Self-incompatibility (SI) in plants has evolved independently multiple times and S-RNase-based gametophytic self-incompatibility (GSI) is most common. The Rosaceae family possesses both self-recognition (Prunus) and nonself-recognition (Malus) GSI systems, and the latter is widespread in flowering plants. Gillenia trifoliata is a Rosaceae species related to Prunus and Malus, providing utility for understanding SI evolution. Gillenia is sister taxon to Malus, but unlike Malus, has not undergone polyploidisation. In addition, the common ancestor of Gillenia and Prunus is close to the origin of the subfamily. Using a highly contiguous Gillenia genome, orthologous regions to both Malus and Prunus S-loci were identified. Only the Prunus-like S-locus was highly polymorphic and had signatures of a functional S-locus including positive selection of the S-RNase. This suggests a self-recognition system controls SI in Gillenia, and the common ancestors of Gillenia and Prunus, and Gillenia and the apple tribe, likely had a self-recognition SI system. Comparative genomics between Gillenia and Malus suggest apple lost the self-recognition mechanism, and a nonself-recognition mechanism evolved independently from a rudimentary locus with at least one male S-determinant. Repetitive sequences in the Malus-like S-locus in Gillenia may facilitate illegitimate recombination, suggesting putative mechanisms of evolution of nonself-recognition S-loci.

The over-expression of the apple SEP1/2 like gene (MADS8) supports its role as a key regulator of fleshy fruit patterning and ripening in pome fruit. Fruit flesh patterning in accessory fruits such as apple and strawberry has been associated with the SEPALLATA (SEP) class of genes. Antisense suppression of multiple SEP homologs greatly reduced fruit fleshy tissue growth and ripening in strawberries and apple, suggesting a co-evolution of fleshy fruit growth and its ability to ripen. In the present study, transgenic apple trees were generated that overexpressed the apple SEP1/2-like gene MADS8 (MADS8ox). These lines produced precocious flowers in the second year of growth, but subsequent flower numbers were not increased. Overexpression MdMADS8 in Arabidopsis promoted flowering and caused curled leaves and a conversion of sepals to petals. In apple, MADS8ox fruit were larger than wild type ‘Royal Gala’ fruit with a higher proportion of flesh tissue compared to core tissue. MADS8ox fruit ripened earlier and faster with higher expression of ripening-related genes such as ACS1 and ACO1. Together, these results add further insights into the role that SEP-like genes have in controlling flowering and fruit patterning and provide further evidence that MdMADS8 is one of the key regulators of fruit flesh development and ripening in apples.

The global trade in kiwifruit started in the 1960s and was built on the good postharvest performance of a single cultivar, Actinidia chinensis var. deliciosa 'Hayward', even though alternative cultivars were available. Even now 'Hayward' is still the dominant traded cultivar. So why is it that the 'Hayward' fruit has maintained its dominant position in the market? One key trait of 'Hayward' fruit is the ability to be successfully stored for long periods. In this study the storage performance of 'Hayward' fruit was compared with fruit of two other A. chinensis var. deliciosa cultivars, 'Bruno' and 'Allison', which were available at the start of the global kiwifruit industry. There are two key aspects for kiwifruit storage performance; firmness maintenance and resistance to chilling damage. Studies coupled investigations of the fruit structure (tissue zones) and maturation with softening and chilling expression in storage. Data showed that the good storage performance of 'Hayward' kiwifruit is linked to its softening pattern, whereby it does not soften too fast to a too low firmness. This may be associated with fruit structure. In addition, at an equivalent maturity, 'Hayward' fruit had a greater tolerance of low storage temperatures without developing chilling damage.

