A cryptic variation in a member of the Ovate Family Proteins is underlying the melon fruit shape QTL fsqs8.1.

KEY MESSAGE: The gene underlying the melon fruit shape QTL fsqs8.1 is a member of the Ovate Family Proteins. Variation in fruit morphology is caused by changes in gene expression likely due to a cryptic structural variation in this locus. Melon cultivars have a wide range of fruit morphologies. Quantitative trait loci (QTL) have been identified underlying such diversity. This research focuses on the fruit shape QTL fsqs8.1, previously detected in a cross between the accession PI 124112 (CALC, producing elongated fruit) and the cultivar 'Piel de Sapo' (PS, producing oval fruit). The CALC fsqs8.1 allele induced round fruit shape, being responsible for the transgressive segregation for this trait observed in that population. In fact, the introgression line CALC8-1, carrying the fsqs8.1 locus from CALC into the PS genetic background, produced perfect round fruit. Following a map-based cloning approach, we found that the gene underlying fsqs8.1 is a member of the Ovate Family Proteins (OFP), CmOFP13, likely a homologue of AtOFP1 and SlOFP20 from Arabidopsis thaliana and tomato, respectively. The induction of the round shape was due to the higher expression of the CALC allele at the early ovary development stage. The fsqs8.1 locus showed an important structural variation, being CmOFP13 surrounded by two deletions in the CALC genome. The deletions are present at very low frequency in melon germplasm. Deletions and single nucleotide polymorphisms in the fsqs8.1 locus could not be not associated with variation in fruit shape among different melon accessions, what indicates that other genetic factors should be involved to induce the CALC fsqs8.1 allele effects. Therefore, fsqs8.1 is an example of a cryptic variation that alters gene expression, likely due to structural variation, resulting in phenotypic changes in melon fruit morphology.

Regulatory circuits involving bud dormancy factor PpeDAM6.

DORMANCY-ASSOCIATED MADS-BOX (DAM) genes have recently emerged as key potential regulators of the dormancy cycle and climate adaptation in perennial species. Particularly, PpeDAM6 has been proposed to act as a major repressor of bud dormancy release and bud break in peach (Prunus persica). PpeDAM6 expression is downregulated concomitantly with the perception of a given genotype-dependent accumulation of winter chilling time, and the coincident enrichment in H3K27me3 chromatin modification at a specific genomic region. We have identified three peach BASIC PENTACYSTEINE PROTEINs (PpeBPCs) interacting with two GA-repeat motifs present in this H3K27me3-enriched region. Moreover, PpeBPC1 represses PpeDAM6 promoter activity by transient expression experiments. On the other hand, the heterologous overexpression of PpeDAM6 in European plum (Prunus domestica) alters plant vegetative growth, resulting in dwarf plants tending toward shoot meristem collapse. These alterations in vegetative growth of transgenic lines associate with impaired hormone homeostasis due to the modulation of genes involved in jasmonic acid, cytokinin, abscisic acid, and gibberellin pathways, and the downregulation of shoot meristem factors, specifically in transgenic leaf and apical tissues. The expression of many of these genes is also modified in flower buds of peach concomitantly with PpeDAM6 downregulation, which suggests a role of hormone homeostasis mechanisms in PpeDAM6-dependent maintenance of floral bud dormancy and growth repression.

Ten Years of VINQUEST: First Insight for Breeding New Apple Cultivars With Durable Apple Scab Resistance.

Apple scab, caused by Venturia inaequalis, is a major fungal disease worldwide. Cultivation of scab-resistant cultivars would reduce the chemical footprint of apple production. However, new apple cultivars carrying durable resistances should be developed to prevent or at least slow the breakdown of resistance against races of V. inaequalis. One way to achieve durable resistance is to pyramid multiple scab resistance genes in a cultivar. The choice of the resistance genes to be combined in the pyramids should take into account the frequency of resistance breakdown and the geographical distribution of apple scab isolates able to cause such breakdowns. In order to acquire this information and to make it available to apple breeders, the VINQUEST project (www.vinquest.ch) was initiated in 2009. Ten years after launching this project, 24 partners from 14 countries regularly contribute data. From 2009 to 2018, nearly 9,000 data points have been collected. This information has been used to identify the most promising apple scab resistance genes for developing cultivars with durable resistance, which to date are: Rvi5, Rvi11, Rvi12, Rvi14, and Rvi15. As expected, Rvi1, together with Rvi3 and Rvi8, were often overcome, and have little value for scab resistance breeding. Rvi10 may also belong to this group. On the other hand, Rvi2, Rvi4, Rvi6, Rvi7, Rvi9, and Rvi13 are still useful for breeding, but their use is recommended only in extended pyramids of ≥3 resistance genes.

