Not Only Editing: A Cas-Cade of CRISPR/Cas-Based Tools for Functional Genomics in Plants and Animals.

The advent of CRISPR/Cas9 technology has revolutionized genome editing, enabling the attainment of once-unimaginable goals. CRISPR/Cas's groundbreaking attributes lie in its simplicity, versatility, universality, and independence from customized DNA-protein systems, erasing the need for specialized expertise and broadening its scope of applications. It is therefore more and more used for genome modification including the generation of mutants. Beyond such editing scopes, the recent development of novel or modified Cas-based systems has spawned an array of additional biotechnological tools, empowering both fundamental and applied research. Precisely targeting DNA or RNA sequences, the CRISPR/Cas system has been harnessed in fields as diverse as gene regulation, deepening insights into gene expression, epigenetic changes, genome spatial organization, and chromatin dynamics. Furthermore, it aids in genome imaging and sequencing, as well as effective identification and countering of viral pathogens in plants and animals. All in all, the non-editing aspect of CRISPR/Cas exhibits tremendous potential across diverse domains, including diagnostics, biotechnology, and fundamental research. This article reviews and critically evaluates the primary CRISPR/Cas-based tools developed for plants and animals, underlining their transformative impact.

Past, present, and future of genetic strategies to control tolerance to the main fungal and oomycete pathogens of grapevine.

The production of high-quality wines is strictly related to the correct management of the vineyard, which guarantees good yields and grapes with the right characteristics required for subsequent vinification. Winegrowers face a variety of challenges during the grapevine cultivation cycle: the most notorious are fungal and oomycete diseases such as downy mildew, powdery mildew, and gray mold. If not properly addressed, these diseases can irremediably compromise the harvest, with disastrous consequences for the production and wine economy. Conventional defense methods used in the past involved chemical pesticides. However, such approaches are in conflict with the growing attention to environmental sustainability and shifts from the uncontrolled use of chemicals to the use of integrated approaches for crop protection. Improvements in genetic knowledge and the availability of novel biotechnologies have created new scenarios for possibly producing grapes with a reduced, if not almost zero, impact. Here, the main approaches used to protect grapevines from fungal and oomycete diseases are reviewed, starting from conventional breeding, which allowed the establishment of new resistant varieties, followed by biotechnological methods, such as transgenesis, cisgenesis, intragenesis, and genome editing, and ending with more recent perspectives concerning the application of new products based on RNAi technology. Evidence of their effectiveness, as well as potential risks and limitations based on the current legislative situation, are critically discussed.

Genome-Wide Datasets of Chicories (Cichorium intybus L.) for Marker-Assisted Crop Breeding Applications: A Systematic Review and Meta-Analysis.

Cichorium intybus L. is the most economically important species of its genus and among the most important of the Asteraceae family. In chicory, many linkage maps have been produced, several sets of mapped and unmapped markers have been developed, and dozens of genes linked to traits of agronomic interest have been investigated. This treasure trove of information, properly cataloged and organized, is of pivotal importance for the development of superior commercial products with valuable agronomic potential in terms of yield and quality, including reduced bitter taste and increased inulin production, as well as resistance or tolerance to pathogens and resilience to environmental stresses. For this reason, a systematic review was conducted based on the scientific literature published in chicory during 1980-2023. Based on the results obtained from the meta-analysis, we created two consensus maps capable of supporting marker-assisted breeding (MAB) and marker-assisted selection (MAS) programs. By taking advantage of the recently released genome of C. intybus, we built a 639 molecular marker-based consensus map collecting all the available mapped and unmapped SNP and SSR loci available for this species. In the following section, after summarizing and discussing all the genes investigated in chicory and related to traits of interest such as reproductive barriers, sesquiterpene lactone biosynthesis, inulin metabolism and stress response, we produced a second map encompassing 64 loci that could be useful for MAS purposes. With the advent of omics technologies, molecular data chaos (namely, the situation where the amount of molecular data is so complex and unmanageable that their use becomes challenging) is becoming far from a negligible issue. In this review, we have therefore tried to contribute by standardizing and organizing the molecular data produced thus far in chicory to facilitate the work of breeders.

Impact of Genomic and Transcriptomic Resources on Apiaceae Crop Breeding Strategies.

