (80%-20% distance) were within +/- 1.9% and +/- 1.1 mm, respectively. For the clinical plan measurements, the gamma-passing of LP at 2%/2 mm and the AD differences were 97.7% +/- 4.2% on average and within +/- 1.8%, respectively. The measurements were in good agreement with the calculations of both the cubic and clinical plans inserted in the MLC except for LPs less than 75 mm regions of some cubic and clinical plans. The calculation errors in collimated beams can be mitigated by substituting uncollimated beams.

Prompt X-ray imaging using a low-energy X-ray camera is a promising method for observing the beam shape from outside the subject. However, such imaging has so far been conducted only on static images with relatively long acquisition times without any energy information. Consequently, we performed list-mode prompt X-ray imaging using a newly developed data acquisition system combined with a pinhole YAP(Ce) camera during irradiation of a water phantom with carbon ions. Prompt X-ray imaging was conducted in list mode with a 1-ms time stamp and 128-channel energy bins during irradiation of a water phantom with 241.5 MeV/n carbon ions. After the imaging, list-mode data were sorted to obtain the time-sequential prompt X-ray images and those with different energies. From the images with different energies, we found the energy spectra were different depending on the areas in the images, and the reduction of the background fraction was possible. From the short time-sequential prompt X-ray images, we could even observe the differences in the images depending on the acquisition times, as well as the spill and ripple shapes of the carbon ion beam.

Purpose The luminescence images of water during the irradiation of carbon-ions provide useful information such as the ranges and the widths of carbon-ion beams. However, measured luminescence images show higher intensities in shallow depths and wider lateral profiles than those of the dose distributions. These differences prevent the luminescence imaging of water from being applied to a quality assurance for carbon-ion therapy. We assumed that the differences were due to the contaminations of Cerenkov-light from the secondary electrons of carbon-ions as well as the prompt gamma photons in the measured image. In this study, we applied a correction method to a luminescence image of water during the irradiation of carbon-ion beams. Methods We estimated the distribution of the Cerenkov-light in water during the irradiation of carbon-ions by Monte Carlo simulation and subtracted the simulated Cerenkov-light from the depth and lateral profiles of the measured luminescence image for 241.5 MeV/u-carbon-ions. Results With these corrections, we successfully obtained depth and lateral profiles whose distributions are almost identical to the dose distributions of carbon-ions. The high intensities in the shallow depth areas decreased and the Bragg peak intensity increased. The beam widths of the measured images approached those of the ionization chamber. Conclusions These results indicate that the luminescence imaging of water with our proposed correction has potential to be used for dose distribution measurements for carbon-ion therapy dosimetry.

Citrus peel degreening is promoted by low temperature via modulation of multiple genes associated with chlorophyll degradation, carotenoid biosynthesis, photosystem disassembly, phytohormones, and transcription factors without involving ethylene signalling., Peel degreening is an important aspect of fruit ripening in many citrus fruit, and previous studies have shown that it can be advanced by ethylene treatment or by low-temperature storage. However, the important regulators and pathways involved in natural peel degreening remain largely unknown. To determine how natural peel degreening is regulated in lemon fruit (Citrus limon), we studied transcriptome and physiochemical changes in the flavedo in response to ethylene treatment and low temperatures. Treatment with ethylene induced rapid peel degreening, which was strongly inhibited by the ethylene antagonist, 1-methylcyclopropene (1-MCP). Compared with 25 ºC, moderately low storage temperatures of 5–20 °C also triggered peel degreening. Surprisingly, repeated 1-MCP treatments failed to inhibit the peel degreening induced by low temperature. Transcriptome analysis revealed that low temperature and ethylene independently regulated genes associated with chlorophyll degradation, carotenoid metabolism, photosystem proteins, phytohormone biosynthesis and signalling, and transcription factors. Peel degreening of fruit on trees occurred in association with drops in ambient temperature, and it coincided with the differential expression of low temperature-regulated genes. In contrast, genes that were uniquely regulated by ethylene showed no significant expression changes during on-tree peel degreening. Based on these findings, we hypothesize that low temperature plays a prominent role in regulating natural peel degreening independently of ethylene in citrus fruit.