ETHQV6.3 is involved in melon climacteric fruit ripening and is encoded by a NAC domain transcription factor.

Fruit ripening is divided into climacteric and non-climacteric types depending on the presence or absence of a transient rise in respiration rate and the production of autocatalytic ethylene. Melon is ideal for the study of fruit ripening, as both climacteric and non-climacteric varieties exist. Two introgressions of the non-climacteric accession PI 161375, encompassed in the QTLs ETHQB3.5 and ETHQV6.3, into the non-climacteric 'Piel de Sapo' background are able to induce climacteric ripening independently. We report that the gene underlying ETHQV6.3 is MELO3C016540 (CmNAC-NOR), encoding a NAC (NAM, ATAF1,2, CUC2) transcription factor that is closely related to the tomato NOR (non-ripening) gene. CmNAC-NOR was functionally validated through the identification of two TILLING lines carrying non-synonymous mutations in the conserved NAC domain region. In an otherwise highly climacteric genetic background, both mutations provoked a significant delay in the onset of fruit ripening and in the biosynthesis of ethylene. The PI 161375 allele of ETHQV6.3 is similar to that of climacteric lines of the cantalupensis type and, when introgressed into the non-climacteric 'Piel de Sapo', partially restores its climacteric ripening capacity. CmNAC-NOR is expressed in fruit flesh of both climacteric and non-climacteric lines, suggesting that the causal mutation may not be acting at the transcriptional level. The use of a comparative genetic approach in a species with both climacteric and non-climacteric ripening is a powerful strategy to dissect the complex mechanisms regulating the onset of fruit ripening.

Dual regulation of water retention and cell growth by a stress-associated protein (SAP) gene in Prunus.

We have identified a gene (PpSAP1) of Prunus persica coding for a stress-associated protein (SAP) containing Zn-finger domains A20 and AN1. SAPs have been described as regulators of the abiotic stress response in plant species, emerging as potential candidates for improvement of stress tolerance in plants. PpSAP1 was highly expressed in leaves and dormant buds, being down-regulated before bud dormancy release. PpSAP1 expression was moderately induced by water stresses and heat in buds. In addition, it was found that PpSAP1 strongly interacts with polyubiquitin proteins in the yeast two-hybrid system. The overexpression of PpSAP1 in transgenic plum plants led to alterations in leaf shape and an increase of water retention under drought stress. Moreover, we established that leaf morphological alterations were concomitant with a reduced cell size and down-regulation of genes involved in cell growth, such as GROWTH-REGULATING FACTOR (GRF)1-like, TONOPLAST INTRINSIC PROTEIN (TIP)-like, and TARGET OF RAPAMYCIN (TOR)-like. Especially, the inverse expression pattern of PpSAP1 and TOR-like in transgenic plum and peach buds suggests a role of PpSAP1 in cell expansion through the regulation of TOR pathway.

Insights into the Role of the Berry-Specific Ethylene Responsive Factor VviERF045.

During grape ripening, numerous transcriptional and metabolic changes are required in order to obtain colored, sweet, and flavored berries. There is evidence that ethylene, together with other signals, plays an important role in triggering the onset of ripening. Here, we report the functional characterization of a berry-specific Ethylene Responsive Factor (ERF), VviERF045, which is induced just before véraison and peaks at ripening. Phylogenetic analysis revealed it is close to the SHINE clade of ERFs, factors involved in the regulation of wax biosynthesis and cuticle morphology. Transgenic grapevines lines overexpressing VviERF045 were obtained, in vitro propagated, phenotypically characterized, and analyzed for the content of specific classes of metabolites. The effect of VviERF045 was correlated with the level of transgene expression, with high-expressing lines showing stunted growth, discolored and smaller leaves, and a lower level of chlorophylls and carotenoids. One line with intermediate expression, L15, was characterized at the transcriptomic level and showed 573 differentially expressed genes compared to wild type plants. Microscopy and gene expression analyses point toward a major role of VviERF045 in epidermis patterning by acting on waxes and cuticle. They also indicate that VviERF045 affects phenolic secondary metabolism and induces a reaction resembling a plant immune response with modulation of receptor like-kinases and pathogen related genes. These results suggest also a possible role of this transcription factor in berry ripening, likely related to changes in epidermis and cuticle of the berry, cell expansion, a decrease in photosynthetic capacity, and the activation of several defense related genes as well as from the phenylpropanoid metabolism. All these processes occur in the berry during ripening.