The Apiaceae taxon is one of the most important families of flowering plants and includes thousands of species used for food, flavoring, fragrance, medical and industrial purposes. This study had the specific intent of reviewing the main genomics and transcriptomic data available for this family and their use for the constitution of new varieties. This was achieved starting from the description of the main reproductive systems and barriers, with particular reference to cytoplasmic (CMS) and nuclear (NMS) male sterility. We found that CMS and NMS systems have been discovered and successfully exploited for the development of varieties only in Foeniculum vulgare, Daucus carota, Apium graveolens and Pastinaca sativa; whereas, strategies to limit self-pollination have been poorly considered. Since the constitution of new varieties benefits from the synergistic use of marker-assisted breeding in combination with conventional breeding schemes, we also analyzed and discussed the available SNP and SSR marker datasets (20 species) and genomes (8 species). Furthermore, the RNA-seq studies aimed at elucidating key pathways in stress tolerance or biosynthesis of the metabolites of interest were limited and proportional to the economic weight of each species. Finally, by aligning 53 plastid genomes from as many species as possible, we demonstrated the precision offered by the super barcoding approach to reconstruct the phylogenetic relationships of Apiaceae species. Overall, despite the impressive size of this family, we documented an evident lack of molecular data, especially because genomic and transcriptomic resources are circumscribed to a small number of species. We believe that our contribution can help future studies aimed at developing molecular tools for boosting breeding programs in crop plants of the Apiaceae family.

The Mitochondrial Genome Assembly of Fennel (Foeniculum vulgare) Reveals Two Different atp6 Gene Sequences in Cytoplasmic Male Sterile Accessions.

Cytoplasmic male sterility (CMS) has always aroused interest among researchers and breeders, being a valuable resource widely exploited not only to breed F1 hybrid varieties but also to investigate genes that control stamen and pollen development. With the aim of identifying candidate genes for CMS in fennel, we adopted an effective strategy relying on the comparison between mitochondrial genomes (mtDNA) of both fertile and sterile genotypes. mtDNA raw reads derived from a CMS genotype were assembled in a single molecule (296,483 bp), while a draft mtDNA assembly (166,124 nucleotides, 94 contigs) was performed using male fertile sample (MF) sequences. From their annotation and alignment, two atp6-like sequences were identified. atp6-, the putative mutant copy with a 300 bp truncation at the 5'-end, was found only in the mtDNA of CMS samples, while the wild type copy (atp6+) was detected only in the MF mtDNA. Further analyses (i.e., reads mapping and Sanger sequencing), revealed an atp6+ copy also in CMS samples, probably in the nuclear DNA. However, qPCRs performed on different tissues proved that, despite its availability, atp6+ is expressed only in MF samples, while apt6- mRNA was always detected in CMS individuals. In the light of these findings, the energy deficiency model could explain the pollen deficiency observed in male sterile flower. atp6- could represent a gene whose mRNA is translated into a not-fully functional protein leading to suboptimal ATP production that guarantees essential cellular processes but not a high energy demand process such as pollen development. Our study provides novel insights into the fennel mtDNA genome and its atp6 genes, and paves the way for further studies aimed at understanding their functional roles in the determination of male sterility.

Did apomixis evolve from sex or was it the other way around?

In angiosperms, there are two pathways of reproduction through seeds: sexual, or amphimictic, and asexual, or apomictic. The essential feature of apomixis is that an embryo in an ovule is formed autonomously. It may form from a cell of the nucellus or integuments in an otherwise sexual ovule, a process referred to as adventitious embryony. Alternatively, the embryo may form by parthenogenesis from an unreduced egg that forms in an unreduced embryo sac. The latter may form from an ameiotic megasporocyte, in which case it is referred to as diplospory, or from a cell of the nucellus or integument, in which case it is referred to as apospory. Progeny of apomictic plants are generally identical to the mother plant. Apomixis has been seen over the years as either a gain- or loss-of-function over sexuality, implying that the latter is the default condition. Here, we consider an additional point of view, that apomixis may be anciently polyphenic with sex and that both reproductive phenisms involve anciently canalized components of complex molecular processes. This polyphenism viewpoint suggests that apomixis fails to occur in obligately sexual eukaryotes because genetic or epigenetic modifications have silenced the primitive sex apomixis switch and/or disrupted molecular capacities for apomixis. In eukaryotes where sex and apomixis are clearly polyphenic, apomixis exponentially drives clonal fecundity during reproductively favorable conditions, while stress induces sex for stress-tolerant spore or egg formation. The latter often guarantees species survival during environmentally harsh seasons.

