FRUIT FLESH IN PEACH:CHARACTERIZATION OF THE 'SLOW SOFTENING' TEXTURE

The aim of this research was to deepen the knowledge about the slow softening texture in peach. The texture is a synthesis of several parameters detected by senses, derived from the food structure. The paramount sense in the texture perception is the tactile one, principally perceived by hand and mouth. The tactile perception is a combination of four classes of mechanoreceptors, each one specialized to perceive mechanic deformation with different speed. This combined perception influences the consumer evaluation of food quality, giving the texture importance among food characteristics. The texture could also affect the taste perception through mechanical actions on food structure. The mechanical property linked to the texture is associated with the cellular organization and the cell wall strength. The main cell wall component affecting texture in fresh fruit is pectin, a polymer of galacturonic acid. The disassembly of pectin involves several enzymatic and non-enzymatic activities acting directly in pectin cleavage or indirectly disrupting non-covalent interactions. The gold standard of texture analyses is the sensorial one, however several issues make sensorial analyses inapplicable to breeding programs to select plant with improved fruit texture. Several efforts were made to achieve instrumental analyses capable of substitute humans in texture analyses. To mimic the tactile sense, a discipline studying the material response to an applied force, the rheology, is applied. The easiest instrumental measure of rheology parameters is the penetrometer test, diffused to measure the firmness, but exploitable to collect the Young\u2019s modulus and the slope of yield stress represented respectively elasticity and fracturability. In peach, so far at least four textures were described, melting (M), stony hard (SH), non-melting (NM) and slow softening (SS). Prior to this work, no reliable objective nor fast tool were available to phenotype and select the SS trait in peach germplasm. The only reliable approach was a sensorial assessment done by a texture-trained panel, requiring repeated and time-consuming assessment. An objective, instrumental method, was set up by processing the data of a digital penetrometer test. The penetrometer itself, as reported in paragraph 2, does not support the ability to discriminate among the different texture types, as already reported in other works. In addition, this method appears to be affected by the fruit ripening season, since the early-ripening accessions tend to show faster loss of firmness, while the late-ripening exhibit a slower firmness loss. Using the data collected in our experiment, the texture dynamic (TD) model was developed from the observation of differences in the rheogram shape due to the elasticity and fracturability parameters. The TD model, that excludes the firmness effect on the fracturability and elasticity parameters, was thus developed, after testing it on 20 accessions in three years, allowing for reliable discrimination between SS and M phenotype. Differences in the TD were also found when comparing M vs SH and M vs NM textures. In particular, when comparing M and SS, TD value is explained for the 96% from the texture. The developed method was then applied (together with sensorial evaluation) to genetically dissect the SS trait. Association and QTL mapping approaches were combined by analyzing a germplasm panel and a biparental progeny, and a single locus at the end of chromosome 8 was identified. RNA-seq analysis of 2 SS and 2 M accessions suggested some common features with the SH type described in literature. In both texture types a lower auxin response was found when compared to the M type. This agrees with the already known activity of auxin in the modulation of cell wall rearrangement and expansion. Therefore, slower softening could be associated to slower cell wall rearrangement. In future, comparison of auxin content in slow softening and melting type peaches might provide further insight into the validity of this hypothesis. In detail, by RNA-seq comparing M and SS a total of 64 differentially expressed genes were found in the genomic region harboring the SS locus. Out of these 64 genes, 16 are uncharacterized, while among the characterized ones, 4 are putatively involved in auxin response based on peach genome annotation. Analysis of polymorphisms in these 4 DEGs based on resequencing data of the \u2018Max10\u2019 and \u2018Rebus 028\u2019 parents of biparental population did not uncover any variants in agreement with the observed segregation. Analyzing 2kb gene models flanking regions, 16 genes were associated with polymorphisms outside the coding sequence: the possible regulatory effects of such variants require further evaluation by expression analyses. In summary, the major results are the setup of a reliable tool to score objectively the SS texture and the detection of a major locus and his dominant mendelian inheritance. However, NGS and RNA-seq approaches are presented as a speculative data only, because they are not supported by hormones content in fruit, and the large locus detected did not allow indication of a putative variant. These results will: a) give impetus in exploring SS genetic and physiology; b) support the design of future crosses and experiments; c) increase marker density in the locus; d) point out the possible central role of auxin (to validate the hypothesis of a similarity between SS and SH physiology); e) allow texture assessment of improved cultivars; and f) allow phenotyping of segregating progenies to develop molecular markers associated with the SS trait