Apple (Malus domestica) is self-incompatible and typically requires cross-pollination for seed and fruit development. Parthenocarpy (fruit development without fertilization) and self-compatibility (fruit set without external pollen) are highly desirable traits in apple breeding, as they ensure consistent fruit production and quality without cross-pollination. However, apple parthenocarpic and self-compatible accessions have not been available for practical breeding. To identify these accessions, we analysed 436 accessions of Malus domestica and 84 accessions of wild Malus species by assessing fruit production. Flowers were bagged before opening to prevent cross-pollination. If fruit developed from the bagged flowers, it indicated the presence of self-compatibility or parthenocarpy, depending on whether the fruit contained seeds. We observed and scored a range of phenotypic expressions among accessions, from weak to strong in both parthenocarpy and potential self-compatibility. Strong parthenocarpy was observed in 5.95% of wild Malus species accessions and 3.44% of M. domestica accessions. Similarly, strong self-compatibility was exhibited in 5.95% of wild Malus species accessions and 2.75% of M. domestica accessions. Although bagged flowers showed lower fruit set rates than open-pollinated (OP) flowers, fruit size, weight, firmness, and soluble sugar and starch content showed no significant differences between fruits produced from bagged and OP flowers. Furthermore, a genome-wide association study (GWAS) was conducted with a high-throughput SNP array. This analysis identified several genes potentially associated with these traits. This research provides parthenocarpic and self-compatible apple accessions for breeding, which can generate novel cultivars that eliminate the need for cross-pollination or produce seedless fruit without pollination.

Herkogamy is the spatial separation of anthers and stigmas within complete flowers, and is a key floral trait that promotes outcrossing in many angiosperms. The degree of separation between pollen-producing anthers and receptive stigmas has been shown to influence rates of self-pollination amongst plants, with a reduction in herkogamy increasing rates of successful selfing in self-compatible species. Self-pollination is becoming a critical issue in horticultural crops grown in environments where biotic pollinators are limited, absent, or difficult to utilise. In these cases, poor pollination results in reduced yield and misshapen fruit. Whilst there is a growing body of work elucidating the genetic basis of floral organ development, the genetic and environmental control points regulating herkogamy are poorly understood. A better understanding of the developmental and regulatory pathways involved in establishing varying degrees of herkogamy is needed to provide insights into the production of flowers more adept at selfing to produce consistent, high-quality fruit. This review presents our current understanding of herkogamy from a genetics and hormonal perspective.

There have been a considerable number of studies that have successfully sped up the flowering cycle in woody perennial horticultural species. One particularly successful study in apple (Malus domestica) accelerated flowering using a silver birch (Betula pendula) APETALA1/FRUITFULL MADS-box gene BpMADS4, which yielded a good balance of vegetative growth to support subsequent flower and fruit development. In this study, BpMADS4 was constitutively expressed in European pear (Pyrus communis) to establish whether this could be used as a tool in a rapid pear breeding program. Transformed pear lines flowered within 6-18 months after grafting onto a quince (Cydonia oblonga) rootstock. Unlike the spindly habit of early flowering apples, the early flowering pear lines displayed a normal tree-like habit. Like apple, the flower appearance was normal, and the flowers were fertile, producing fruit and seed upon pollination. Seed from these transformed lines were germinated and 50% of the progeny flowered within 3 months of sowing, demonstrating a use for these in a fast breeding program.

During fruit ripening, polygalacturonases (PGs) are key contributors to the softening process in many species. Apple is a crisp fruit that normally exhibits only minor changes to cell walls and limited fruit softening. Here, we explore the effects of PG overexpression during fruit development using transgenic apple lines overexpressing the ripening-related endo-POLYGALACTURONASE1 gene. MdPG1-overexpressing (PGox) fruit displayed early maturation/ripening with black seeds, conversion of starch to sugars and ethylene production occurring by 80 days after pollination (DAP). PGox fruit exhibited a striking, white-skinned phenotype that was evident from 60 DAP and most likely resulted from increased air spaces and separation of cells in the hypodermis due to degradation of the middle lamellae. Irregularities in the integrity of the epidermis and cuticle were also observed. By 120 DAP, PGox fruit cracked and showed lenticel-associated russeting. Increased cuticular permeability was associated with microcracks in the cuticle around lenticels and was correlated with reduced cortical firmness at all time points and extensive post-harvest water loss from the fruit, resulting in premature shrivelling. Transcriptomic analysis suggested that early maturation was associated with upregulation of genes involved in stress responses, and overexpression of MdPG1 also altered the expression of genes involved in cell wall metabolism (e.g. β-galactosidase, MD15G1221000) and ethylene biosynthesis (e.g. ACC synthase, MD14G1111500). The results show that upregulation of PG not only has dramatic effects on the structure of the fruit outer cell layers, indirectly affecting water status and turgor, but also has unexpected consequences for fruit development.