Structure, target-specificity and expression of PN_LNC_N13, a long non-coding RNA differentially expressed in apomictic and sexual Paspalum notatum.

KEY MESSAGE: ncRNA PN_LNC_N13 shows contrasting expression in reproductive organs of sexual and apomictic Paspalum notatum genotypes. Apomictic plants set genetically maternal seeds whose embryos derive by parthenogenesis from unreduced egg cells, giving rise to clonal offspring. Several Paspalum notatum apomixis related genes were identified in prior work by comparative transcriptome analyses. Here, one of these candidates (namely N13) was characterized. N13 belongs to a Paspalum gene family including 30-60 members, of which at least eight are expressed at moderate levels in florets. The sequences of these genes show no functional ORFs, but include segments of different protein coding genes. Particularly, N13 shows partial identity to maize gene BT068773 (RESPONSE REGULATOR 6). Secondary structure predictions as well as mature miRNA and target cleavage detection suggested that N13 is not a miRNA precursor. Moreover, N13 family members produce abundant 24-nucleotide small RNAs along extensive parts of their sequences. Surveys in the GREENC and CANTATA databases indicated similarity with plant long non-coding RNAs (lncRNAs) involved in splicing regulation; consequently, N13 was renamed as PN_LNC_N13. The Paspalum BT068773 predicted ortholog (N13TAR) originates floral transcript variants shorter than the canonical maize isoform and with possible structural differences between the apomictic and sexual types. PN_LNC_N13 is expressed only in apomictic plants and displays quantitative representation variation across reproductive developmental stages. However, PN_LNC_N13-like homologs and/or its derived sRNAs showed overall a higher representation in ovules of sexual plants at late premeiosis. Our results suggest the existence of a whole family of N13-like lncRNAs possibly involved in splicing regulation, with some members characterized by differential activity across reproductive types.

Combinatorial Regulation of Stilbene Synthase Genes by WRKY and MYB Transcription Factors in Grapevine (Vitis vinifera L.).

Stilbene synthase (STS) is the key enzyme leading to the biosynthesis of resveratrol. Recently we reported two R2R3-MYB transcription factor (TF) genes that regulate the stilbene biosynthetic pathway in grapevine: VviMYB14 and VviMYB15. These genes are strongly co-expressed with STS genes under a range of stress and developmental conditions, in agreement with the specific activation of STS promoters by these TFs. Genome-wide gene co-expression analysis using two separate transcriptome compendia based on microarray and RNA sequencing data revealed that WRKY TFs were the top TF family correlated with STS genes. On the basis of correlation frequency, four WRKY genes, namely VviWRKY03, VviWRKY24, VviWRKY43 and VviWRKY53, were further shortlisted and functionally validated. Expression analyses under both unstressed and stressed conditions, together with promoter-luciferase reporter assays, suggested different hierarchies for these TFs in the regulation of the stilbene biosynthetic pathway. In particular, VviWRKY24 seems to act as a singular effector in the activation of the VviSTS29 promoter, while VviWRKY03 acts through a combinatorial effect with VviMYB14, suggesting that these two regulators may interact at the protein level as previously reported in other species.

Developing a Molecular Identification Assay of Old Landraces for the Genetic Authentication of Typical Agro-Food Products: The Case Study of the Barley 'Agordino'.

The orzo Agordino is a very old local variety of domesticated barley (Hordeum vulgare ssp. distichum L.) that is native to the Agordo District, Province of Belluno, and is widespread in the Veneto Region, Italy. Seeds of this landrace are widely used for the preparation of very famous dishes of the dolomitic culinary tradition such as barley soup, bakery products and local beer. Understanding the genetic diversity and identity of the Agordino barley landrace is a key step to establish conservation and valorisation strategies of this local variety and also to provide molecular traceability tools useful to ascertain the authenticity of its derivatives. The gene pool of the Agordino barley landrace was reconstructed using 60 phenotypically representative individual plants and its genotypic relationships with commercial varieties were investigated using 21 pure lines widely cultivated in the Veneto Region. For genomic DNA analysis, following an initial screening of 14 mapped microsatellite (SSR) loci, seven discriminant markers were selected on the basis of their genomic position across linkage groups and polymorphic marker alleles per locus. The genetic identity of the local barley landrace was determined by analysing all SSR markers in a single multi-locus PCR assay. Extent of genotypic variation within the Agordino barley landrace and the genotypic differentiation between the landrace individuals and the commercial varieties was determined. Then, as few as four highly informative SSR loci were selected and used to develop a molecular traceability system exploitable to verify the genetic authenticity of food products deriving from the Agordino landrace. This genetic authentication assay was validated using both DNA pools from individual Agordino barley plants and DNA samples from Agordino barley food products. On the whole, our data support the usefulness and robustness of this DNA-based diagnostic tool for the orzo Agordino identification, which could be rapidly and efficiently exploited to guarantee the authenticity of local varieties and the typicality of food products.