Imaging of the secondary electron bremsstrahlung (SEB) X-rays emitted during particle-ion irradiation is a promising method for beam range estimation. However, the SEB X-ray images are not directly correlated to the dose images. In addition, limited spatial resolution of the X-ray camera and low-count situation may impede correctly estimating the beam range and width in SEB X-ray images. To overcome these limitations of the SEB X-ray images measured by the X-ray camera, a deep learning (DL) approach was proposed in this work to predict the dose images for estimating the range and width of the carbon-ion beam on the measured SEB X-ray images. To prepare enough data for the DL training efficiently, 10,000 simulated SEB X-ray and dose image pairs were generated by our in-house developed model function for different carbon-ion beam energies and doses. The proposed DL neural network consists of two U-nets for SEB X-ray to dose image conversion and super-resolution. After the network being trained with these simulated X-ray and dose image pairs, the dose images were predicted from simulated and measured SEB X-ray testing images for performance evaluation. For the 500 simulated testing images, the average mean squared error (MSE) was 2.5 × 10^-5 and average structural similarity index (SSIM) was 0.997 while the error of both beam range and width was within 1 mm FWHM. For the three measured SEB X-ray images, the MSE was no worse than 5.5 × 10^-3 and SSIM was no worse than 0.980 while the error of the beam range and width was 2 mm and 5 mm FWHM, respectively. We have demonstrated the advantages of predicting dose images from not only simulated data but also measured data using our deep learning approach.

This study investigated variations in the relative biological effectiveness (RBE) values among various sarcoma and normal-tissue-derived cell lines (normal cell line) in proton beam and carbon-ion irradiations. We used a consistent protocol that specified the timing of irradiation after plating cells and detailed the colony formation assay. We examined the cell type dependence of RBE for proton beam and carbon-ion irradiations using four human sarcoma cell lines (MG63 osteosarcoma, HT1080 fibrosarcoma, SW872 liposarcoma, and SW1353 chondrosarcoma) and three normal cell lines (HDF human dermal fibroblast, hTERT-HME1 mammary gland, and NuLi-1 bronchus epithelium). The cells were irradiated with gamma rays, proton beams at the center of the spread-out Bragg peak, or carbon-ion beams at 54.4 keV/μm linear energy transfer. In all sarcoma and normal cell lines, the average RBE values in proton beam and carbon-ion irradiations were 1.08 ± 0.11 and 2.08 ± 0.36, which were consistent with the values of 1.1 and 2.13 used in current treatment planning systems, respectively. Up to 34% difference in the RBE of the proton beam was observed between MG63 and HT1080. Similarly, a 32% difference in the RBE of the carbon-ion beam was observed between SW872 and the other sarcoma cell lines. In proton beam irradiation, normal cell lines had less variation in RBE values (within 10%), whereas in carbon-ion irradiation, RBE values differed by up to 48% between hTERT-HME1 and NuLi-1. Our results suggest that specific dose evaluations for tumor and normal tissues are necessary for treatment planning in both proton and carbon-ion therapies.

In contrast to the progress in the research on physiological disorders relating to shelf life in fruit crops, it has been difficult to non-destructively predict their occurrence. Recent high-tech instruments have gradually enabled non-destructive predictions for various disorders in some crops, while there are still issues in terms of efficiency and costs. Here, we propose application of a deep neural network (or simply deep learning) to simple RGB images to predict a severe fruit disorder in persimmon, rapid over-softening. With 1,080 RGB images of ‘Soshu’ persimmon fruits, three convolutional neural networks (CNN) were examined to predict rapid over-softened fruits with a binary classification and the date to fruit softening. All of the examined CNN models worked successfully for binary classification of the rapid over-softened fruits and the controls with > 80% accuracy using multiple criteria. Furthermore, the prediction values (or confidence) in the binary classification were correlated to the date to fruit softening. Although the features for classification by deep learning have been thought to be in a black box by conventional standards, recent feature visualization methods (or “explainable” deep learning) has allowed identification of the relevant regions in the original images. We applied Grad-CAM, Guided backpropagation, and layer-wise relevance propagation (LRP), to find early symptoms for CNNs classification of rapid over-softened fruits. The focus on the relevant regions tended to be on color unevenness on the surface of the fruit, especially in the peripheral regions. These results suggest that deep learning frameworks could potentially provide new insights into early physiological symptoms of which researchers are unaware.