Variability of candidate genes, genetic structure and association with sugar accumulation and climacteric behavior in a broad germplasm collection of melon (Cucumis melo L.).

BACKGROUND: A collection of 175 melon (Cucumis melo L.) accessions (including wild relatives, feral types, landraces, breeding lines and commercial cultivars) from 50 countries was selected to study the phenotypic variability for ripening behavior and sugar accumulation. The variability of single nucleotide polymorphisms (SNPs) at 53 selected candidate genes involved in sugar accumulation and fruit ripening processes was studied, as well as their association with phenotypic variation of related traits. RESULTS: The collection showed a strong genetic structure, defining seven groups plus a number of accessions that could not be associated to any of the groups (admixture), which fitted well with the botanical classification of melon varieties. The variability in candidate genes for ethylene, cell wall and sugar-related traits was high and similar to SNPs located in reference genes. Variability at ripening candidate genes had an important weight on the genetic stratification of melon germplasm, indicating that traditional farmers might have selected for ripening traits during cultivar diversification. A strong relationship was also found between the genetic structure and phenotypic diversity, which could hamper genetic association studies. Accessions belonging to the ameri group are the most appropriate for association analysis given the high phenotypic and molecular diversity within the group, and lack of genetic structure. The most remarkable association was found between sugar content and SNPs in LG III, where a hotspot of sugar content QTLs has previously been defined. By studying the differences in allelic variation of SNPs within horticultural groups with specific phenotypic features, we also detected differential variation in sugar-related candidates located in LGIX and LGX, and in ripening-related candidates located in LGII and X, all in regions with previously mapped QTLs for the corresponding traits. CONCLUSIONS: In the current study we have found an important variability at both the phenotypic and candidate gene levels for ripening behavior and sugar accumulation in melon fruit. By combination of differences in allelic diversity and association analysis, we have identified several candidate genes that may be involved in the melon phenotypic diversity.

Epigenetic regulation of bud dormancy events in perennial plants.

Release of bud dormancy in perennial plants resembles vernalization in Arabidopsis thaliana and cereals. In both cases, a certain period of chilling is required for accomplishing the reproductive phase, and several transcription factors with the MADS-box domain perform a central regulatory role in these processes. The expression of DORMANCY-ASSOCIATED MADS-box (DAM)-related genes has been found to be up-regulated in dormant buds of numerous plant species, such as poplar, raspberry, leafy spurge, blackcurrant, Japanese apricot, and peach. Moreover, functional evidence suggests the involvement of DAM genes in the regulation of seasonal dormancy in peach. Recent findings highlight the presence of genome-wide epigenetic modifications related to dormancy events, and more specifically the epigenetic regulation of DAM-related genes in a similar way to FLOWERING LOCUS C, a key integrator of vernalization effectors on flowering initiation in Arabidopsis. We revise the most relevant molecular and genomic contributions in the field of bud dormancy, and discuss the increasing evidence for chromatin modification involvement in the epigenetic regulation of seasonal dormancy cycles in perennial plants.

Prediction of components of the sporopollenin synthesis pathway in peach by genomic and expression analyses.

BACKGROUND: The outer cell wall of the pollen grain (exine) is an extremely resistant structure containing sporopollenin, a mixed polymer made up of fatty acids and phenolic compounds. The synthesis of sporopollenin in the tapetal cells and its proper deposition on the pollen surface are essential for the development of viable pollen. The beginning of microsporogenesis and pollen maturation in perennial plants from temperate climates, such as peach, is conditioned by the duration of flower bud dormancy. In order to identify putative genes involved in these processes, we analyzed the results of previous genomic experiments studying the dormancy-dependent gene expression in different peach cultivars. RESULTS: The expression of 50 genes induced in flower buds after the endodormancy period (flower-bud late genes) was compared in ten cultivars of peach with different dormancy behaviour. We found two co-expression clusters enriched in putative orthologs of sporopollenin synthesis and deposition factors in Arabidopsis. Flower-bud late genes were transiently expressed in anthers coincidently with microsporogenesis and pollen maturation processes. We postulated the participation of some flower-bud late genes in the sporopollenin synthesis pathway and the transcriptional regulation of late anther development in peach. CONCLUSIONS: Peach and the model plant Arabidopsis thaliana show multiple elements in common within the essential sporopollenin synthesis pathway and gene expression regulatory mechanisms affecting anther development. The transcriptomic analysis of dormancy-released flower buds proved to be an efficient procedure for the identification of anther and pollen development genes in perennial plants showing seasonal dormancy.

