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

Tecnología y educación en estudiantes universitarios

Presentamos el avance de los resultados del proyecto UBACyT: "Usos de las TICs en estudiantes universitarios y su relación con las estrategias de aprendizaje y estudio", donde se desarrolló y perfeccionó el instrumento sobre usos de las TICS, que fuera iniciado en el Proyecto UBACyT P413. Este instrumento nos permitió profundizar, actualizar e indagar otras instancias de consumo e interacción de los jóvenes como las redes sociales. También hemos correlacionados los resultados con el inventario de Estrategias de Aprendizaje y Estudio LASSI (Liporace; 2009). El objetivo general fue el de identificar las posibles relaciones entre los usos de las TICS y las estrategias de estudio y aprendizaje en estudiantes universitarios de la UBA; en función de corroborar si los usos espontáneos de la tecnología y las estrategias y competencias desplegadas a partir de ello tienen alguna relación con las estrategias de estudio y aprendizaje que se despliegan en el ámbito académico. Con esta finalidad se seleccionó una muestra de tipo intencional, no probabilística. Se utilizó para el análisis de los datos estadística descriptiva e inferencial.Facultad de Psicologí

Tecnología y educación en estudiantes universitarios

Presentamos el avance de los resultados del proyecto UBACyT: "Usos de las TICs en estudiantes universitarios y su relación con las estrategias de aprendizaje y estudio", donde se desarrolló y perfeccionó el instrumento sobre usos de las TICS, que fuera iniciado en el Proyecto UBACyT P413. Este instrumento nos permitió profundizar, actualizar e indagar otras instancias de consumo e interacción de los jóvenes como las redes sociales. También hemos correlacionados los resultados con el inventario de Estrategias de Aprendizaje y Estudio LASSI (Liporace; 2009). El objetivo general fue el de identificar las posibles relaciones entre los usos de las TICS y las estrategias de estudio y aprendizaje en estudiantes universitarios de la UBA; en función de corroborar si los usos espontáneos de la tecnología y las estrategias y competencias desplegadas a partir de ello tienen alguna relación con las estrategias de estudio y aprendizaje que se despliegan en el ámbito académico. Con esta finalidad se seleccionó una muestra de tipo intencional, no probabilística. Se utilizó para el análisis de los datos estadística descriptiva e inferencial.Facultad de Psicologí

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

Disease Resistant Citrus Breeding Using Newly Developed High Resolution Melting and CAPS Protocols for Alternaria Brown Spot Marker Assisted Selection

Alternaria alternata is a fungus that causes a serious disease in susceptible genotypes of citrus, particularly in mandarins. The Alternaria citri toxin (ACT) produced by the pathogen induces necrotic lesions on young leaves and fruits, defoliation and fruit drop. Here, we describe two methods of marker-assisted selection (MAS) that could be used for the early identification of Alternaria brown spot (ABS)-resistant mandarin hybrids. The first method is based on a nested PCR coupled to high resolution melting (HRM) analysis at the SNP08 locus, which is located at 0.4 cM from the ABS resistance locus, and was previously indicated as the most suitable for the selection of ABS-resistant hybrids. The method was validated on 41 mandarin hybrids of the CREA germplasm collection, and on 862 progenies generated from five crosses involving different susceptible parents. Four out of five populations showed Mendelian segregation at the analyzed locus, while a population involving Murcott tangor as male parent showed distorted segregation toward the susceptible hybrids. The second method is based on a cleaved amplified polymorphic sequences (CAPS) marker that was developed using the same primers as the nested PCR at the SNP08 locus, coupled with BccI restriction enzyme digestion. To verify the reliability of the two genotyping methods, in vitro leaf phenotyping was carried out by inoculating A. alternata spores onto young leaves of 101 hybrids, randomly chosen among the susceptible and resistant progenies. The phenotyping confirmed the SNP08 genotyping results, so the proposed method of selection based on HRM or CAPS genotyping could be routinely used as an alternative to KBioscience competitive allele specific polymerase chain reaction (KASPar) single nucleotide polymorphism (SNP) genotyping system to improve citrus breeding programs. While the study confirmed that the SNP08 marker is a reliable tool for MAS of new citrus hybrids with different genetic backgrounds, it also identified a small group of genotypes where the resistance mechanism requires further investigation

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

A dual sgRNA-directed CRISPR/Cas9 construct for editing the fruit-specific β-cyclase 2 gene in pigmented citrus fruits

CRISPR/Cas9 genome editing is a modern biotechnological approach used to improve plant varieties, modifying only one or a few traits of a specific variety. However, this technology cannot be easily used to improve fruit quality traits in citrus, due to the lack of knowledge of key genes, long juvenile stage, and the difficulty regenerating whole plants of specific varieties. Here, we introduce a genome editing approach with the aim of producing citrus plantlets whose fruits contain both lycopene and anthocyanins. Our method employs a dual single guide RNA (sgRNA)-directed genome editing approach to knockout the fruit-specific β-cyclase 2 gene, responsible for the conversion of lycopene to beta-carotene. The gene is targeted by two sgRNAs simultaneously to create a large deletion, as well as to induce point mutations in both sgRNA targets. The EHA105 strain of Agrobacterium tumefaciens was used to transform five different anthocyanin-pigmented sweet oranges, belonging to the Tarocco and Sanguigno varietal groups, and ‘Carrizo’ citrange, a citrus rootstock as a model for citrus transformation. Among 58 plantlets sequenced in the target region, 86% of them were successfully edited. The most frequent mutations were deletions (from -1 to -74 nucleotides) and insertions (+1 nucleotide). Moreover, a novel event was identified in six plantlets, consisting of the inversion of the region between the two sgRNAs. For 20 plantlets in which a single mutation occurred, we excluded chimeric events. Plantlets did not show an altered phenotype in vegetative tissues. To the best of our knowledge, this work represents the first example of the use of a genome editing approach to potentially improve qualitative traits of citrus fruit

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