Apple scab, caused by Venturia inaequalis, is a major disease in Malus & times; domestica. Although resistance genes such as Rvi6 have been introduced through breeding, the emergence of virulent pathogen races has raised concerns over the durability of single-gene resistance. Nonhost resistance (NHR), a broad-spectrum and durable form of immunity found in nonhost species, offers a promising alternative. This study assessed resistance to V. inaequalis in three Pyrus species and three intergeneric hybrids with M. & times; domestica. Following inoculation, macroscopic and microscopic evaluations revealed that susceptible Malus cultivars showed extensive lesion development and hyphal elongation. In contrast, no typical scab symptoms were observed in Pyrus, and hyphal growth was inhibited after initial conidial germination, indicating the presence of NHR in Pyrus species, including both European and Asian pears, against V. inaequalis. The intergeneric hybrids exhibited resistance phenotypes consistent with Pyrus-derived NHR. Our findings demonstrated that NHR can be inherited by Malus-Pyrus hybrids, suggesting that Pyrus could serve as a genetic source of novel, durable resistance for scab-resistant apple breeding.

Abstract Wide hybridization between related species and genera provides valuable opportunities for broadening genetic diversity and introducing desirable traits. In the tribe Maleae (Rosaceae), Malus (apple) and Pyrus (pear) are phylogenetically closely related, and apple × pear hybrids represent promising materials such as for disease-resistance breeding. However, the effective utilization of such hybrids in breeding programs is constrained by long juvenile period. In this study, we established a tissue culture-based regeneration and genetic transformation platform for apple x pear hybrids. Key stages affecting adventitious shoot regeneration were optimized, and appropriate ranges of antibiotic selection pressure and bacterial elimination conditions were systematically evaluated. Regeneration capacity was predominantly genotype-dependent and became further restricted under Agrobacterium infection, necessitating precise balancing between regeneration competence and selection pressure. Using the highly competence line and the established transformation system, MdFT1 gene was successfully introduced and over-expressed in intergeneric hybrids, resulting in transgenic plants exhibiting floral bud initiation approximately six months after infection under in vitro conditions. This study provides a practical and efficient regeneration–transformation framework for apple × pear hybrids and demonstrates its applicability for FT- mediated early flowering. The established system offers technical support for accelerated breeding strategies and facilitates the utilization of novel resources in genetic improvement of pome fruit.

Floral morphological traits, such as double-flowering, are important aspects of the commercial value of ornamental plants. Commercially available double-flower Madagascar periwinkle (Catharanthus roseus), cv. ’Sunny Princess’, exhibits a unique morphology in which normal floral organs, including pistils and stamens, are retained, while additional petals develops at the base of the corolla. The aim of this study was to elucidate the molecular basis of double-flowering in C. roseus. Quantitative trait locus (QTL) analysis of F₂ populations derived from crosses between single- and double-flower plants revealed that the PHANTASTICA-like (PHAL) gene is associated with the trait. Two PHAN homologs were identified in C. roseus (CrPHAN and PHAN-like (PHAL)). Double-flower plants consistently harbored a frameshift mutation immediately downstream of the PHAL initiation codon, likely resulting in a loss of function. Additionally, a nonsense mutation in PHAL was identified in double-flower cultivars with different genetic backgrounds, as further evidence of the role of this gene. These findings demonstrate that the loss of PHAL function is associated with double flowering in C. roseus. The results provide new insights into the genetic regulation of floral morphology in ornamental cultivars.