Although the luminescence images of water and an acrylic block during irradiation of proton and carbon ion beams were measured recently, they were not measured for helium ions. Because the mass of helium ions is four times that of protons and one-third that of carbon ions, different results from protons or carbon ions will be obtained by luminescence imaging. We measured the luminescence images of water and an acrylic block during irradiation of helium ion beams. Imaging was conducted using a cooled charge-coupled device (CCD) camera during the irradiation of helium ion pencil beams at three different energies to a water phantom and an acrylic block in addition to mini-beams of helium ions at four different energies. From the measured luminescence images during irradiation of helium ion pencil beams, we could estimate the ranges of the beams for water and the acrylic block. In the luminescence images of the acrylic block during irradiation of mini-beams of helium ions, we observed the luminescence of mini-beams with Bragg peaks in the images. Luminescence imaging of water and an acrylic block during the irradiation of helium ion beams was possible and could be used for range determination of the beams.

Although luminescence imaging of water during irradiation by particle ions is a promising method for dose estimation, it has only been tried for static images in which temporal information is not included. In addition to the positional distribution of the beam, temporal information is also important because the beams from a synchrotron-based therapy system have short pulse shapes called spills. The temporal information is also important for high dose rate, short-time radiotherapy, or so-called FLASH radiotherapy. To measure the particle ion beam distributions with precise temporal information, we conducted short time sequential luminescence imaging of protons. First, we measured short time sequential luminescence images during irradiation of a water phantom by 150-MeV protons using a cooled charge-coupled device (CCD) camera at 0.143-s intervals. With this imaging, the images showed beam distributions, but the shapes of the spill were not precisely evaluated in time intensity curves due to the insufficient sampling rate of the imaging. Then we measured short time sequential optical images with 0.053-s intervals. With this imaging, the images showed that the beam distributions in the spill shape could be measured, but the image and depth profiles evaluated from the images were noisy due to the insufficient light intensity. Consequently, we measured short time sequential luminescence images during irradiation of fluorescein (FS) water by 150-MeV protons. Since FS water produced ∼10 times higher luminescence, we could obtain high-intensity images enabling us to evaluate the time intensity curves based on the shape of the spills during measurement with 0.053-s intervals. The depth profiles of the beam were also obtained from the measured images. With these results, we confirmed that time sequential luminescence imaging was possible and, in such cases, FS water images measured at 0.053-s intervals are most promising to measure the short time sequential luminescence images during irradiation of protons.

Imaging of positrons induced by nuclear reactions with a proton beam is a possible method for observing the beam shape from outside the subject. However, such imaging of induced positrons has so far been conducted for solid materials. The induced positron distribution in liquid water has not been measured or reported. To clarify the distribution of induced positrons in liquid water, we conducted Cherenkov-light imaging after irradiation by protons to a water phantom. After irradiation by a 117-MeV proton beam to a phantom containing liquid water, Cherenkov-light imaging of the induced positrons was conducted using a cooled charge-coupled device (CCD) camera following the decay of the positrons. We also imaged the luminescence of water during irradiation by the proton beam to compare the distributions. We could measure the distribution of Cherenkov-light from the induced positrons in liquid water. Positron distributions kept their beam shapes in water but were different from that of the luminescence image; positron distribution was wider in the deep area of the beams in the lateral as well as depth direction. The distributions' shapes were only slightly changed with time. We conclude that Cherenkov-light imaging from the induced positrons after irradiation by a proton beam in water was possible, and we found that the induced positrons kept their beam shape in water with different shape from that of dose. These findings may provide new insights for imaging in particle therapy.

