Molecular Mechanism of the Self-incompatibility in the Rosaceous Species

Self-incompatibility(GSI) in the rosaceous species is controlled by the S locus consisting of S-RNase gene and an unidentified 'pollen S' gene. A～200kbp of cosmid contig for the Sc haplotype of almond was constructed. Genomic Southern blot analyses showed that most cosmid end probes, except those near the Sc-RNase gene, cross-hybridized with DNA fragments from different S haplotypes, implying that the cosmid contig extends to the borders of the S locus. A～70kbp segment of the Sc haplotype, the S haplotype-specific region containing the S-RNase gene, was complentely sequenced. This regin was found to contain two pollen-expressed F-box genes that are likely candidates for pollen S genes. One of them, named SFB(S haplotype-specific F-Box protein), was specifically expressed in pollen, and showed high level of S haplotype-specific sequence polymorphism, comparable to that of the S-RNases. The other is unlikely to determine the S specificity of pollen, because it showed little allelic sequence polymorphism and was expressed also in pistil. Three other S haplotypes were cloned and the pollen-expressed genes were physically mapped. In all four cases, SFBs were physically linked to the S-RNase genes, and were located at the S haplotype-specific region, suggesting that the two genes are inherited as aunit. These features are consistent with the hypothrsis that SFB is the pollen S gene. This hypothesis predicts involvement of the ubiquitin/26S proteasome proteolytic pathway in the RNase-based SI system

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 Y-monoecy) was derived, which involved silencing of the sex-determining gene, OGI, approximately 2 million years ago. Analyses of the entire X and Y-monoecy 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

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

Examining the Role of Low Temperature in Satsuma Mandarin Fruit Peel Degreening via Comparative Physiological and Transcriptomic Analysis

Peel degreening is the most conspicuous aspect of fruit ripening in many citrus fruits because of its importance for marketability. In this study, peel degreening in response to propylene (an ethylene analog) and at varying storage temperatures was characterized in Satsuma mandarin (Citrus unshiu Marc.) fruit. Propylene treatment triggered rapid peel degreening (within 4-6 days), indicated by an increase in the citrus color index (CCI) and chlorophyll loss. Peel degreening was also observed in fruit at 10 degrees C and 15 degrees C after 28-42 days, with gradual CCI increase and chlorophyll reduction. However, fruit at 5 degrees C, 20 degrees C, and 25 degrees C remained green, and no substantial changes in peel CCI and chlorophyll content were recorded during the 42-day storage duration. The transcriptomes of peels of fruit treated with propylene for 4 days and those stored at varying temperatures for 28 days were then analyzed by RNA-Seq. We identified three categories of differentially expressed genes that were regulated by (i) propylene (and by analogy, ethylene) alone, (ii) low temperature (5 degrees C, 10 degrees C, or 15 degrees C vs. 25 degrees C) alone, and (iii) either propylene or low temperature. Gene-encoding proteins associated with chlorophyll degradation (such as CuSGR1, CuNOL, CuACD2, CuCAB2, and CuLHCB2) and a transcription factor (CuERF114) were differentially expressed by propylene or low temperature. To further examine temperature-induced pathways, we also monitored gene expression during on-tree fruit maturation vs. postharvest. The onset of on-tree peel degreening coincided with autumnal drops in field temperatures, and it was accompanied by differential expression of low temperature-regulated genes. On the contrary, genes that were exclusively regulated by propylene (such as CuCOPT1 and CuPOX-A2) displayed insignificant expression changes during on-tree peel degreening. These findings indicate that low temperatures could be involved in the fruit ripening-related peel degreening independently of ethylene

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

Deep Learning Predicts Rapid Over-softening and Shelf Life in Persimmon Fruits

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

MIG-seq is an effective method for high-throughput genotyping in wheat (Triticum spp.)

MIG-seq (Multiplexed inter-simple sequence repeats genotyping by sequencing) has been developed as a low cost genotyping technology, although the number of polymorphisms obtained is assumed to be minimal, resulting in the low application of this technique to analyses of agricultural plants. We applied MIG-seq to 12 plant species that include various crops and investigated the relationship between genome size and the number of bases that can be stably sequenced. The genome size and the number of loci, which can be sequenced by MIG-seq, are positively correlated. This is due to the linkage between genome size and the number of simple sequence repeats (SSRs) through the genome. The applicability of MIG-seq to population structure analysis, linkage mapping, and quantitative trait loci (QTL) analysis in wheat, which has a relatively large genome, was further evaluated. The results of population structure analysis for tetraploid wheat showed the differences among collection sites and subspecies, which agreed with previous findings. Additionally, in wheat biparental mapping populations, over 3, 000 SNPs/indels with low deficiency were detected using MIG-seq, and the QTL analysis was able to detect recognized flowering-related genes. These results revealed the effectiveness of MIG-seq for genomic analysis of agricultural plants with large genomes, including wheat

Postharvest Properties of Ultra-Late Maturing Peach Cultivars and Their Attributions to Melting Flesh (M) Locus: Re-evaluation of M Locus in Association With Flesh Texture

The postharvest properties of two ultra-late maturing peach cultivars, "Tobihaku" (TH) and "Daijumitsuto" (DJ), were investigated. Fruit were harvested at commercial maturity and held at 25 degrees C. TH exhibited the characteristics of normal melting flesh (MF) peach, including rapid fruit softening associated with appropriate level of endogenous ethylene production In contrast, DJ did not soften at all during 3 weeks experimental period even though considerable ethylene production was observed. Fruit of TH and DJ were treated with 5,000 ppm of propylene, an ethylene analog, continuously for 7 days. TH softened rapidly whereas DJ maintained high flesh firmness in spite of an increase in endogenous ethylene production, suggesting that DJ but not TH lacked the ability to be softened in response to endogenous and exogenous ethylene/propylene. DNA-seq analysis showed that tandem endo-polygalacturonase (endoPG) genes located at melting flesh (M) locus, Pp-endoPGM (PGM), and Pp-endoPGF (PGF), were deleted in DJ. The endoPG genes at M locus are known to control flesh texture of peach fruit, and it was suggested that the non-softening property of DJ is due to the lack of endoPG genes. On the other hand, TH possessed an unidentified M haplotype that is involved in determination of MF phenotype. Structural identification of the unknown M haplotype, designated as M-0, through comparison with previously reported M haplotypes revealed distinct differences between PGM on M-0 haplotype (PGM-M-0) and PGM on other haplotypes (PGM-M-1). Peach M haplotypes were classified into four main haplotypes: M-0 with PGM-M-0; M-1 with both PGM-M-1 and PGF; M-2 with PGM-M-1; and M-3 lacking both PGM and PGF. Re-evaluation of M locus in association with MF/non-melting flesh (NMF) phenotypes in more than 400 accessions by using whole genome shotgun sequencing data on database and/or by PCR genotyping demonstrated that M-0 haplotype was the common haplotype in MF accessions, and M-0 and M-1 haplotypes were dominant over M-2 and M-3 haplotypes and co-dominantly determined the MF trait. It was also assumed on the basis of structural comparison of M haplotypes among Prunus species that the ancestral haplotype of M-0 diverged from those of the other haplotypes before the speciation of Prunus persica

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 F-1 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 degrees C, moderately low storage temperatures of 5-20 degrees 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