Mitochondrial DNA editing in potato through mitoTALEN and mitoTALECD: molecular characterization and stability of editing events.

BACKGROUND: The aim of this study was to evaluate and characterize the mutations induced by two TALE-based approaches, double-strand break (DSB) induction by the FokI nuclease (mitoTALEN) and targeted base editing by the DddA cytidine deaminase (mitoTALECD), to edit, for the first time, the mitochondrial genome of potato, a vegetatively propagated crop. The two methods were used to knock out the same mitochondrial target sequence (orf125). RESULTS: Targeted chondriome deletions of different sizes (236-1066 bp) were induced by mitoTALEN due to DSB repair through ectopic homologous recombination of short direct repeats (11-12 bp) present in the target region. Furthermore, in one case, the induced DSB and subsequent repair resulted in the amplification of an already present substoichiometric molecule showing a 4288 bp deletion spanning the target sequence. With the mitoTALECD approach, both nonsense and missense mutations could be induced by base substitution. The deletions and single nucleotide mutations were either homoplasmic or heteroplasmic. The former were stably inherited in vegetative offspring. CONCLUSIONS: Both editing approaches allowed us to obtain plants with precisely modified mitochondrial genomes at high frequency. The use of the same plant genotype and mtDNA region allowed us to compare the two methods for efficiency, accuracy, type of modifications induced and stability after vegetative propagation.

Simultaneous improvement of grain yield and grain protein concentration in durum wheat by using association tests and weighted GBLUP.

KEY MESSAGE: Simultaneous improvement for GY and GPC by using GWAS and GBLUP suggested a significant application in durum wheat breeding. Despite the importance of grain protein concentration (GPC) in determining wheat quality, its negative correlation with grain yield (GY) is still one of the major challenges for breeders. Here, a durum wheat panel of 200 genotypes was evaluated for GY, GPC, and their derived indices (GPD and GYD), under eight different agronomic conditions. The plant material was genotyped with the Illumina 25 k iSelect array, and a genome-wide association study was performed. Two statistical models revealed dozens of marker-trait associations (MTAs), each explaining up to 30%. phenotypic variance. Two markers on chromosomes 2A and 6B were consistently identified by both models and were found to be significantly associated with GY and GPC. MTAs identified for phenological traits co-mapped to well-known genes (i.e., Ppd-1, Vrn-1). The significance values (p-values) that measure the strength of the association of each single nucleotide polymorphism marker with the target traits were used to perform genomic prediction by using a weighted genomic best linear unbiased prediction model. The trained models were ultimately used to predict the agronomic performances of an independent durum wheat panel, confirming the utility of genomic prediction, although environmental conditions and genetic backgrounds may still be a challenge to overcome. The results generated through our study confirmed the utility of GPD and GYD to mitigate the inverse GY and GPC relationship in wheat, provided novel markers for marker-assisted selection and opened new ways to develop cultivars through genomic prediction approaches.

Corrigendum to "In silico and in vitro approaches allow the identification of the Prosystemin molecular network" [Comput Struct Biotechnol J 21 (2023) 212-223].

[This corrects the article DOI: 10.1016/j.csbj.2022.12.006.].

Phenolic Compounds and Capsaicinoids in Three Capsicum annuum Varieties: From Analytical Characterization to In Silico Hypotheses on Biological Activity.

The affinity of specific phenolic compounds (PCs) and capsaicinoids (CAPs) present in three Capsicum annuum varieties (Friariello, Cayenne and Dzuljunska Sipka) to the transient receptor potential vanilloid member 1 (TRPV1) was investigated by integrating an analytic approach for the simultaneous extraction and analysis through high-performance liquid chromatography coupled with ion trap mass spectrometry (HPLC/ITMS) and UV detection (HPLC-UV) of PCs and CAPs and structural bioinformatics based on the protein modelling and molecular simulations of protein-ligand docking. Overall, a total of 35 compounds were identified in the different samples and CAPs were quantified. The highest content of total polyphenols was recorded in the pungent Dzuljunska Sipka variety (8.91 ± 0.05 gGAE/Kg DW) while the lowest was found in the non-pungent variety Friariello (3.58 ± 0.02 gGAE/Kg DW). Protein modelling generated for the first time a complete model of the homotetrameric human TRPV1, and it was used for docking simulations with the compounds detected via the analytic approach, as well as with other compounds, as an inhibitor reference. The simulations indicate that different capsaicinoids can interact with the receptor, providing details on the molecular interaction, with similar predicted binding energy values. These results offer new insights into the interaction of capsaicinoids with TRPV1 and their possible actions.