Following successful pollination, Dendrobium orchid flowers rapidly undergo senescence. In Dendrobium cv. Khao Chaimongkol, compatible pollination resulted in faster ethylene production and more rapid development of senescence symptoms, such as drooping, epinasty, venation and yellowing, compared with non-pollinated controls or pollination with incompatible pollinia. The DenACS1 and DenACO1 genes in the perianth of florets that had been pollinated with compatible pollinia were expressed more highly than those in non-pollinated open florets. Incompatible pollinia reduced the expression of DenACS1 and DenACO1 genes in the perianth. Transcript levels of the ethylene receptor gene DenERS1 and signaling genes DenEIL1 and DenERF1 showed differential spatial regulation with greater expression in the perianth than in the column plus ovary following compatible pollination. Compatible pollinia increased ethylene production concomitant with premature senescence and the increased expression of the DenACS1 and DenACO1 genes, and suppressed the ethylene receptor gene DenERS1, whereas incompatible pollinia did not stimulate ethylene production nor induce premature senescence but induced higher expression of DenERS1 both in the perianth and in the column plus ovary. These results suggest that the increased ethylene production in open florets pollinated with compatible pollen was partially due to an increase in the expression of DenACS1 and DenACO1 genes. The compatible pollinia induced a negative regulation of DenERS1 which may play an important role in ethylene perception and in modulating ethylene signaling transduction during pollinia-induced flower senescence.

DATA REPORT article Front. Genet., 16 May 2022Sec. Plant Genomics https://doi.org/10.3389/fgene.2022.852161

SummaryAllele‐specific expression (ASE) can lead to phenotypic diversity and evolution. However, the mechanisms regulating ASE are not well understood, particularly in woody perennial plants. In this study, we investigated ASE genes in the apple cultivar ‘Royal Gala’ (RG). A high quality chromosome‐level genome was assembled using a homozygous tetra‐haploid RG plant, derived from anther cultures. Using RNA‐sequencing (RNA‐seq) data from RG flower and fruit tissues, we identified 2091 ASE genes. Compared with the haploid genome of ‘Golden Delicious’ (GD), a parent of RG, we distinguished the genomic sequences between the two alleles of 817 ASE genes, and further identified allele‐specific presence of a transposable element (TE) in the upstream region of 354 ASE genes. These included MYB110a that encodes a transcription factor regulating anthocyanin biosynthesis. Interestingly, another ASE gene, MYB10 also showed an allele‐specific TE insertion and was identified using genome data of other apple cultivars. The presence of the TE insertion in both MYB genes was positively associated with ASE and anthocyanin accumulation in apple petals through analysis of 231 apple accessions, and thus underpins apple flower colour evolution. Our study demonstrated the importance of TEs in regulating ASE on a genome‐wide scale and presents a novel method for rapid identification of ASE genes and their regulatory elements in plants.

Fruit development, following pollination, involves a cell division stage, followed by cell expansion . A major challenges to study each individual stages is the limitations imposed by analyses of fruit development on the plant. Rapid growing cell culture systems that differentiate could be used to identify and understand the molecular control of each of these processes in vitro. In this study, an apple cell culture was initiated from the widely characterised 'Royal Gala' cultivar. The established apple cell culture was used to develop a screening system to assess the effect of different hormones on cell differentiation, which then were characterised at molecular and cellular levels. Three different hormone pairs were screened to determine the optimal for cell expansion on actively dividing cells . Using this strategy, we were able to reproduce, in vitro, the first two stages of apple fruit cell differentiation, cell division and expansion, which was correlated with changes in genes expression of specific markers for those two stages, a cyclin-dependent kinase gene (CDKB2;2) and an expansion gene (EXP3). The cell culture provides a unique tool and opportunity to study the effect of plant hormones on cell differentiation and will allow to decipher the molecular mechanism of apple fruit development irrespective of y.