Prompt secondary electron bremsstrahlung X-ray (prompt X-ray) imaging using a low-energy X-ray camera is a promising method for observing a beam shape from outside the subject. However, the images measured within short times suffer from statistical noise. Consequently, we performed prompt X-ray imaging with higher sensitivity using a larger-diameter pinhole collimator and compared the results with those of a conventional collimator. Prompt X-ray imaging was conducted during irradiation with pencil beams of 241.5-MeV/n carbon ions to a water phantom. A newly developed X-ray camera with a 4-mm diameter as well as conventional 1.5-mm-diameter pinhole collimators was used for the imaging in list mode, and we compared the prompt X-ray images, energy spectra, and time count rate curves between 1.5-mm-diameter and 4-mm-diameter pinhole collimators. The prompt X-ray images taken with the 4-mm-diameter pinhole collimators had ∼7 times higher sensitivity with 70 % lower offset fractions originating from the prompt gamma photons. Furthermore, the ranges were more precisely estimated with the 4-mm collimator than with the 1.5-mm collimator. The energy spectra showed less contamination by tungsten-characteristic X-rays for the 4-mm pinhole collimator. Even for images measured with 0.1-s intervals, the beam shapes and time count rate curves could be obtained with less statistical noise using the 4-mm-diameter pinhole collimators. The use of the 4-mm-diameter pinhole collimator attached to the X-ray camera had advantages for prompt X-ray imaging with high sensitivity and low background, enabling us to image the beams even with short-time measurements.

In the evolutionary paths of plants, variations of the cis-regulatory elements (CREs) resulting in expression diversification have played a central role in driving the establishment of lineage-specific traits. However, it is difficult to predict expression behaviors from the CRE patterns to properly harness them, mainly because the biological processes are complex. In this study, we used cistrome datasets and explainable convolutional neural network (CNN) frameworks to predict genome-wide expression patterns in tomato fruits from the DNA sequences in gene regulatory regions. By fixing the effects of trans-elements using single cell-type spatiotemporal transcriptome data for the response variables, we developed a prediction model of a key expression pattern for the initiation of tomato fruit ripening. Feature visualization of the CNNs identified nucleotide residues critical to the objective expression pattern in each gene and their effects, were validated experimentally in ripening tomato fruits. This cis-decoding framework will not only contribute to understanding the regulatory networks derived from CREs and transcription factor interactions, but also provide a flexible way of designing alleles with optimized expression.

The shapes of plant organs reflect the evolution of each lineage and have been diversified according to lineage-specific adaptations to environment. Research on the molecular pathways responsible for organ shapes has traditionally been focused mainly on leaves or flowers. Thus, little is known about the pathways controlling fruit shapes, despite their diversity in some plant species. In this study, we analyzed oriental persimmon (Diospyros kaki), which exhibits considerable diversity in fruit shapes among cultivars, to elucidate the underlying molecular mechanism using transcriptomic data and quantitative evaluation. First, to filter the candidate genes associated with persimmon fruit shapes, the whole gene expression patterns obtained using mRNA-Seq analysis from 100 individuals, including a segregated population and various cultivars, were assessed to detect correlations with principal component scores for fruit shapes characterized with elliptic Fourier descriptors. Next, a gene co-expression network analysis with weighted gene co-expression network analysis (WGCNA) package revealed that class 1 KNOX family genes and SEEDSTICK function as integrators along with some phytohormone-related genes, to regulate the fruit shape diversity. On the other hand, the OVATE family genes also contribute to fruit shape diversity, of which pathway would be potentially shared with other plant species. Evolutionary aspects suggest that acquisition of a high lineage-specific and variable expression of class 1 KNOX gene, knotted-like homeobox of Arabidopsis thaliana 1 (KNAT1), in young fruit is important for establishing the persimmon-specific mechanism that determines fruit shape diversity.