Genetic diversity and signature of divergence in the genome of grapevine clones of Southern Italy varieties.

Sexual reproduction has contributed to a significant degree of variability in cultivated grapevine populations. However, the additional influence of spontaneous somatic mutations has played a pivotal role in shaping the diverse landscape of grapevine agrobiodiversity. These naturally occurring selections, termed 'clones,' represent a vast reservoir of potentially valuable traits and alleles that hold promise for enhancing grape quality and bolstering plant resilience against environmental and biotic challenges. Despite their potential, many of these clones remain largely untapped.In light of this context, this study aims to delve into the population structure, genetic diversity, and distinctive genetic loci within a collection of 138 clones derived from six Campanian and Apulian grapevine varieties, known for their desirable attributes in viticulture and winemaking. Employing two reduced representation sequencing methods, we extracted Single-Nucleotide Polymorphism (SNP) markers. Population structure analysis and fixation index (FST) calculations were conducted both between populations and at individual loci. Notably, varieties originating from the same geographical region exhibited pronounced genetic similarity.The resulting SNP dataset facilitated the identification of approximately two hundred loci featuring divergent markers (FST ≥ 0.80) within annotated exons. Several of these loci exhibited associations with essential traits like phenotypic adaptability and environmental responsiveness, offering compelling opportunities for grapevine breeding initiatives. By shedding light on the genetic variability inherent in these treasured traditional grapevines, our study contributes to the broader understanding of their potential. Importantly, it underscores the urgency of preserving and characterizing these valuable genetic resources to safeguard their intra-varietal diversity and foster future advancements in grapevine cultivation.

In silico and in vitro approaches allow the identification of the Prosystemin molecular network.

Tomato Prosystemin (ProSys), the precursor of Systemin, a small peptidic hormone, is produced at very low concentration in unchallenged plants, while its expression greatly increases in response to several different stressors triggering an array of defence responses. The molecular mechanisms that underpin such a wide array of defence barriers are not fully understood and are likely correlated with the intrinsically disordered (ID) structure of the protein. ID proteins interact with different protein partners forming complexes involved in the modulation of different biological mechanisms. Here we describe the ProSys-protein network that shed light on the molecular mechanisms underpinning ProSys associated defence responses. Three different approaches were used. In silico prediction resulted in 98 direct interactors, most clustering in phytohormone biosynthesis, transcription factors and signal transduction gene classes. The network shows the central role of ProSys during defence responses, that reflects its role as central hub. In vitro ProSys interactors, identified by Affinity Purification-Mass Spectrometry (AP-MS), revealed over three hundred protein partners, while Bimolecular Fluorescent Complementation (BiFC) experiments validated in vivo some interactors predicted in silico and in vitro. Our results demonstrate that ProSys interacts with several proteins and reveal new key molecular events in the ProSys-dependent defence response of tomato plant.

Whole-exome sequencing of selected bread wheat recombinant inbred lines as a useful resource for allele mining and bulked segregant analysis.

Although wheat (Triticum aestivum L.) is the main staple crop in the world and a major source of carbohydrates and proteins, functional genomics and allele mining are still big challenges. Given the advances in next-generation sequencing (NGS) technologies, the identification of causal variants associated with a target phenotype has become feasible. For these reasons, here, by combining sequence capture and target-enrichment methods with high-throughput NGS re-sequencing, we were able to scan at exome-wide level 46 randomly selected bread wheat individuals from a recombinant inbred line population and to identify and classify a large number of single nucleotide polymorphisms (SNPs). For technical validation of results, eight randomly selected SNPs were converted into Kompetitive Allele-Specific PCR (KASP) markers. This resource was established as an accessible and reusable molecular toolkit for allele data mining. The dataset we are making available could be exploited for novel studies on bread wheat genetics and as a foundation for starting breeding programs aimed at improving different key agronomic traits.