De novo sequencing of the Hypericum perforatum L. flower transcriptome to identify potential genes that are related to plant reproduction sensu lato.

BACKGROUND: St. John's wort (Hypericum perforatum L.) is a medicinal plant that produces important metabolites with antidepressant and anticancer activities. Recently gained biological information has shown that this species is also an attractive model system for the study of a naturally occurring form of asexual reproduction called apomixis, which allows cloning plants through seeds. In aposporic gametogenesis, one or multiple somatic cells belonging to the ovule nucellus change their fate by dividing mitotically and developing functionally unreduced embryo sacs by mimicking sexual gametogenesis. Although the introduction of apomixis into agronomically important crops could have revolutionary implications for plant breeding, the genetic control of this mechanism of seed formation is still not well understood for most of the model species investigated so far. We used Roche 454 technology to sequence the entire H. perforatum flower transcriptome of whole flower buds and single flower verticils collected from obligately sexual and unrelated highly or facultatively apomictic genotypes, which enabled us to identify RNAs that are likely exclusive to flower organs (i.e., sepals, petals, stamens and carpels) or reproductive strategies (i.e., sexual vs. apomictic). RESULTS: Here we sequenced and annotated the flower transcriptome of H. perforatum with particular reference to reproductive organs and processes. In particular, in our study we characterized approximately 37,000 transcripts found expressed in male and/or female reproductive organs, including tissues or cells of sexual and apomictic flower buds. Ontological annotation was applied to identify major biological processes and molecular functions involved in flower development and plant reproduction. Starting from this dataset, we were able to recover and annotate a large number of transcripts related to meiosis, gametophyte/gamete formation, and embryogenesis, as well as genes that are exclusively or preferentially expressed in sexual or apomictic libraries. Real-Time RT-qPCR assays on pistils and anthers collected at different developmental stages from accessions showing alternative modes of reproduction were used to identify potential genes that are related to plant reproduction sensu lato in H. perforatum. CONCLUSIONS: Our approach of sequencing flowers from two fully obligate sexual genotypes and two unrelated highly apomictic genotypes, in addition to different flower parts dissected from a facultatively apomictic accession, enabled us to analyze the complexity of the flower transcriptome according to its main reproductive organs as well as for alternative reproductive behaviors. Both annotation and expression data provided original results supporting the hypothesis that apomixis in H. perforatum relies upon spatial or temporal mis-expression of genes acting during female sexual reproduction. The present analyses aim to pave the way toward a better understanding of the molecular basis of flower development and plant reproduction, by identifying genes or RNAs that may differentiate or regulate the sexual and apomictic reproductive pathways in H. perforatum.

Soil salinization in agriculture: Mitigation and adaptation strategies combining nature-based solutions and bioengineering.

Soil salinization is among the most critical threats to agriculture and food security. Excess of salts adversely affects soil structure and fertility, plant growth, crop yield, and microorganisms. It is caused by natural processes, such as dry climates and low precipitations, high evaporation rate, poor waterlogging, and human factors, such as inappropriate irrigation practices, poor drainage systems, and excessive use of fertilizers. The growing extremization of climate with prolonged drought conditions is worsening the phenomenon. Nature-based solutions (NBS), combined with precision or conservation agriculture, represent a sustainable response, and offer benefits through revitalizing ecosystem services. This perspective explores NBS that can be adopted, along with their challenges and implementation limitations. We also argue that NBS could not be enough to combat hunger in the world's most vulnerable regions and fully achieve the Sustainable Development Goal - Zero Hunger (SDG2). We therefore discuss their possible combination with salt-tolerant crops based on bioengineering.

Revitalizing agriculture: next-generation genotyping and -omics technologies enabling molecular prediction of resilient traits in the Solanaceae family.