The ability to quantify the colour of fruit is extremely important for a number of applied fields including plant breeding, postharvest assessment, and consumer quality assessment. Fruit and other plant organs display highly complex colour patterning. This complexity makes it challenging to compare and contrast colours in an accurate and time efficient manner. Multiple methodologies exist that attempt to digitally quantify colour in complex images but these either require a priori knowledge to assign colours to a particular bin, or fit the colours present within segment of the colour space into a single colour value using a thresholding approach. A major drawback of these methodologies is that, through the process of averaging, they tend to synthetically generate values that may not exist within the context of the original image. As such, to date there are no published methodologies that assess colour patterning using a data driven approach. In this study we present a methodology to acquire and process digital images of biological samples that contain complex colour gradients. The CIE (Commission Internationale de l’Eclairage/International Commission on Illumination) ΔE2000 formula was used to determine the perceptually unique colours (PUC) within images of fruit containing complex colour gradients. This process, on average, resulted in a 98% reduction in colour values from the number of unique colours (UC) in the original image. This data driven procedure summarised the colour data values while maintaining a linear relationship with the normalised colour complexity contained in the total image. A weighted ΔE2000 distance metric was used to generate a distance matrix and facilitated clustering of summarised colour data. Clustering showed that our data driven methodology has the ability to group these complex images into their respective binomial families while maintaining the ability to detect subtle colour differences. This methodology was also able to differentiate closely related images. We provide a high quality set of complex biological images that span the visual spectrum that can be used in future colorimetric research to benchmark colourimetric method development.

Apple (Malus x domestica) fruit size is dependent on cell division and cell expansion, processes that are subsequently regulated by plant hormones such as auxins, gibberellins, and cytokinins. In this study, we investigated the role of cell division and cell expansion in apple growth and identified which of the two was more deterministic of final fruit size. Three cultivars of different sizes were selected, namely, “Twenty Ounce” (large-sized), “Royal Gala” (medium-sized), and “Crabapple” (small-sized). Gene expression and cell size analyses were conducted over the course of two consecutive seasons. The expression patterns of three classes of genes were markedly similar across all cultivars. Two cell division markers, namely MdCDKB2;2 and MdANT2, were discovered to be correlatively expressed, as both displayed initially high expression levels, which gradually declined from the early to late stages of the growth time course. For cell expansion markers, MdEXP3 was upregulated as the cells expanded, while MdARF106 was expressed in both the cell division and expansion stages. Meanwhile, the ripening-related gene MdACO1 was expectedly expressed only during the ending stages associated with ripening. Interestingly, the cell measurements taken regularly from each cultivar throughout the same experimental timespan showed that cell sizes were unaltered and remained constant from initial pollination at the zeroth Day After Pollination (DAP) to ripening at 120 Days After Full Bloom (DAFB).

The postharvest performance of kiwifruit is largely assessed on the basis of firmness retention and the development of disorders such as chilling damage. In a number of fruit species, nitric oxide (NO) is considered to play a role in the signaling pathways associated with fruit ripening and chilling response. The effects of exogenous NO on fruit softening, chilling injury (CI) and rots in Actinidia chinensis var. deliciosa 'Hayward' kiwifruit have been investigated at 20 and 0 degrees C. Fruit were immersed in 0 (distilled water), 1 or 5 mM sodium nitroprusside dehydrate (SNP) solution, a NO donor, for 10 min, before drying in air and holding for up to 3 weeks at 20 degrees C and up to 24 weeks at 0 degrees C. 'Hayward' kiwifruit responded to NO treatment with a significant slowing of softening at 20 degrees C, whereas at 0 degrees C, the slowing of softening was observed only in the 5 mM treatment, and the effect was transitory, there being no effect on firmness for the majority of the storage period past the first 3 weeks. In addition, in the treated fruit at 0 degrees C there was a significant incidence of skin damage, and no reduction in the incidence of CI. It is concluded that while treatment with NO may have a significant effect in slowing fruit softening at 20 degrees C, in the concentrations applied here, it had only a short-term effect on slowing softening at 0 degrees C. Contrary to other published results, NO appears not to reduce the incidence of CI, and may increase the incidence of fruit damage and rots during long-term storage at 0 degrees C.

