Ongoing Rapid Evolution of a Post-Y Region Revealed by Chromosome-Scale Genome Assembly of a Hexaploid Monoecious Persimmon (Diospyros kaki).

Plants have evolved sex chromosomes independently in many lineages, and loss of separate sexes can also occur. In this study, we assembled a monoecious recently hexaploidized persimmon (Diospyros kaki), in which the Y chromosome has lost the maleness-determining function. Comparative genomic analysis of D. kaki and its dioecious relatives uncovered the evolutionary process by which the nonfunctional Y chromosome (or Ymonoecy) was derived, which involved silencing of the sex-determining gene, OGI, approximately 2 million years ago. Analyses of the entire X and Ymonoecy chromosomes suggested that D. kaki's nonfunctional male-specific region of the Y chromosome (MSY), which we call a post-MSY, has conserved some characteristics of the original functional MSY. Specifically, comparing the functional MSY in Diospyros lotus and the nonfunctional "post-MSY" in D. kaki indicated that both have been rapidly rearranged, mainly via ongoing transposable element bursts, resembling structural changes often detected in Y-linked regions, some of which can enlarge the nonrecombining regions. The recent evolution of the post-MSY (and possibly also MSYs in dioecious Diospyros species) therefore probably reflects these regions' ancestral location in a pericentromeric region, rather than the presence of male-determining genes and/or genes controlling sexually dimorphic traits.

Evolution of sex in crops: recurrent scrap and rebuild.

Sexuality is the main strategy for maintaining genetic diversity within a species. In flowering plants (angiosperms), sexuality is derived from ancestral hermaphroditism and multiple sexualities can be expressed in an individual. The mechanisms conferring chromosomal sex determination in plants (or dioecy) have been studied for over a century by both biologists and agricultural scientists, given the importance of this field for crop cultivation and breeding. Despite extensive research, the sex determining gene(s) in plants had not been identified until recently. In this review, we dissect plant sex evolution and determining systems, with a focus on crop species. We introduced classic studies with theoretical, genetic, and cytogenic approaches, as well as more recent research using advanced molecular and genomic techniques. Plants have undergone very frequent transitions into, and out of, dioecy. Although only a few sex determinants have been identified in plants, an integrative viewpoint on their evolutionary trends suggests that recurrent neofunctionalization events are potentially common, in a "scrap and (re)build" cycle. We also discuss the potential association between crop domestication and transitions in sexual systems. We focus on the contribution of duplication events, which are particularly frequent in plant taxa, as a trigger for the creation of new sexual systems.

Genetic basis of lineage-specific evolution of fruit traits in hexaploid persimmon.

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 in applying genetic approaches. Hexaploid Oriental persimmon (Diospyros kaki) has evolved specific fruit characteristics, 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 the genetic mechanisms by which fruit traits are independently established, possibly due to polyploidization events.

Transcriptomic Interpretation on Explainable AI-Guided Intuition Uncovers Premonitory Reactions of Disordering Fate in Persimmon Fruit.

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 DNNs is still mostly limited to rapid and effective phenotyping. The recent development of explainable CNN frameworks has allowed visualization of the features in the prediction by a 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 Gradient-weighted Class Activation Mapping (Grad-Class Activation Mapping (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 the 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 the 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.

Recurrent neo-sex chromosome evolution in kiwifruit.

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 lineages1,2 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.

Polyploidy before and after domestication of crop species.

Recent advances in the genomics of polyploid species answer some of the long-standing questions about the role of polyploidy in crop species. Here, we summarize the current literature to reexamine scenarios in which polyploidy played a role both before and after domestication. The prevalence of polyploidy can help to explain environmental robustness in agroecosystems. This review also clarifies the molecular basis of some agriculturally advantageous traits of polyploid crops, including yield increments in polyploid cotton via subfunctionalization, modification of a separated sexuality to selfing in polyploid persimmon via neofunctionalization, and transition to a selfing system via nonfunctionalization combined with epistatic interaction between duplicated S-loci. The rapid progress in genomics and genetics is discussed along with how this will facilitate functional studies of understudied polyploid crop species.

Genome-wide cis-decoding for expression design in tomato using cistrome data and explainable deep learning.

In the evolutionary history of plants, variation in cis-regulatory elements (CREs) resulting in diversification of gene expression has played a central role in driving the evolution of lineage-specific traits. However, it is difficult to predict expression behaviors from 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 (Solanum lycopersicum) fruit from the DNA sequences in gene regulatory regions. By fixing the effects of trans-acting factors using single cell-type spatiotemporal transcriptome data for the response variables, we developed a prediction model for crucial expression patterns in 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 fruit. This cis-decoding framework will not only contribute to the understanding of the regulatory networks derived from CREs and transcription factor interactions, but also provides a flexible means of designing alleles for optimized expression.

Reinvention of hermaphroditism via activation of a RADIALIS-like gene in hexaploid persimmon.