Fruit growth has long been described using single- or double-sigmoid curves; however, these temporal models cannot fully capture the spatial heterogeneity that ultimately shapes a fruit. Here, we present a three-dimensional analysis pipeline that non-destructively tracks spatial fruit growth dynamics from field-collected imagery. Surface landmarks were drawn, and video recordings were taken throughout development for three pome fruits, apple (Malus × domestica), Japanese pear (Pyrus pyrifolia) and European pear (Pyrus communis), and two drupe fruits, peach (Prunus persica) and Japanese apricot (Prunus mume), to track their motion. Using 3D Gaussian splatting, we successfully reconstructed 3D models of the fruits, and the landmark displacement could be measured with high accuracy, with R2 ≥ 0.98 when compared to manual recordings. We found a common spatial growth gradient in the longitudinal growth shared in the pomes and drupes of the Rosaceae; proximal (stem-end) regions exhibited more pronounced growth than the distal (stylar) end. An exception was found in European pear ‘Bartlett,’ which showed relatively vigorous growth in the distal region, explaining its distinct shape with expanded distal end. Transverse expansion varied far less than longitudinal expansion, with a possible association with initial fruit morphology. Inter-fruit growth variability peaked in the fastest-growing regions, particularly in the distal area of the European pear, highlighting the link between growth vigor and phenotypic variance. These results provide foundational insights into the developmental dynamics of both pome and drupe fruits of the Rosaceae family, contributing to the optimization of fruit size, shape, and uniformity.

The objective of this study was to investigate how fruit texture and cell wall composition change during the ripening stage of ‘Shine Muscat’. The physical properties of the berries, including adhesive strength between flesh and skin, were measured at different stages of maturity. The cell wall components were analyzed by dividing the berries into three parts: central berry flesh, flesh around skin, and skin. The adhesive strength between the flesh and skin increased from 1.2 N at 28 days after the veraison to 1.6 N at 49 days after the veraison. In addition, at 56 days after veraison, the second fracture strain rate was <100 %, indicating that the texture was suitable for eating without peeling the skin.With respect to the central berry flesh content of cell wall components changed slightly with maturation. However, in the flesh around the skin, both pectin and hemicellulose showed an increasing trend with maturity, and their content increased significantly 49 days after veraison, when the adhesive strength between the skin and flesh increased. In pectin, arabinose was found to be the most prominent monosaccharide. Contents of cell wall components in the flesh varied by site of berry. Specifically, changes in the adhesion of skin and flesh at different maturity can be attributed to changes in the pectin and hemicellulose structures in the flesh around the skin. As the fruit ripened, changes were observed in the composition of the cell walls in the flesh around the skin, suggesting that these changes may have affected the texture of the berry.

Volatile compounds are important components for determining consumer preference for fruits and their market value, as well as fruit quality. Generally, volatile esters and lactones contribute to characteristic fruity aroma attributes, and their biosynthesis is catalyzed by alcohol acyltransferase (AAT) in various fruits. In this study, we investigated the transcriptomes of the fruit of Prunus mume cultivars to identify the key genes associated with differences in volatile ester and γ-decalactone contents. Based on the results of co-expression network analysis, AAT1 was classified into the module showing highly positive correlations with the content of volatile compounds. Among the AAT1 homologs, only AAT1 expression was correlated with the volatile content in P. mume cultivars and was remarkably higher in mature fruit. These results indicated that AAT1 plays a central role in the production of volatile esters and γ-decalactone in P. mume fruit. Our phylogenetic and transcriptome analyses also showed evidence of gene duplications of the AAT1 homologs in the ancestral rosaceous genome and of transcription factors that may be involved in volatile compound production. The identification of key genes for aroma development in P. mume fruit provides a basis for breeding programs focusing on flavor improvement.

Floral morphology is a crucial trait that determines the commercial value of ornamental plants and pollinator preference. Scaevola aemula (Fan Flower) typically develops bilaterally symmetrical flowers, but a recessive mutant with a radially symmetrical flower (peloric) has been isolated from a breeding population. In the present study, we explored the molecular basis of the floral traits of S. aemula. We found a genetic-phenotypic association for the CYCLOIDEA (CYC) gene SaCYC2, which is known as a regulatory gene for floral symmetry in various plants. The peloric flower line had a nonsense mutation immediately downstream of the start codon of SaCYC2 causing a loss of function. RNA-sequencing analysis revealed three paralogous CYC genes in S. aemula, but only SaCYC2 was preferentially expressed in the dorsal side of the petals. Moreover, we developed a co-dominant cleaved amplified polymorphic sequence (CAPS) marker based on the nonsense mutation to verify floral traits, thereby accelerating the selection and breeding of Scaevola spp. Our findings support the conserved function of CYC genes that determine aspects of floral morphology in Asteraceae.

