Lantana camara L. is sold worldwide for ornamental purposes, although it is also characterized by high invasiveness potential. Genetic and molecular data available for L. camara are still poor, and breeding is performed through conventional methods. This study focused on a molecular genotyping analysis through the ddRADseq method on an experimental collection of lantana clonal lines to evaluate the potential of molecular techniques in performing marker-assisted breeding, in favour of variety registration and in guaranteeing plant variety protection for the species. Although high genetic uniformity was observed in the population, a unique molecular profile was assigned to every line, indicating the effectiveness of the approach used. Interestingly, low degrees of heterozygosity were observed. In addition, the possibility of inferring ploidy levels through SNP profiles was assessed since it would avoid the necessity of previous biological knowledge and the use of fresh materials. Ploidy analysis is of high interest for lantana breeding to obtain less invasive triploids. Flow cytometry and chromosome counting were used for inference assessment. An nQuack framework provided correct results for the majority of the clonal lines, confirming its effectiveness. These findings encourage the adoption of molecular systems to help breed minor species such as L. camara.

Background: The overwhelming majority of the antimicrobial peptides (AMPs) studied in mussels (Mytilus spp.) so far are specifically expressed by hemocytes and display compact disulfide-stabilized structures. However, gill-specific myticalins play a role in mucosal immunity and are one of the very few examples of known molluscan AMPs lacking cysteine residues. Methods: We investigate the molecular evolution of myticalins, compiling a collection of sequences obtained by carefully annotating 169 genome assemblies of different Mytilus species. We determine the gene presence/absence patterns and gene expression profiles for the five myticalin subfamilies, including the newly reported myticalin E. Results: All sequences are deposited in MyticalinDB, a novel database that includes a total of 100 unique mature myticalin peptides encoded by 215 protein precursors, greatly enriching the compendium of these molecules from previous reports. Among the five subfamilies, myticalin A and C are the most widespread and highly expressed across all Mytilus species. Interestingly, structural prediction reveals a previously unreported strong amphipathic nature for some myticalins, which may be highly relevant for their biological activity. Conclusions: The results reported in this work support the role of myticalins in gill-associated mucosal immunity and highlight the importance of inter-individual molecular diversity in establishing an efficient response to microbial infections. The newly established MyticalinDB provides a valuable resource for investigating the evolution and extraordinary molecular diversity of this AMP family.

Molecular genotyping is a key factor for plant breeding programming and plant variety protection (PVP). However, its potential still remains to be elucidated when considering ornamental plants like Petunia × hybrida. In this study, a petunia breeding clone collection, including sister line groups, was genotyped through double digest Restriction-site Associated DNA sequencing (ddRADseq), and its genetic diversity and structure were studied. In addition to estimating the high genetic similarity observed among sister lines, this approach allowed the unique discrimination of each clone too. Molecular results agreed with genealogy data, supporting the assessment of genotyping effectiveness. In addition, the minimal number of variants able to uniquely discriminate and/or correctly cluster the experimental lines was investigated. The loci number could be reduced to eight to achieve line discrimination, and a method to identify the specific variant sets is presented. Conversely, to preserve the original clustering with minor adjustments, one hundred loci were required and were obtained through minor allele frequency (MAF) filtering. Moreover, analysis of the chromosomal distribution of variants revealed a predominant accumulation in distal regions. Genetic analyses were repeated considering only variants located in coding sequences and results were in agreement with what previously observed, disclosing the potential of the expressed regions for genotyping purposes. Eventually, the applied approach enabled the investigation of SNPs within genes putatively involved in traits of interest. Our findings encourage the adoption of high-throughput and cost-effective sequencing techniques for petunia genotyping aimed at achieving PVP, supporting new variety registration, and developing marker-assisted breeding (MAB) and marker-assisted selection (MAS) strategies.

Genome editing technologies, especially those based on the CRISPR/Cas9 system, have revolutionized crop breeding by enabling precise genetic modifications. Specifically, delivering preassembled ribonucleoprotein (RNP) complexes-consisting of the Cas9 endonuclease coupled to specific single guide RNAs (sgRNAs)-into protoplasts offers an effective DNA-free method that prevents the integration of foreign genetic material. Despite the availability of detailed protocols, establishing a standardized and efficient in vitro regeneration procedure-from protoplast isolation to whole plant regeneration-remains challenging due to significant variability in regeneration efficiency across different varieties and biotypes. Therefore, optimizing each step is essential to maximize the recovery of successful edited plants. In this study, we developed an efficient protocol for regenerating whole plants from protoplasts isolated from 12 representative Italian varieties of chicory and endive. We focused on leaf chicory and endive biotypes with high horticultural value, including Radicchio types, which are important targets for quality improvement. Our optimized platform supports protoplast isolation, PEG-mediated transfection, and plant regeneration, demonstrating promising potential for future genome editing applications. Notably, the high responsiveness of protoplasts to PEG-mediated transfection suggests that coupling this method with our regeneration procedure could facilitate the use of advanced biotechnological strategies. The combination of high transient transformation efficiency, versatile encapsulation techniques, and successful plant regeneration establishes chicory and endive as promising candidates for DNA-free genome editing via protoplasts, providing a technically precise approach with reduced environmental and economic impacts compared to conventional breeding methods.