In flowering plants, different lineages have independently transitioned from the ancestral hermaphroditic state into and out of various sexual systems1. Polyploidizations are often associated with this plasticity in sexual systems2,3. Persimmons (the genus Diospyros) have evolved dioecy via lineage-specific palaeoploidizations. More recently, hexaploid D. kaki has established monoecy and also exhibits reversions from male to hermaphrodite flowers in response to natural environmental signals (natural hermaphroditism, NH), or to artificial cytokinin treatment (artificial hermaphroditism, AH). We sought to identify the molecular pathways underlying these polyploid-specific reversions to hermaphroditism. Co-expression network analyses identified regulatory pathways specific to NH or AH transitions. Surprisingly, the two pathways appeared to be antagonistic, with abscisic acid and cytokinin signalling for NH and AH, respectively. Among the genes common to both pathways leading to hermaphroditic flowers, we identified a small-Myb RADIALIS-like gene, named DkRAD, which is specifically activated in hexaploid D. kaki. Consistently, ectopic overexpression of DkRAD in two model plants resulted in hypergrowth of the gynoecium. These results suggest that production of hermaphrodite flowers via polyploidization depends on DkRAD activation, which is not associated with a loss-of-function within the existing sex determination pathway, but rather represents a new path to (or reinvention of) hermaphroditism.

Explainable Deep Learning Reproduces a 'Professional Eye' on the Diagnosis of Internal Disorders in Persimmon Fruit.

Recent rapid progress in deep neural network techniques has allowed recognition and classification of various objects, often exceeding the performance of the human eye. In plant biology and crop sciences, some deep neural network frameworks have been applied mainly for effective and rapid phenotyping. In this study, beyond simple optimizations of phenotyping, we propose an application of deep neural networks to make an image-based internal disorder diagnosis that is hard even for experts, and to visualize the reasons behind each diagnosis to provide biological interpretations. Here, we exemplified classification of calyx-end cracking in persimmon fruit by using five convolutional neural network models with various layer structures and examined potential analytical options involved in the diagnostic qualities. With 3,173 visible RGB images from the fruit apex side, the neural networks successfully made the binary classification of each degree of disorder, with up to 90% accuracy. Furthermore, feature visualizations, such as Grad-CAM and LRP, visualize the regions of the image that contribute to the diagnosis. They suggest that specific patterns of color unevenness, such as in the fruit peripheral area, can be indexes of calyx-end cracking. These results not only provided novel insights into indexes of fruit internal disorders but also proposed the potential applicability of deep neural networks in plant biology.

Genome-wide study on the polysomic genetic factors conferring plasticity of flower sexuality in hexaploid persimmon.

Sexuality is one of the fundamental mechanisms that work towards maintaining genetic diversity within a species. In diploid persimmons (Diospyros spp.), separated sexuality, the presence of separate male and female individuals (dioecy), is controlled by the Y chromosome-encoded small-RNA gene, OGI. On the other hand, sexuality in hexaploid Oriental persimmon (Diospyros kaki) is more plastic, with OGI-bearing genetically male individuals, able to produce both male and female flowers (monoecy). This is thought to be linked to the partial inactivation of OGI by a retrotransposon insertion, resulting in DNA methylation of the OGI promoter region. To identify the genetic factors regulating branch sexual conversion, genome-wide correlation/association analyses were conducted using ddRAD-Seq data from an F1 segregating population, and using both quantitative and diploidized genotypes, respectively. We found that allelic ratio at the Y-chromosomal region, including OGI, was correlated with male conversion based on quantitative genotypes, suggesting that OGI can be activated in cis in a dosage-dependent manner. Genome-wide association analysis based on diploidized genotypes, normalized for the effect of OGI allele dosage, detected three fundamental loci associated with male conversion. These loci underlie candidate genes, which could potentially act epigenetically for the activation of OGI expression.

Low temperature modulates natural peel degreening in lemon fruit independently of endogenous ethylene.

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.

Molecular Mechanism Underlying Derepressed Male Production in Hexaploid Persimmon.

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.

Epigenetic Flexibility Underlies Somaclonal Sex Conversions in Hexaploid Persimmon.

Epigenetic regulation adds a flexible layer to genetic variations, potentially enabling long-term, but reversible, changes to a trait, while maintaining genetic information. In the hexaploid Oriental persimmon (Diospyros kaki), genetically monoecious cultivars bearing male flowers require the Y-encoded small RNA (smRNA) gene, OGI. This gene represses the expression of its autosomal counterpart gene, MeGI, as part of the canonical male production system. However, a D. kaki cultivar, Saijo, which lacks the OGI gene and originally bears only female flowers, occasionally produces somaclonal mutant male and revertant female (RF) branches. In this study, we investigated the mechanisms underlying these somaclonal sex conversions in persimmon. Specifically, we aimed to unravel how a genetically female tree without the OGI gene can produce male flowers and RF flowers. Applying multi-omics approaches, we revealed that this noncanonical male production system is basically consistent with the canonical system, in which the accumulation of smRNA targeting MeGI and the considerable DNA methylation of MeGI are involved. The epigenetic status of MeGI on CGN and CHG was synchronized to the genome-wide methylation patterns, both in transition to and from the male production system. These results suggest that the somaclonal sex conversions in persimmon are driven by the genome-wide epigenetic regulatory activities. Moreover, flexibility in the epigenetic layers of long-lived plant species (e.g. trees) is important for overcoming genetic robustness.