Wide hybridizations across species and genera have been employed to enhance agriculturally important traits in crops. Within the tribe Maleae of the Rosaceae family, different genera and species exhibit several traits useful for increasing diversity and gene pool through hybridization. This study aimed to develop and characterize intergeneric hybrid individuals between Malus and Pyrus. Through seed germination, shoot multiplication, and rooting in vitro, acclimatized seedlings showing vegetative growth on their own roots were obtained from crosses of Malus × domestica pollinated by Pyrus communis, P. bretschneideri, and the Pyrus interspecific hybrid (P. communis × P. pyrifolia). Comparative analysis of leaf morphology, flow cytometry, and molecular genotyping confirmed the hybrid status of the individuals. Genome-wide genotyping revealed that all the hybrid individuals inherited genomic fragments symmetrically from the Malus and Pyrus parents. To the best of our knowledge, this is the first report on the development of intergeneric hybrid seedlings between Malus × domestica and P. bretschneideri. Furthermore, the Pyrus interspecific hybrid individual served as a bridge plant for introducing the genetic background of P. pyrifolia into Malus × domestica. The results of this study provided a crucial foundation for breeding through intergeneric hybridization between Malus and Pyrus, facilitating the incorporation of valuable traits from diverse gene pools.

In the long history of human relations with flowering cherry trees in Japan, 'Somei-Yoshino' occupies an exceptional position among a variety of flowering trees: it is a self-incompatible interspecific hybrid but has been enthusiastically planted by grafting throughout Japan, due most likely to its flamboyant appearance upon full bloom. Thus, 'Somei-Yoshino' gives us a rare opportunity to trace and investigate the occurrence and distribution of somatic mutations within a single plant species through analysis of the genomes of the clonally propagated trees grown under a variety of geographical and artificial environments. In the studies presented here, a total of 46 samples of 'Somei-Yoshino' trees were collected and their genomes were analysed. We identified 684 single nucleotide mutations, of which 71 were present in more than two samples. Clustering analysis of the mutations indicated that the 46 samples were classified into eight groups, four of which included 36 of the 46 samples analysed. Interestingly, all the four tree samples collected in Ueno Park of Tokyo were members of the four groups mentioned above. Based on comparative analysis of their mutations, one of the four trees growing in Ueno Park was concluded to be the closest to the original ancestor. We propose that somatic mutations may be used as tracers to establish the ancestral relationship amongst clonally propagated individuals.

Hybridization across species and genus has been utilized to improve agriculturally important traits in crops. These interspecific and intergeneric hybridizations are often inhibited by reproductive barriers at the prezygotic and postzygotic stages. In this study, we used intergeneric hybridization between apple and pear to investigate the effects of species and varietal differences on pollen tube growth, seed germination, and seedling survival in a wide range of germplasms. In apple pistils pollinated by pears, inferior pollen tube growth was often observed for European pear cultivars, whereas hybrid seedlings derived from a cross between apple and European pear showed higher survival rate. In contrast, Japanese pear and Chinese pear pollination resulted in higher fruit setting which likely resulted from a higher rate of compatible pistils but a lower rate of seedling survival. Our results suggest the presence of both prezygotic and postzygotic barriers that occur independently in different crossing combinations. We also developed a Cleaved Amplified Polymorphic Sequence marker for detecting intergeneric hybrids derived from a wide range of crosses between apples and pears. The results of the present study will help in the development of intergeneric hybrids between apple and pear for introducing valuable traits from a diverse gene pool.