Commercially grown kiwifruit (genus Actinidia) are generally of two sub-species which have a base haploid genome of 29 chromosomes. The yellow-fleshed Actinidia chinensis var. chinensis, is either diploid (2n = 2x = 58) or tetraploid (2n = 4x = 116) and the green-fleshed cultivar A. chinensis var. deliciosa "Hayward," is hexaploid (2n = 6x = 174). Advances in breeding green kiwifruit could be greatly sped up by the use of molecular resources for more efficient and faster selection, for example using marker-assisted selection (MAS). The key genetic marker that has been implemented for MAS in hexaploid kiwifruit is for gender testing. The limited marker-trait association has been reported for other polyploid kiwifruit for fruit and production traits. We have constructed a high-density linkage map for hexaploid green kiwifruit using genotyping-by-sequence (GBS). The linkage map obtained consists of 3686 and 3940 markers organized in 183 and 176 linkage groups for the female and male parents, respectively. Both parental linkage maps are co-linear with the A. chinensis "Red5" reference genome of kiwifruit. The linkage map was then used for quantitative trait locus (QTL) mapping, and successfully identified QTLs for king flower number, fruit number and weight, dry matter accumulation, and storage firmness. These are the first QTLs to be reported and discovered for complex traits in hexaploid kiwifruit.

Seedless fruits have high consumer appeal and have made seeded varieties obsolete in some crops. In seedless apple varieties, core tissues which normally contain the seed can be unpalatable, reducing the seedless appeal. Apples are accessory fruit with edible flesh derived from hypanthial tissue – a floral tube fused to a compound ovary. Here we show that through suppression of AGAMOUS-like carpel identity genes and hormone induced fruit set, it is possible to generate coreless and therefore seedless apples. Suppression of AGAMOUS-like genes increased petal whorls and fully eliminated carpel development. Treatments with a combination of gibberellin, cytokinin and auxin, rather than single treatments, were required for fruit initiation in these lines. Transcriptomic analysis of agamous RNAi lines suggested conservation of AGAMOUS-dependent gene networks between apple and Arabidopsis. In the absence of all sexual tissues, the developing fruit continues to grow and follow a ripening process similar to that of a regular apple. The coreless phenotype offers a new concept for pipfruit consumers improving convenience and reducing food waste.

The Rosaceae family has striking phenotypic diversity and high syntenic conservation. Gillenia trifoliata is sister species to the Maleae tribe of apple and ~1000 other species. Gillenia has many putative ancestral features, such as herb/sub-shrub habit, dry fruit-bearing and nine base chromosomes. This coalescence of ancestral characters in a phylogenetically important species, positions Gillenia as a 'rosetta stone' for translational science within Rosaceae. We present genomic and phenological resources to facilitate the use of Gillenia for this purpose. The Gillenia genome is the first fully annotated chromosome-level assembly with an ancestral genome complement (x = 9), and with it we developed an improved model of the Rosaceae ancestral genome. MADS and NAC gene family analyses revealed genome dynamics correlated with growth and reproduction and we demonstrate how Gillenia can be a negative control for studying fleshy fruit development in Rosaceae.