Genetic Dissection of Complex Fruit Quantitative Traits in Peach Progen

Major research efforts in peach are dedicated to the discovery of genomic variants causing phenotypic effects in complex fruit traits such as: maturity date (MD), fruit size (FW), sugar (SSC) and acid content (TA), flesh texture (slow softening, SSf) and resistance to brown rot by Monilinia spp. (BRr). Five segregating progenies showing phenotypic variation for at least one of these traits are available in our experimental fields. For SSC and TA, an already validated approach based on Near-InfraRed spectroscopy (NIR), is being applied to phenotype some segregating progenies. For SSf and BRr instead, trait characterization has been performed, resulting in the identification of co-factor traits and definition of standardized phenotyping tools, which are currently applied in the characterization of segregating material (in the context of FruitBreedomics EU project). High-density linkage maps have been constructed with genotypic data obtained from IPSC Illumina 9K SNP chip (Italian Drupomics and FruitBreedomics frameworks) and Genotyping-by-Sequencing (GBS). Additionally, parents of these progenies have been re-sequenced (30-40x) and genetic variants present along their genomes have been identified. Multiple-QTL models (MQM) coupled with the use of co-factor traits is leading to the discovery of significant QTLs. Genomic variants are explored within QTL intervals on the genomes of progeny parents, in order to identify possible mutations causing phenotypic differences, and develop markers for marker-assisted selection approaches

Deletion of the miR172 target site in a TOE-type gene is a strong candidate variant for dominant double-flower trait in Rosaceae

Double flowers with supernumerary petals have been selected by humans for their attractive appearance and commercial value in several ornamental plants, including Prunus persica (peach), a recognized model for Rosaceae genetics and genomics. Despite the relevance of this trait, knowledge of the underlying genes is limited. Of two distinct loci controlling the double-flower phenotype in peach, we focused on the dominant Di2 locus. High-resolution linkage mapping in five segregating progenies delimited Di2 to an interval spanning 150858bp and 22 genes, including Prupe.6G242400 encoding an euAP2 transcription factor. Analyzing genomic resequencing data from single- and double-flower accessions, we identified a deletion spanning the binding site for miR172 in Prupe.6G242400 as a candidate variant for the double-flower trait, and we showed transcript expression for both wild-type and deleted alleles. Consistent with the proposed role in controlling petal number, Prupe.6G242400 is expressed in buds at critical times for floral development. The indelDi2 molecular marker designed on this sequence variant co-segregated with the phenotype in 621 progenies, accounting for the dominant inheritance of the Di2 locus. Further corroborating the results in peach, we identified a distinct but similar mutation in the ortholog of Prupe.6G242400 in double-flower roses. Phylogenetic analysis showed that these two genes belong to a TARGET OF EAT (TOE)-type clade not represented in Arabidopsis, indicating a divergence of gene functions between AP2-type and TOE-type factors in Arabidopsis and other species. The identification of orthologous candidate genes for the double-flower phenotype in two important Rosaceae species provides valuable information to understand the genetic control of this trait in other major ornamental plants. Significance Statement We used peach as a model to gain insight into the molecular basis of double flowers, an important trait in many ornamental plants. We propose that a deletion causes a TOE-type transcription factor to escape miR172-mediated repression, in turn resulting in an increased number of petals, as corroborated by results on the orthologous gene in rose