Muscadine grape (Vitis rotundifolia Michx.) is native to southeastern USA. Although it has not been commercially cultivated in Japan, the unique taste of its berries makes muscadine grape potentially useful for expanding the table grape market in Japan. To satisfy consumer demands, there has been an increase in the production of seedless Euvitis table grape (V. vinifera, V. labrusca, and V. labruscana) berries in Japan. Moreover, seedless grape berries are generally produced by treating normally seeded cultivars ( e.g., 'Delaware') with gibberellic acid (GA) and streptomycin (SM). Additionally, the synthetic cytokinin forchlorfenuron [N-(2-chloro-4-pyridyl)-N '-phenylurea; CPPU] is often applied along with GA or SM to enhance berry set and the growth of seedless berries. Although these chemicals have been used to produce seedless Euvitis grape berries, their effects on muscadine grapes remain relatively unknown. The objective of this study was to clarify the effects of these chemicals on muscadine grape seed formation and development. We used three hermaphrodite cultivars ('Carlos', 'Bountiful', and 'Doreen') in this study. In contrast to the well-known effects of GA on Euvitis seedless berry set, the GA treatments at 0 and 2 weeks before anthesis did not affect seed formation and development. However, the SM treatment significantly decreased the seed weight, whereas the CPPU treatment increased the berry size. We also observed that the SM treatment at 1 week before anthesis was more effective than the SM treatment at 2 weeks before anthesis for producing berries with small seeds. Histological examinations of the female reproductive organs suggested that the SM treatment may have inhibited endosperm development, leading to the degeneration of the endosperm and embryo and the production of berries with shriveled seeds.

The development of intergeneric hybrids for horticultural crops has been attempted to introduce new quality and resistance traits and to enlarge the gene pool. Interspecific and intergeneric hybridization are often hindered by incompatibility reactions occurring at various stages of hybridization, from early pollination to initial growth, and the reproductive stages of the progeny. In this study, we investigated intergeneric and interspecific cross-compatibility among six species in the tribe Maleae (Rosaceae), namely, Pyrus communis (European pear), P. pyrifolia (Japanese pear), Malus × domestica (apple), Eriobotrya japonica (loquat), Cydonia oblonga (quince), and Pseudocydonia sinensis (Chinese quince). In vivo pollen tube growth tests showed the presence of a postmating, prezygotic barrier in many cross-combinations, in which cross-compatibility was regulated by both genetic distance and crossing direction. Strong hybridization barriers were observed in pollen tube growth and fruit setting when intergeneric hybridization was performed with E. japonica, a species phylogenetically distant from the others studied. Different compatibility reactions in reciprocal crosses were observed in some intergeneric hybridizations; C. oblonga as a pollen donor was incompatible with P. sinensis, whereas the reciprocal cross was compatible, resulting in the development of hybrid seedlings. Furthermore, the pollen tube growth rate differed among Pyrus species when pollinated on the apple pistils, suggesting divergence of cross-compatibility response in a specific linage. Factors affecting intergeneric hybridization are discussed with reference to the genetic distance between species and morphological characteristics such as pistil length. Our comprehensive assessment of intergeneric cross-compatibility will help provide a way to overcome crossing barriers and develop new hybrid crops in the tribe Maleae.

For the first time, stone cells in pear and apple pedicel were studied. The lignification of the pedicel outer part was correlated with flesh, and the secondary cell wall biosynthesis genes were activated. Fruit pedicels act as bridges between the fruit and the shoot. They have secondary thickened cell walls that presumably function in mechanical support, water and nutrient transport. Stone cells are cells with a secondary cell wall thickening. In pears, yet not in apples, the stone cells affect the flesh texture. There have been few reports on stone cell formation in pear and apple pedicels; therefore, we studied these cells for the first time. The apple pedicel had few stone cells in the cortex. The formation of stone cells in pear continued until seven weeks after flowering (WAF), and the density was significantly higher than in apple. The stone cell formation degree (SFD) of pear was 3.6–7.1 times higher than that of apple. Total lignin and lignin non-condensed structure (G and S units) content in the pear pedicle outer part was 1.5–2.7 times higher than that of the apple at harvest. The SFD of the pedicel outer part had a positive correlation with the G and S units content of the flesh. The total lignin and G and S units content between flesh and the pedicel outer part were positively correlated. Correlation analysis revealed a positive relationship between fruit and pedicel formation of the stone cells. The WGCNA showed that NST3 was linked to NAC028, MYB46, CESA, POD, LAC, and VSR6. These genes were highly expressed in the outer part of the pear pedicel, while they were suppressed in that issue of the apple at 4 WAF.