Key message A novel locus, qCSS3, involved in the non-seed-shattering behaviour of Japonica rice cultivar, 'Nipponbare', was detected by QTL-seq analysis using the segregating population with the fixed known seed-shattering loci. Abstract Asian cultivated rice, Oryzasativa, was domesticated from its wild ancestor, O.rufipogon. Loss of seed shattering is one of the most recognisable traits selected during rice domestication. Three quantitative trait loci (QTLs), qSH1, qSH3, and sh4, were previously reported to be involved in the loss of seed shattering of Japonica cultivated rice, O.sativa 'Nipponbare'. However, the introgression line (IL) carrying 'Nipponbare' alleles at these three loci in the genetic background of wild rice, O.rufipogon W630, showed a lower value for detaching a grain from the pedicel than 'Nipponbare'. Here, we investigated abscission layer formation in the IL and found a partially formed abscission layer in the central region between the epidermis and vascular bundles. Based on QTL-seq analysis using the F-2 population obtained from a cross between 'Nipponbare' and the IL, we detected two novel loci qCSS3 and qCSS9 (QTL for the Control of Seed Shattering in rice on chromosomes 3 and 9), which were found to be involved in the difference in seed-shattering degree between 'Nipponbare' and W630. Then, we further focused on qCSS3 in order to understand its potential role on the loss of seed shattering. The candidate region of qCSS3 was found to be located within a 526-kb region using substitution mapping analysis. Interestingly, the qCSS3 candidate region partially overlaps the selective sweep detected for Japonica but not for Indica rice cultivars, suggesting that this region harbours the mutation at a novel seed-shattering locus specifically selected for non-seed-shattering behaviour in Japonica cultivars.

Interspecific hybridization is often hindered by incompatibility reactions occurring at various stages of hybridization, from early pollination to initial growth and reproductive stages of the progeny. Characterization of reproductive barriers leads to further understanding of how each species recognizes its own identity and supports the development of methods to overcome the barrier and produce interspecific hybrids. We investigated interspecific cross-(in)compatibility among five species in Prunus (Rosaceae), namely P. armeniaca, P. avium, P. mume, P. persica and P. salicina. In vivo pollen tube growth tests showed the presence of a post-mating pre-zygotic barrier in many cross combinations. Irrespective of the crossing direction, reduced pollen tube growth was observed in interspecific hybridization with P. avium, a species phylogenetically remote from the other species. Different compatibility reactions in reciprocal crosses were observed in some interspecific hybridizations. P. persica as a pollen donor was compatible with P. mume and P. salicina, while the reciprocal cross was incompatible. Furthermore, P. salicina as a pistil parent accepted pollen from P. armeniaca, P. mume and P. persica, while P. salicina was cross-incompatible with those species in reciprocal crosses. These results suggest that not only the phylogenetic relationship but also other mechanisms may be involved in interspecific cross-(in)compatibility. Embryo rescue culture showed the presence of a post-zygotic barrier in the cross between P. mume as seed parent and P. persica as pollen parent. Factors affecting interspecific hybridization are discussed with special reference to genetic distance between parents and morphological characters such as pistil length.

The imaging of the luminescence of water and Cherenkov-light with fluorescein during irradiation is a useful method to increase intensity; however, the magnitudes of the increase in intensity for Cherenkov-light and luminescence of water have been neither evaluated nor compared. Therefore, we measured the Cherenkov-light and luminescence of water with and without fluorescein during irradiation, and then we compared the magnitudes of the intensity increase with fluorescein. For 6 and 10 MV X-rays as well as 6 and 12 MeV electrons, the intensity of the Cherenkov-light images increased only 2 to 3 times with fluorescein water compared with that of water. Meanwhile, for 100 kV X-rays and 150 MeV protons, the intensity of the luminescence images increased more than 10 times with fluorescein water compared with that of water. The difference could probably be attributed to the difference in the light-emission mechanisms and coherency between Cherenkov-light and the luminescence of water.