This review highlights -omics research in Solanaceae family, with a particular focus on resilient traits. Extensive research has enriched our understanding of Solanaceae genomics and genetics, with historical varietal development mainly focusing on disease resistance and cultivar improvement but shifting the emphasis towards unveiling resilience mechanisms in genebank-preserved germplasm is nowadays crucial. Collecting such information, might help researchers and breeders developing new experimental design, providing an overview of the state of the art of the most advanced approaches for the identification of the genetic elements laying behind resilience. Building this starting point, we aim at providing a useful tool for tackling the global agricultural resilience goals in these crops.

The Arabidopsis thaliana Mob1A gene is required for organ growth and correct tissue patterning of the root tip.

BACKGROUND AND AIMS: The Mob1 family includes a group of kinase regulators conserved throughout eukaryotes. In multicellular organisms, Mob1 is involved in cell proliferation and apoptosis, thus controlling appropriate cell number and organ size. These functions are also of great importance for plants, which employ co-ordinated growth processes to explore the surrounding environment and respond to changing external conditions. Therefore, this study set out to investigate the role of two Arabidopsis thaliana Mob1-like genes, namely Mob1A and Mob1B, in plant development. METHODS: A detailed spatio-temporal analysis of Mob1A and Mob1B gene expression was performed by means of bioinformatic tools, the generation of expression reporter lines and in situ hybridization of gene-specific probes. To explore the function of the two genes in plant development, knock-out and knock-down mutants were isolated and their phenotype quantitatively characterized. KEY RESULTS: Transcripts of the two genes were detected in specific sets of cells in all plant organs. Mob1A was upregulated by several stress conditions as well as by abscisic acid and salicylic acid. A knock-out mutation in Mob1B did not cause any visible defect in plant development, whereas suppression of Mob1A expression affected organ growth and reproduction. In the primary root, reduced levels of Mob1A expression brought about severe defects in tissue patterning of the stem cell niche and columella and led to a decrease in meristem size. Moreover, loss of Mob1A function resulted in a higher sensitivity of root growth to abscisic acid. CONCLUSIONS: Taken together, the results indicate that arabidopsis Mob1A is involved in the co-ordination of tissue patterning and organ growth, similarly to its orthologues in other multicellular eukaryotes. In addition, Mob1A serves a plant-specific function by contributing to growth adjustments in response to stress conditions.

Pipeline to Design Inbred Lines and F1 Hybrids of Leaf Chicory (Radicchio) Using Male Sterility and Genotyping-by-Sequencing.

Chicory, a horticultural crop cultivated worldwide, presents many botanical varieties and local biotypes. Among these, cultivars of the Italian radicchio group of the pure species Cichorium intybus L. and its interspecific hybrids with Cichorium endivia L.-as the "Red of Chioggia" biotype-includes several phenotypes. This study uses a pipeline to address the marker-assisted breeding of F1 hybrids: it presents the genotyping-by-sequencing results of four elite inbred lines using a RADseq approach and an original molecular assay based on CAPS markers for screening mutants with nuclear male sterility in the radicchio of Chioggia. A total of 2953 SNP-carrying RADtags were identified and used to compute the actual estimates of homozygosity and overall genetic similarity and uniformity of the populations, as well as to determine their genetic distinctiveness and differentiation. Molecular data were further used to investigate the genomic distribution of the RADtags among the two Cichorium species, allowing their mapping in 1131 and 1071 coding sequences in chicory and endive, respectively. Paralleling this, an assay to screen the genotype at the male sterility locus Cims-1 was developed to discriminate wild-type and mutant alleles of the causative gene myb80-like. Moreover, a RADtag mapped close to this genomic region proved the potential application of this method for future marker-assisted selection tools. Finally, after combining the genotype information of the core collection, the best 10 individuals from each inbred line were selected to compute the observed genetic similarity as a measure of uniformity as well as the expected homozygosity and heterozygosity estimates scorable by the putative progenies derived from selfing (pollen parent) and full-sibling (seed parent) or pair-wise crossing (F1 hybrids). This predictive approach was conducted as a pilot study to understand the potential application of RADseq in the fine tuning of molecular marker-assisted breeding strategies aimed at the development of inbred lines and F1 hybrids in leaf chicory.

Current insights and advances into plant male sterility: new precision breeding technology based on genome editing applications.