To gain insights into the genetic mechanisms underlying blooming and petal movement in flowering cherry (Cerasus × yedoensis), we performed time-course RNA-seq analysis of the floral buds and open-flowers of the most popular flowering cherry cultivar, 'Somei-Yoshino.' Independent biological duplicate samples of floral buds and open-flowers were collected from 'Somei-Yoshino' trees grown at three different locations in Japan. RNA-seq reads obtained from floral bud and open-flower samples collected in the current study (in 2019) and in a previous study (in 2017) were aligned against the genome sequence of 'Somei-Yoshino' to quantify gene transcript levels. Clustering analysis of RNA-seq reads revealed dynamic changes in the transcriptome, with genes in seven modules predominantly expressed at specific time points, ranging from 5 weeks before flowering to 2 weeks after flowering. Based on the identified gene modules and Gene Ontology (GO) terms enriched at different floral stages, we speculate that the genetic mechanisms underlying petal movement and flower opening in cherry involve the processes of development, cell wall organization, reproduction, and metabolism, which are executed by genes encoding transcription factors, phytohormones, transporters, and polysaccharide metabolic enzymes. Furthermore, we established a statistical model for cherry bloom forecasting, based on gene expression levels as RNA markers at different time points before flowering.

Gibberellin-regulated protein (GRP) is a fruit severe allergen. The amounts of GRP expression normalized against actin in peach were determined by reverse transcription-quantitative PCR (RT-qPCR). The results were consistent with those determined by enzyme-linked immunosorbent assay (ELISA). The GRP expression was more evident in flesh than peel and increased rapidly in the maturing period. This approach is applicable to estimate the amount of GRP in other plants.

Japanese apricot (Prunus mume) cultivars have been classified into several subpopulations, such as fruiting, fruiting-small, and ornamental, based on a variety of morphological traits and human preference. A recent genome-wide analysis indicated that these subpopulations show some genetic differentiation, but the fruit traits remain to be investigated. Therefore, we investigated phenotypic diversity of several fruit traits, such as sugar, organic acid, and volatile compound contents and compositions, in a variety of cultivars of Japanese apricot (P. mume), apricot (P. armeniaca) and their interspecific hybrids. We found that Japanese apricot cultivars show diverse phenotypic variation and that some fruit traits appeared to be associated with the differing subpopulations. The fruiting cultivars investigated appear to have higher contents of sugars and favorable volatile compounds such as lactones and esters. We also found that 'Joshuhaku', a local cultivar in Kyoto Prefecture, has desirable fruit traits including large fruit size, high contents of organic acid and favorable volatile compounds. Moreover, interspecific hybridization was found to have large effects on fruit traits. Our results indicated a wide diversity of fruit traits in Japanese apricot, in which sugars and volatile compounds were associated with fruiting cultivars. These observations may assist with the breeding of new Japanese apricot cultivars.

In Japan, seedless grapes are obtained by applications of plant growth regulators such as gibberellic acid (GA 3 ) and 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU). To analyze the effect of these chemicals on the palatability of the texture of unpeeled ‘Shine Muscat’ grape berries in the mature stage, we treated plants with GA 3 plus CPPU on 1 day after full bloom and GA 3 at 10–12 days after full bloom (GA 3 10DAFB). Then, we investigated the rheological properties and plant cell wall components. For texture analysis, mechanical properties were examined using a puncture test, and the peeling ability as a skin property was determined a tensile test. To assay cell wall components, berries were divided into three parts (central part of mesocarp, pericarp around skin, and skin). CPPU treatment of more than 5 ppm at full bloom led to inferior mechanical properties of berries. Furthermore, CPPU treatment of more than 5 ppm at full bloom increased cellulose contents of the skin and pericarp around the skin. GA 3 treatment increased berry firmness and strengthened adhesive forces between the skin and pulp. Simultaneously, uronic acid contents in the 0.05 N sodium carbonate solution-soluble fraction and total sugar contents in the 4 M KOH soluble fraction of the pericarp around the skin on GA 3 treatment were higher than those of the control. Therefore, the increase of cell wall components by GA 3 treatment made the berry texture firm and strengthened adhesive forces between the skin and pulp. These results suggest that each plant growth regulator affects the cell wall components of grape berries and varies their texture.