The commonly accepted kiwifruit harvest index based solely on soluble solids content (SSC) has lost its original significance owing to the increased sophistication of marketing coupled with the commercialisation of new cultivars, many with yellow flesh when ripe. The precision of novel harvest indices may be improved by a molecular understanding of fruit maturation changes in commonly monitored fruit attributes, including SSC, flesh colour and firmness. Transcriptional changes in the early-maturing Actinidia chinensis var. chinensis ‘Zesy002’ and later maturing A. chinensis var. deliciosa ‘Hayward’, grown at a single site, have been quantified weekly in the period leading up to and past the commercial harvest period. Transcriptomic data highlighted numerous maturation related changes in the fruit, and differences between the two cultivars. Examples of gene changes of relevance for non-structural carbohydrates included the large sub-unit 4 of ADP-glucose pyrophosphorylase (APL4) indicative of starch synthesis, BETA AMYLASE 3.2 (BAM3.2) for starch breakdown and the sugar transporter (SWEET9a). The association between STAYGREEN2 (SGR2) with flesh degreening was strong in ‘Zesy002’, although a later increase in ‘Hayward’ to levels similar to those in ‘Zesy002’ was not accompanied by an equivalent flesh colour change. In ‘Zesy002’ there were numerous individual cell wall gene changes associated with the change to rapid softening, including EXPANSIN7 (EXP7), POLYGALACTURONASE1 (PG1), PECTATE LYASE (PL), PECTIN METHYL ESTERASE (PME) and XYLOGLUCAN TRANSGLYCOSYLASE/ HYDROLASE (XTH). However, among these genes, it is possible to see similar changes in ‘Hayward’ which were not associated with a marked change in softening rate, including for EXP7 and PG1. The most obvious start points for changes in transcription of these genes were the seed coat colour change, the cessation of growth, and the change to ripening (rapid softening and starch breakdown). The findings are discussed with respect to fruit maturation and the possible use of gene markers as harvest indices.

Background The phytohormone ethylene controls many processes in plant development and acts as a key signaling molecule in response to biotic and abiotic stresses: it is rapidly induced by flooding, wounding, drought, and pathogen attack as well as during abscission and fruit ripening. In kiwifruit (Actinidia spp.), fruit ripening is characterized by two distinct phases: an early phase of system-1 ethylene biosynthesis characterized by absence of autocatalytic ethylene, followed by a late burst of autocatalytic (system-2) ethylene accompanied by aroma production and further ripening. Progress has been made in understanding the transcriptional regulation of kiwifruit fruit ripening but the regulation of system-1 ethylene biosynthesis remains largely unknown. The aim of this work is to better understand the transcriptional regulation of both systems of ethylene biosynthesis in contrasting kiwifruit organs: fruit and leaves. Results A detailed molecular study in kiwifruit (A. chinensis) revealed that ethylene biosynthesis was regulated differently between leaf and fruit after mechanical wounding. In fruit, wound ethylene biosynthesis was accompanied by transcriptional increases in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), ACC oxidase (ACO) and members of the NAC class of transcription factors (TFs). However, in kiwifruit leaves, wound-specific transcriptional increases were largely absent, despite a more rapid induction of ethylene production compared to fruit, suggesting that post-transcriptional control mechanisms in kiwifruit leaves are more important. One ACS member, AcACS1, appears to fulfil a dominant double role; controlling both fruit wound (system-1) and autocatalytic ripening (system-2) ethylene biosynthesis. In kiwifruit, transcriptional regulation of both system-1 and -2 ethylene in fruit appears to be controlled by temporal up-regulation of four NAC (NAM, ATAF1/2, CUC2) TFs (AcNAC1-4) that induce AcACS1 expression by directly binding to the AcACS1 promoter as shown using gel-shift (EMSA) and by activation of the AcACS1 promoter in planta as shown by gene activation assays combined with promoter deletion analysis. Conclusions Our results indicate that in kiwifruit the NAC TFs AcNAC2-4 regulate both system-1 and -2 ethylene biosynthesis in fruit during wounding and ripening through control of AcACS1 expression levels but not in leaves where post-transcriptional/translational regulatory mechanisms may prevail.