Phyto-Friendly Soil Bacteria and Fungi Provide Beneficial Outcomes in the Host Plant by Differently Modulating Its Responses through (In)Direct Mechanisms.

Sustainable agricultural systems based on the application of phyto-friendly bacteria and fungi are increasingly needed to preserve soil fertility and microbial biodiversity, as well as to reduce the use of chemical fertilizers and pesticides. Although there is considerable attention on the potential applications of microbial consortia as biofertilizers and biocontrol agents for crop management, knowledge on the molecular responses modulated in host plants because of these beneficial associations is still incomplete. This review provides an up-to-date overview of the different mechanisms of action triggered by plant-growth-promoting microorganisms (PGPMs) to promote host-plant growth and improve its defense system. In addition, we combined available gene-expression profiling data from tomato roots sampled in the early stages of interaction with Pseudomonas or Trichoderma strains to develop an integrated model that describes the common processes activated by both PGPMs and highlights the host's different responses to the two microorganisms. All the information gathered will help define new strategies for the selection of crop varieties with a better ability to benefit from the elicitation of microbial inoculants.

Integration of high-throughput phenotyping with anatomical traits of leaves to help understanding lettuce acclimation to a changing environment.

MAIN CONCLUSION: The combination of image-based phenotyping with in-depth anatomical analysis allows for a thorough investigation of plant physiological plasticity in acclimation, which is driven by environmental conditions and mediated by anatomical traits. Understanding the ability of plants to respond to fluctuations in environmental conditions is critical to addressing climate change and unlocking the agricultural potential of crops both indoor and in the field. Recent studies have revealed that the degree of eco-physiological acclimation depends on leaf anatomical traits, which show stress-induced alterations during organogenesis. Indeed, it is still a matter of debate whether plant anatomy is the bottleneck for optimal plant physiology or vice versa. Here, we cultivated 'Salanova' lettuces in a phenotyping chamber under two different vapor pressure deficits (VPDs; low, high) and watering levels (well-watered, low-watered); then, plants underwent short-term changes in VPD. We aimed to combine high-throughput phenotyping with leaf anatomical analysis to evaluate their capability in detecting the early stress signals in lettuces and to highlight the different degrees of plants' eco-physiological acclimation to the change in VPD, as influenced by anatomical traits. The results demonstrate that well-watered plants under low VPD developed a morpho-anatomical structure in terms of mesophyll organization, stomatal and vein density, which more efficiently guided the acclimation to sudden changes in environmental conditions and which was not detected by image-based phenotyping alone. Therefore, we emphasized the need to complement high-throughput phenotyping with anatomical trait analysis to unveil crop acclimation mechanisms and predict possible physiological behaviors after sudden environmental fluctuations due to climate changes.

Multi-omics data integration provides insights into the post-harvest biology of a long shelf-life tomato landrace.

In this study we investigated the transcriptome and epigenome dynamics of the tomato fruit during post-harvest in a landrace belonging to a group of tomatoes (Solanum lycopersicum L.) collectively known as "Piennolo del Vesuvio", all characterized by a long shelf-life. Expression of protein-coding genes and microRNAs as well as DNA methylation patterns and histone modifications were analysed in distinct post-harvest phases. Multi-omics data integration contributed to the elucidation of the molecular mechanisms underlying processes leading to long shelf-life. We unveiled global changes in transcriptome and epigenome. DNA methylation increased and the repressive histone mark H3K27me3 was lost as the fruit progressed from red ripe to 150 days post-harvest. Thousands of genes were differentially expressed, about half of which were potentially epi-regulated as they were engaged in at least one epi-mark change in addition to being microRNA targets in ~5% of cases. Down-regulation of the ripening regulator MADS-RIN and of genes involved in ethylene response and cell wall degradation was consistent with the delayed fruit softening. Large-scale epigenome reprogramming that occurred in the fruit during post-harvest likely contributed to delayed fruit senescence.

Merging genotyping-by-sequencing data from two ex situ collections provides insights on the pea evolutionary history.