Postharvest water loss in pepper fruit (Capsicum annuum L.) reduces its shelf life. Fruit texture is one of the most important components of fruit quality for consumers. In this study, the anatomical traits of pepper fruit related to postharvest water loss and texture were assessed. There was a strong positive relationship between postharvest water loss and the thickness of the cuticular membrane, cuticular weight, total cutin weight, and polysaccharide-cutan weight. An amorphous fibrous structure that forms a path for diffusion and increases water loss was observed in the thick cuticle of the pericarp. In addition, positive correlations between the hardness of the exocarp and the weight of cuticular membrane, cutin content, and polysaccharide-cutan content were found. These results indicate that the thickness of the cuticular membrane wedged between subepidermal cells may influence water loss through the pericarp of pepper fruit and fruit with a high cutin and polysaccharide content have a hard tough texture.

To gain genetic insights into the early-flowering phenotype of ornamental cherry, also known as sakura, we determined the genome sequences of two early-flowering cherry (Cerasus × kanzakura) varieties, ‘Kawazu-zakura’ and ‘Atami-zakura’. Since the two varieties are interspecific hybrids, likely derived from crosses between Cerasus campanulata (early-flowering species) and Cerasus speciosa, we employed the haplotype-resolved sequence assembly strategy. Genome sequence reads obtained from each variety by single molecule real-time sequencing (SMRT) were split into two subsets, based on the genome sequence information of the two probable ancestors, and assembled to obtain haplotype-phased genome sequences. The resultant genome assembly of ‘Kawazu-zakura’ spanned 519.8 Mb with 1,544 contigs and an N50 value of 1,220.5 kb, while that of ‘Atami-zakura’ totaled 509.6 Mb with 2,180 contigs and an N50 value of 709.1 kb. A total of 72,702 and 72,528 potential protein-coding genes were predicted in the genome assemblies of ‘Kawazu-zakura’ and ‘Atami-zakura’, respectively. Gene clustering analysis identified 2,634 clusters uniquely presented in the C. campanulata haplotype sequences, which might contribute to its early-flowering phenotype. Genome sequences determined in this study provide fundamental information for elucidating the molecular and genetic mechanisms underlying the early-flowering phenotype of ornamental cherry tree varieties and their relatives.

KEY MESSAGE:Characterization of hybrid seed failure in Prunus provides insight into conserved or lineage-specific hybrid incompatibility mechanisms in plant species. Postzygotic hybrid incompatibility resulting from a cross between different species involves complex mechanisms occurring at various developmental stages. Embryo arrest, followed by seed abortion, is the first stage of such incompatibility reactions and inhibits hybrid seed development. In Prunus, a rosaceous woody species, some interspecific crosses result in fruit drop during the early stage of fruit development, in which inferior seed development may be accounted for the observed hybrid incompatibility. In this study, we investigated ovule development and the transcriptomes of developing ovules in inter-subgeneric crosses of Prunus. We conducted a cross of Prunus mume (subgenus Prunus), pollinated by P. persica (subgenus Amygdalus), and found that ovule and seed coat degeneration occurs before fruit drop. Transcriptome analysis identified differentially expressed genes enriched in several GO pathways, including organelle development, stimulus response, and signaling. Among these pathways, the organelle-related genes were actively regulated during ovule development, as they showed higher expression in the early stage of interspecific crosses and declined in the later stage, suggesting that the differential regulation of organelle function may induce the degeneration of hybrid ovules. Additionally, genes related to ovule and seed coat development, such as genes encoding AGL-like and auxin response, were differentially regulated in Prunus interspecific crosses. Our results provide histological and molecular information on hybrid seed abortion in Prunus that could be utilized to develop new hybrid crops. Additionally, we compared and discussed transcriptome responses to hybrid seed failure in Prunus and other plant species, which provides insight into conserved or lineage-specific hybrid incompatibility mechanisms in some plant species.