Plant male sterility (MS) represents the inability of the plant to generate functional anthers, pollen, or male gametes. Developing MS lines represents one of the most important challenges in plant breeding programs, since the establishment of MS lines is a major goal in F1 hybrid production. For these reasons, MS lines have been developed in several species of economic interest, particularly in horticultural crops and ornamental plants. Over the years, MS has been accomplished through many different techniques ranging from approaches based on cross-mediated conventional breeding methods, to advanced devices based on knowledge of genetics and genomics to the most advanced molecular technologies based on genome editing (GE). GE methods, in particular gene knockout mediated by CRISPR/Cas-related tools, have resulted in flexible and successful strategic ideas used to alter the function of key genes, regulating numerous biological processes including MS. These precision breeding technologies are less time-consuming and can accelerate the creation of new genetic variability with the accumulation of favorable alleles, able to dramatically change the biological process and resulting in a potential efficiency of cultivar development bypassing sexual crosses. The main goal of this manuscript is to provide a general overview of insights and advances into plant male sterility, focusing the attention on the recent new breeding GE-based applications capable of inducing MS by targeting specific nuclear genic loci. A summary of the mechanisms underlying the recent CRISPR technology and relative success applications are described for the main crop and ornamental species. The future challenges and new potential applications of CRISPR/Cas systems in MS mutant production and other potential opportunities will be discussed, as generating CRISPR-edited DNA-free by transient transformation system and transgenerational gene editing for introducing desirable alleles and for precision breeding strategies.

Is apomixis occurring in walnut (Juglans regia L.)? New data from progeny molecular tests and cytological investigations shed light on its reproductive system.

Introduction: Persian walnut (Juglans regia) is an economically important nut fruit species cultivated worldwide for its nutritious kernel and timber quality wood. Walnut trees are mostly hetero-dichogamous and, depending on the genotype, some cultivars are protogynous, while others are protandrous. Although selfing is possible when male and female blooms overlap, the dichogamy of the species promotes outcrossing. In addition to sexual reproduction, some reports indicate that elements of apomixis may occur in commercial orchards of walnut varieties and in the last two decades, nut production by apomixis has been reported in walnut. However, there are no reliable studies on the occurrence of apomictic reproduction based on cytoembryological observations and/or molecular marker-progeny tests. This study addresses the combined use of molecular and cytological analyses to gain new insights into the population genetics and reproduction systems of J. regia. Methods: We systematically analyzed the reproductive origin of individual progeny plants from 8 different cultivated walnut genotypes using microsatellite genotyping and carried out cytohistological investigations of 5 cultivated walnut genotypes arising seed sets from isolated flowers, to shed light on the mode of reproduction. Results and discussion: These cytometric and genotyping analyses did not support any asexual mode of reproduction or asexual propagation by seed and all individuals studied were identified as zygotic plants produced by crossing. Likewise, the cytological findings did not confirm completely the first component of apomixis, namely apomeiosis. On the other hand, according to histological evidence, adventitious embryony seems to take place at low frequency. Overall, our findings suggest that the occurrence of gametophytic apomixis is unlikely in J. regia, but sporophytic apomixis cannot be completely ruled out.

MIK2 is a candidate gene of the S-locus for sporophytic self-incompatibility in chicory (Cichorium intybus, Asteraceae).

The Cichorium genus offers a unique opportunity to study the sporophytic self-incompatibility (SSI) system, being composed of species characterized by highly efficient self-incompatibility (e.g., C. intybus) and complete self-compatibility (e.g., C. endivia). To this end, the chicory genome was used to map seven previously identified SSI locus-associated markers. The region containing the S-locus was therefore restricted to an ~4 M bp window on chromosome 5. Among the genes predicted in this region, MDIS1 INTERACTING RECEPTOR LIKE KINASE 2 (ciMIK2) was particularly promising as a candidate for SSI. Its ortholog in Arabidopsis (atMIK2) is involved in pollen-stigma recognition reactions, and its protein structure is similar to that of S-receptor kinase (SRK), a key component of the SSI system in the Brassica genus. The amplification and sequencing of MIK2 in chicory and endive accessions revealed two contrasting scenarios. In C. endivia, MIK2 was fully conserved even when comparing different botanical varieties (i.e., smooth and curly endive). In C. intybus, 387 polymorphic positions and 3 INDELs were identified when comparing accessions of different biotypes all belonging to the same botanical variety (i.e., radicchio). The polymorphism distribution throughout the gene was uneven, with hypervariable domains preferentially localized in the LRR-rich extracellular region, putatively identified as the receptor domain. The gene was hypothesized to be under positive selection, as the nonsynonymous mutations were more than double the synonymous ones (dN/dS = 2.17). An analogous situation was observed when analyzing the first 500 bp of the MIK2 promoter: no SNPs were observed among the endive samples, whereas 44 SNPs and 6 INDELs were detected among the chicory samples. Further analyses are needed to confirm the role of MIK2 in SSI and to demonstrate whether the 23 species-specific nonsynonymous SNPs in the CDS and/or the species-specific 10 bp-INDEL found in a CCAAT box region of the promoter are responsible for the contrasting sexual behaviors of chicory and endive.