Pea (Pisum sativum L. subsp. sativum) is one of the oldest domesticated species and a widely cultivated legume. In this study, we combined next generation sequencing (NGS) data referring to two genotyping-by-sequencing (GBS) libraries, each one prepared from a different Pisum germplasm collection. The selection of single nucleotide polymorphism (SNP) loci called in both germplasm collections caused some loss of information; however, this did not prevent the obtainment of one of the largest datasets ever used to explore pea biodiversity, consisting of 652 accessions and 22 127 markers. The analysis of population structure reflected genetic variation based on geographic patterns and allowed the definition of a model for the expansion of pea cultivation from the domestication centre to other regions of the world. In genetically distinct populations, the average decay of linkage disequilibrium (LD) ranged from a few bases to hundreds of kilobases, thus indicating different evolutionary histories leading to their diversification. Genome-wide scans resulted in the identification of putative selective sweeps associated with domestication and breeding, including genes known to regulate shoot branching, cotyledon colour and resistance to lodging, and the correct mapping of two Mendelian genes. In addition to providing information of major interest for fundamental and applied research on pea, our work describes the first successful example of integration of different GBS datasets generated from ex situ collections - a process of potential interest for a variety of purposes, including conservation genetics, genome-wide association studies, and breeding.

Sweet Cherry Diversity and Relationships in Modern and Local Varieties Based on SNP Markers.

The sweet cherry is an important fruit species that is widespread globally. In addition to the well-known traditional and modern varieties, a myriad of landraces is present in Europe, as well as in southern Italy. This study aims to evaluate the population structure, genetic relationships, and cases of duplicate samples in a collection of 143 accessions using GBS-derived SNP markers. The genetic material under investigation includes modern commercial varieties, ancient European and American varieties, landraces, and individuals retrieved from small orchards. Some of the known varieties were genetically analyzed here for the first time. In addition, several genotypes were collected from the Basilicata region (southern Italy), an area largely unexplored for sweet cherry genetic resources. The relationships among genotypes were assessed using four different methods: allele frequency and ancestry estimation, principal component analysis, Neighbor-Joining tree, and identity-by-state estimation. The analyses returned quite congruent results and highlighted the presence of four main genetic groups, namely: (i) American varieties, (ii) the 'Germersdorfer-Ferrovia' cluster, (iii) the 'Burlat' group, and (iv) the group of Italian landraces. The main drivers of clustering were ancestry, geographical distribution, and some important traits such as self-compatibility. The sweet cherries from Basilicata, herewith examined for the first time, were mostly distributed within the group of Italian landraces, being particularly linked to the autochthonous varieties of the Campania region. However, some genotypes were outside this group, thus suggesting the introduction of genetic material from other Italian regions or from European countries. The considerable amount of American and European modern varieties analyzed are genetically very closely related, suggesting a reduced genetic basis. In addition, we highlighted the discriminating ability of SNP markers to distinguish between an original variety and its mutant. Overall, our results may be useful in defining conservation strategies for sweet cherry germplasm and developing future breeding programs to enlarge the genetic basis of commercial varieties.

The Tomato Interspecific NB-LRR Gene Arsenal and Its Impact on Breeding Strategies.

Tomato (Solanum lycopersicum L.) is a model system for studying the molecular basis of resistance in plants. The investigation of evolutionary dynamics of tomato resistance (R)-loci provides unique opportunities for identifying factors that promote or constrain genome evolution. Nucleotide-binding domain and leucine-rich repeat (NB-LRR) receptors belong to one of the most plastic and diversified families. The vast amount of genomic data available for Solanaceae and wild tomato relatives provides unprecedented insights into the patterns and mechanisms of evolution of NB-LRR genes. Comparative analysis remarked a reshuffling of R-islands on chromosomes and a high degree of adaptive diversification in key R-loci induced by species-specific pathogen pressure. Unveiling NB-LRR natural variation in tomato and in other Solanaceae species offers the opportunity to effectively exploit genetic diversity in genomic-driven breeding programs with the aim of identifying and introducing new resistances in tomato cultivars. Within this motivating context, we reviewed the repertoire of NB-LRR genes available for tomato improvement with a special focus on signatures of adaptive processes. This issue is still relevant and not thoroughly investigated. We believe that the discovery of mechanisms involved in the generation of a gene with new resistance functions will bring great benefits to future breeding strategies.