Integrative genomics approaches validate PpYUC11-like as candidate gene for the stony hard trait in peach (P. persica L. Batsch)

Texture is one of the most important fruit quality attributes. In peach, stony hard (SH) is a recessive monogenic trait (hd/hd) that confers exceptionally prolonged firm flesh to fully ripe fruit. Previous studies have shown that the SH mutation affects the fruit ability to synthesize appropriate amounts of indol-3-acetic acid (IAA), which orchestrates the ripening processes through the activation of system 2 ethylene pathway. Allelic variation in a TC microsatellite located within the first intron of PpYUC11-like (a YUCCA-like auxin-biosynthesis gene) has been recently proposed as the causal mutation of the SH phenotype

The role of Italy in the use of advanced plant breeding techniques on fruit trees: state of the art and future perspectives

Climate change is deeply impacting the food chain production, lowering quality and yield. In this context, the international scientific community has dedicated many efforts to enhancing resilience and sustainability in agriculture. Italy is among the main European producers of several fruit trees; therefore, national research centers and universities undertook several initiatives to maintain the specificity of the ‘Made in Italy’ label. Despite their importance, fruit crops are suffering from difficulties associated with the conventional breeding approaches, especially in terms of financial commitment, land resources availability, and long generation times. The ‘new genomic techniques’ (NGTs), renamed in Italy as ‘technologies for assisted evolution’ (TEAs), reduce the time required to obtain genetically improved cultivars while precisely targeting specific DNA sequences. This review aims to illustrate the role of the Italian scientific community in the use of NGTs, with a specific focus on Citrus, grapevine, apple, pear, chestnut, strawberry, peach, and kiwifruit. For each crop, the key genes and traits on which the scientific community is working, as well as the technological improvements and advancements on the regeneration of local varieties, are presented. Lastly, a focus is placed on the legal aspects in the European and in Italian context

PeachVar-DB : Curated Collection of Genetic Variations for the Interactive Analysis of Peach Genome Data

Applying next-generation sequencing (NGS) technologies to species of agricultural interest has the potential to accelerate the understanding and exploration of genetic resources. The storage, availability and maintenance of huge quantities of NGS-generated data remains a major challenge. The PeachVar-DB portal, available at http://hpc-bioinformatics.cineca.it/peach, is an open-source catalog of genetic variants present in peach (Prunus persica L. Batsch) and wild-related species of Prunus genera, annotated from 146 samples publicly released on the Sequence Read Archive (SRA). We designed a user-friendly web-based interface of the database, providing search tools to retrieve single nucleotide polymorphism (SNP) and InDel variants, along with useful statistics and information. PeachVar-DB results are linked to the Genome Database for Rosaceae (GDR) and the Phytozome database to allow easy access to other external useful plant-oriented resources. In order to extend the genetic diversity covered by the PeachVar-DB further, and to allow increasingly powerful comparative analysis, we will progressively integrate newly released data

QTL mapping for brown rot (Monilinia fructigena) resistance in an intraspecific peach (Prunus persica L. Batsch) F1 progeny