Although the luminescence of water at lower energy than the Cerenkov-light threshold during carbon-ion irradiation was found and imaging was possible, the temporal response has not been measured, and so the difference from Cerenkov-light remains unclear. To clarify this point, we measured the temporal response of the luminescence of water at lower energy than the Cerenkov-light threshold and compared it with that of Cerenkov-light. We used silicon photomultiplier (Si-PM) modules to measure the temporal response at the Bragg peak area of a water phantom during irradiation of the carbon ion where the Cerenkov-light was not included. We also measured the temporal response at the shallow depth of the water phantom where the Cerenkov-light was included. In both areas, we measured the temporal waveforms of the light produced by the irradiation of the carbon ions in which the ripples of spills were clearly observed. We found no difference in the waveforms between the Bragg peak and the shallow depths of water. Our results do not contradict the hypothesis that the luminescence of water and Cerenkov-light are produced by the same mechanism.

Sex expression in plants is often flexible and contributes to the maintenance of genetic diversity within a species. In diploid persimmons (the genus Diospyros), the sexuality is controlled by the Y chromosome-encoded small-RNA gene, OGI, and its autosomal counterpart, MeGI. Hexaploid Oriental persimmon (Diospyros kaki) evolved more flexible sex expression, where genetically male individuals carrying OGI can produce both male and female flowers (monoecy). This is due to (semi-)inactivation of OGI by the Kali-SINE retrotransposon insertion on the promoter region and the resultant DNA methylations. Instead, flower sex determination in Oriental persimmon is also dependent on DNA methylation states of MeGI. Here, we focused on a cultivar, Kumemaru, which shows stable male flower production. Our results demonstrated that cv. Kumemaru carries OGI with Kali-SINE, which was highly methylated as well as in other monoecious cultivars; nevertheless, OGI gene could have a basal expression level. Transcriptomic analysis between cv. Kumemaru and 14 cultivars that predominantly produce female flowers showed differentially expressed genes (DEGs) specific to cv. Kumemaru, which is mainly involved in stress responses. Co-expression gene networks focusing on the DEGs also suggested the involvement of stress signals, mainly via gibberellin (GA), salicylic acid (SA), and especially jasmonic acid (JA) signal pathways. We also identified potential regulators of this co-expression module, represented by the TCP4 transcription factor. Furthermore, we attempted to identify cv. Kumemaru-specific transcript polymorphisms potentially contributing to derepressed OGI expression by cataloging subsequences (k-mers) in the transcriptomic reads from cv. Kumemaru and the other 14 female cultivars. Overall, although the direct genetic factor to activate OGI remains to be solved, our results implied the involvement of stress signals in the release of silenced OGI and the resultant continuous male production.

Kiwifruit (Actinidia chinensis) is a dioecious, long-living woody perennial vine. Reduced generation time and induction of hermaphroditism can accelerate crop improvement and facilitate alternative farming for better food security in the face of climate change. Previous studies identified that CENTRORADIALIS genes CEN and CEN4 act to repress flowering, whilst the male-specific Shy Girl (SyGl) gene with homology to type-C cytokinin response regulators could repress gynoecium development in model plants. Here we use CRISPR/Cas9 to mutagenize CEN, CEN4 and SyGl in the male kiwifruit A. chinensis 'Bruce'. Biallelic mutations of CEN and CEN4 generated rapid-flowering male plants, and simultaneous targeting of CEN4 and SyGl gave rise to rapid-flowering hermaphrodites with restored gynoecial function and viable pollen, providing functional evidence for the role of SyGl in suppression of feminization. Analysis of ovary tissues identified genes that contribute to carpel development and revealed that SyGl affected both cytokinin profiles and the expression of genes involved in cytokinin metabolism and signalling. The plant lines generated by CEN4/SyGl knockout could self-pollinate and produce fast-flowering offspring. These results establish that SyGI acts as the suppressor of feminization in kiwifruit and demonstrate the potential for accelerated breeding in an outcrossing horticultural woody perennial.