Almond diversity and homozygosity define structure, kinship, inbreeding, and linkage disequilibrium in cultivated germplasm, and reveal genomic associations with nut and seed weight.

Almond [Prunus dulcis Miller (D.A. Webb)] is the main tree nut species worldwide. Here, genotyping-by-sequencing (GBS) was applied to 149 almond cultivars from the ex situ collections of the Italian Council for Agricultural Research (CREA) and the Spanish National Research Council (CSIC), leading to the detection of 93,119 single-nucleotide polymorphisms (SNPs). The study of population structure outlined four distinct genetic groups and highlighted diversification between the Mediterranean and Californian gene pools. Data on SNP diversity and runs of homozygosity (ROHs) allowed the definition of kinship, inbreeding, and linkage disequilibrium (LD) decay in almond cultivated germplasm. Four-year phenotypic observations, gathered on 98 cultivars of the CREA collection, were used to perform a genome-wide association study (GWAS) and, for the first time in a crop species, homozygosity mapping (HM), resulting in the identification of genomic associations with nut, shell, and seed weight. Both GWAS and HM suggested that loci controlling nut and seed weight are mostly independent. Overall, this study provides insights on the almond cultivation history and delivers information of major interest for almond genetics and breeding. In a broader perspective, our results encourage the use of ROHs in crop science to estimate inbreeding, choose parental combinations minimizing the risk of inbreeding depression, and identify genomic footprints of selection for specific traits.

Guidelines for Setting Up a mRNA Sequencing Experiment and Best Practices for Bioinformatic Data Analysis.

RNA-sequencing, commonly referred to as RNA-seq, is the most recently developed method for the analysis of transcriptomes. It uses high-throughput next-generation sequencing technologies and has revolutionized our understanding of the complexity and dynamics of whole transcriptomes.In this chapter, we recall the key developments in transcriptome analysis and dissect the different steps of the general workflow that can be run by users to design and perform a mRNA-seq experiment as well as to process mRNA-seq data obtained by the Illumina technology. The chapter proposes guidelines for completing a mRNA-seq study properly and makes available recommendations for best practices based on recent literature and on the latest developments in technology and algorithms. We also remark the large number of choices available (especially for bioinformatic data analysis) in front of which the scientist may be in trouble.In the last part of the chapter we discuss the new frontiers of single-cell RNA-seq and isoform sequencing by long read technology.

Cultivated Tomato (Solanum lycopersicum L.) Suffered a Severe Cytoplasmic Bottleneck during Domestication: Implications from Chloroplast Genomes.

In various crops, genetic bottlenecks occurring through domestication can limit crop resilience to biotic and abiotic stresses. In the present study, we investigated nucleotide diversity in tomato chloroplast genome through sequencing seven plastomes of cultivated accessions from the Campania region (Southern Italy) and two wild species among the closest (Solanum pimpinellifolium) and most distantly related (S. neorickii) species to cultivated tomatoes. Comparative analyses among the chloroplast genomes sequenced in this work and those available in GenBank allowed evaluating the variability of plastomes and defining phylogenetic relationships. A dramatic reduction in genetic diversity was detected in cultivated tomatoes, nonetheless, a few de novo mutations, which still differentiated the cultivated tomatoes from the closest wild relative S. pimpinellifolium, were detected and are potentially utilizable as diagnostic markers. Phylogenetic analyses confirmed that S. pimpinellifolium is the closest ancestor of all cultivated tomatoes. Local accessions all clustered together and were strictly related with other cultivated tomatoes (S. lycopersicum group). Noteworthy, S. lycopersicum var. cerasiforme resulted in a mixture of both cultivated and wild tomato genotypes since one of the two analyzed accessions clustered with cultivated tomato, whereas the other with S. pimpinellifolium. Overall, our results revealed a very reduced cytoplasmic variability in cultivated tomatoes and suggest the occurrence of a cytoplasmic bottleneck during their domestication.