Brown rot (BR) caused by Monilinia spp. leads to significant post-harvest losses in stone fruit production, especially peach. Previous genetic analyses in peach progenies suggested that BR resistance segregates as a quantitative trait. In order to uncover genomic regions associated with this trait and identify molecular markers for assisted selection (MAS) in peach, an F1 progeny from the cross "Contender" (C, resistant) 7 "Elegant Lady" (EL, susceptible) was chosen for quantitative trait loci (QTL) analysis. Over two phenotyping seasons, skin (SK) and flesh (FL) artificial infections were performed on fruits using a Monilinia fructigena isolate. For each treatment, infection frequency (if) and average rot diameter (rd) were scored. Significant seasonal and intertrait correlations were found. Maturity date (MD) was significantly correlated with disease impact. Sixty-three simple sequence repeats (SSRs) plus 26 single-nucleotide polymorphism (SNP) markers were used to genotype the C 7 EL population and to construct a linkage map. C 7 EL map included the eight Prunus linkage groups (LG), spanning 572.92 cM, with an average interval distance of 6.9 cM, covering 78.73 % of the peach genome (V1.0). Multiple QTL mapping analysis including MD trait as covariate uncovered three genomic regions associated with BR resistance in the two phenotyping seasons: one containing QTLs for SK resistance traits near M1a (LG C 7 EL-2, R2 = 13.1-31.5 %) and EPPISF032 (LG C 7 EL-4, R2 = 11-14 %) and the others containing QTLs for FL resistance, near markers SNP_IGA_320761 and SNP_IGA_321601 (LG3, R2 = 3.0-11.0 %). These results suggest that in the C 7 EL F1 progeny, skin resistance to fungal penetration and flesh resistance to rot spread are distinguishable mechanisms constituting BR resistance trait, associated with different genomic regions. Discovered QTLs and their associated markers could assist selection of new cultivars with enhanced resistance to Monilinia spp. in fruit

Fruit ripening evolution in diverse commercial apricots by conventional and non-destructive methods: preliminary results

Cultivated apricot (Prunus armeniaca L.) is represented by a growing number of commercial cultivars featuring a wide variability of flesh texture and ripening evolution, affecting the entire chain from harvest to the final consumer. In order to establish fast and reliable tools easy applicable in the field to determine the harvest time, diverse commercial apricots were evaluated over 2 years for fruit ripening evolution by conventional and non-destructive methods. All fruit were harvested from a single tree at physiological ripening onset, then fruit were graded with a DA-meter\uae device, according to three ripening stages (physiological and commercial ripening, veraison), 30 fruit each. Fruit from each stage, after being scored by a near-infrared analyzer, were than randomly divided into three batches, each of which was subjected to standard ripening assessment: firmness (Fi) by using a digital penetrometer, soluble solids content (SSC) by using a digital refractometer, and total acidity (TA) by titration and pH measurement. Fi and SSC were assessed on both fruit sides, while TA and pH were determined on 10 homogenized fruits. The curves produced after optimization showed good predictability, particularly for TA, SSC and pH: R2 was 78.57, 79.17 and 80.56 (in cross-validation, respectively), despite the diversity of the assessed cultivars and the different stages of ripening. Some more work still has to be done to improve the predictability of Fi (R2=38.53), and further investigation has to be carried out to better fit the data from the penetrometer, or other devices measuring flesh texture. These results are to be considered as preliminary for the setting up of cultivar-specific harvest parameters

Stem Xylem Characterization for Vitis Drought Tolerance

Together with stomatal conductance and root conductivity, the stem water reserve and transport systems could be regulatory mechanisms able to participate in the regulation of the plant water status. Lianas, such as Vitis spp., minimize the trunk support role, and stems have evolved to improve their ability in water transport. In this work, stems of 10 different Vitis species were studied in relation to their expected drought tolerance using reflectance spectroscopy. Spectra were measured before (T0) and after coloration with Sudan IV dye. The T0 spectral signature showed characteristic species features. The partial least squares (PLS) regression and the self-organizing map (SOM) neural network analysis were able to predict the expected drought tolerance score; thus, reflectance spectroscopy was demonstrated to be a useful technique for drought tolerance phenotyping. These methods could be applied for the preliminary selection of new rootstocks/cultivars. Wood composition variation appeared to be correlated with the water stress susceptibility. To clarify this relationship, the attention was focused on the wood hydrophobicity. Sudan IV is a microscopy dye traditionally used to underline suberin, waxes, and, in general, hydrophobic substances. Differences between rough and colored spectra evidenced the absorption band of Sudan IV with a maximum at 539 nm. The coloration intensity was used to develop a hydrophobicity index. The obtained values were correlated with the expected drought tolerance score. Therefore, hydrophobic compounds seem to play an important role in water use efficiency, and an hydrophobic barrier in the xylem tissue appears to be a protective mechanism against water stress