Dissecting the effect of soil on plant phenology and berry transcriptional plasticity in two Italian grapevine varieties (Vitis vinifera L.).

Grapevine embodies a fascinating species as regards phenotypic plasticity and genotype-per-environment interactions. The terroir, namely the set of agri-environmental factors to which a variety is subjected, can influence the phenotype at the physiological, molecular, and biochemical level, representing an important phenomenon connected to the typicality of productions. We investigated the determinants of plasticity by conducting a field-experiment where all terroir variables, except soil, were kept as constant as possible. We isolated the effect of soils collected from different areas, on phenology, physiology, and transcriptional responses of skin and flesh of a red and a white variety of great economic value: Corvina and Glera. Molecular results, together with physio-phenological parameters, suggest a specific effect of soil on grapevine plastic response, highlighting a higher transcriptional plasticity of Glera in respect to Corvina and a marked response of skin compared to flesh. Using a novel statistical approach, we identified clusters of plastic genes subjected to the specific influence of soil. These findings could represent an issue of applicative value, posing the basis for targeted agricultural practices to enhance the desired characteristics for any soil/cultivar combination, to improve vineyards management for a better resource usage and to valorize vineyards uniqueness maximizing the terroir-effect.

Boosting grapevine breeding for climate-smart viticulture: from genetic resources to predictive genomics.

The multifaceted nature of climate change is increasing the urgency to select resilient grapevine varieties, or generate new, fitter cultivars, to withstand a multitude of new challenging conditions. The attainment of this goal is hindered by the limiting pace of traditional breeding approaches, which require decades to result in new selections. On the other hand, marker-assisted breeding has proved useful when it comes to traits governed by one or few genes with great effects on the phenotype, but its efficacy is still restricted for complex traits controlled by many loci. On these premises, innovative strategies are emerging which could help guide selection, taking advantage of the genetic diversity within the Vitis genus in its entirety. Multiple germplasm collections are also available as a source of genetic material for the introgression of alleles of interest via adapted and pioneering transformation protocols, which present themselves as promising tools for future applications on a notably recalcitrant species such as grapevine. Genome editing intersects both these strategies, not only by being an alternative to obtain focused changes in a relatively rapid way, but also by supporting a fine-tuning of new genotypes developed with other methods. A review on the state of the art concerning the available genetic resources and the possibilities of use of innovative techniques in aid of selection is presented here to support the production of climate-smart grapevine genotypes.

Assessment of the Genetic Distinctiveness and Uniformity of Pre-Basic Seed Stocks of Italian Ryegrass Varieties.

Lolium multiflorum Lam., commonly known as Italian ryegrass, is a forage grass mostly valued for its high palatability and digestibility, along with its high productivity. However, Italian ryegrass has an outbreeding nature and therefore has high genetic heterogeneity within each variety. Consequently, the exclusive use of morphological descriptors in the existing varietal identification and registration process based on the Distinctness, Uniformity, and Stability (DUS) test results in an inadequately precise assessment. The primary objective of this work was to effectively test whether the uniformity observed at the phenological level within each population of Italian ryegrass was confirmed at the genetic level through an SSR marker analysis. In this research, using 12 polymorphic SSR loci, we analyzed 672 samples belonging to 14 different Italian ryegrass commercial varieties to determine the pairwise genetic similarity (GS), verified the distribution of genetic diversity within and among varieties, and investigated the population structure. Although the fourteen commercial varieties did not show elevated genetic differentiation, with only 13% of the total variation attributable to among-cultivar genetic variation, when analyzed as a core, each variety constitutes a genetic cluster on its own, resulting in distinct characteristics from the others, except for two varieties. In this way, by combining a genetic tool with the traditional morphological approach, we were able to limit biases linked to the environmental effect of field trials, assessing the real source of diversity among varieties and concretely answering the key requisites of the Plant Variety Protection (PVP) system.