Chilling-dependent release of seed and bud dormancy in peach associates to common changes in gene expression.

Reproductive meristems and embryos display dormancy mechanisms in specialized structures named respectively buds and seeds that arrest the growth of perennial plants until environmental conditions are optimal for survival. Dormancy shows common physiological features in buds and seeds. A genotype-specific period of chilling is usually required to release dormancy by molecular mechanisms that are still poorly understood. In order to find common transcriptional pathways associated to dormancy release, we analyzed the chilling-dependent expression in embryos of certain genes that were previously found related to dormancy in flower buds of peach. We propose the presence of short and long-term dormancy events affecting respectively the germination rate and seedling development by independent mechanisms. Short periods of chilling seem to improve germination in an abscisic acid-dependent manner, whereas the positive effect of longer cold treatments on physiological dwarfing coincides with the accumulation of phenylpropanoids in the seed.

Histone modifications and expression of DAM6 gene in peach are modulated during bud dormancy release in a cultivar-dependent manner.

• Bud dormancy release in many woody perennial plants responds to the seasonal accumulation of chilling stimulus. MADS-box transcription factors encoded by DORMANCY ASSOCIATED MADS-box (DAM) genes in peach (Prunus persica) are implicated in this pathway, but other regulatory factors remain to be identified. In addition, the regulation of DAM gene expression is not well known at the molecular level. • A microarray hybridization approach was performed to identify genes whose expression correlates with the bud dormancy-related behaviour in 10 different peach cultivars. Histone modifications in DAM6 gene were investigated by chromatin immunoprecipitation in two different cultivars. • The expression of DAM4-DAM6 and several genes related to abscisic acid and drought stress response correlated with the dormancy behaviour of peach cultivars. The trimethylation of histone H3 at K27 in the DAM6 promoter, coding region and the second large intron was preceded by a decrease in acetylated H3 and trimethylated H3K4 in the region of translation start, coinciding with repression of DAM6 during dormancy release. • Analysis of chromatin modifications reinforced the role of epigenetic mechanisms in DAM6 regulation and bud dormancy release, and highlighted common features with the vernalization process in Arabidopsis thaliana and cereals.

Identification of genes associated with bud dormancy release in Prunus persica by suppression subtractive hybridization.

To better understand the molecular and physiological mechanisms underlying maintenance and release of seasonal bud dormancy in perennial trees, we identified differentially expressed genes during dormancy progression in reproductive buds from peach (Prunus persica [L.] Batsch) by suppression subtractive hybridization (SSH) and microarray hybridization. Four SSH libraries were constructed, which were respectively enriched in cDNA highly expressed in dormant buds (named DR), in dormancy-released buds (RD) and in the cultivars with different chilling requirement, 'Zincal 5' (ZS) and 'Springlady' (SZ), sampled after dormancy release. About 2500 clones picked from the four libraries were loaded on a glass microarray. Hybridization of microarrays with the final products of SSH procedure was performed in order to validate the selected clones that were effectively enriched in their respective sample. Nearly 400 positive clones were sequenced, which corresponded to 101 different unigenes with diverse functional annotation. We obtained DAM4, 5 and 6 genes coding for MADS-box transcription factors previously related to growth cessation and terminal bud formation in the evergrowing mutant of peach. Several other cDNAs are similar to dormancy factors described in other species, and others have been related to bud dormancy for the first time in this study. Quantitative reverse transcription polymerase chain reaction analysis confirmed differential expression of cDNAs coding for a Zn-finger transcription factor, a GRAS-like regulator, a DNA-binding protein and proteins similar to forisome subunits involved in the reversible occlusion of sieve elements in Fabaceae, among others.