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.

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.

First genomic insights into the Mandevilla genus.

Mandevilla (Apocynaceae) is a greatly appreciated genus in the world ornamental market. In this study, we attempted to address the poor genetic knowledge and the huge taxonomic gaps existing in this genus by analyzing a collection of 55 accessions. After cytometrically determining the triploid genome size (1,512.64 Mb) of a reference sample (variety "Mandevilla 2001"), the plastidial genome (cpDNA, 0.18 Mb) and a draft of the nuclear genome (nuDNA, 207 Mb) were assembled. While cpDNA was effective in reconstructing the phylogenesis of the Apocynaceae family based on a DNA superbarcoding approach, the nuDNA assembly length was found to be only 41% of the haploid genome size (506 Mb, predicted based on the K-mer frequency distribution). Its annotation enabled the prediction of 37,811 amino acid sequences, of which 10,562 resulted full length proteins. Among them, we identified nine proteins whose orthologs (in Catharanthus roseus) are involved in the biosynthesis of monoterpene indole alkaloids (MIAs), including catharanthine, tabersonine, and vincadifformine. The nuclear genome draft was also useful to develop a highly informative (average polymorphism information content, PIC = 0.62) set of 23 simple sequence repeat (SSR) markers that was validated on the Mandevilla collection. These results were integrated with cytometric measurements, nuclear ITS1 haplotyping and chloroplast DNA barcoding analyses to assess the origin, divergence and relationships existing among the 55 accessions object of the study. As expected, based on the scarce information available in the literature, the scenario was extremely intricate. A reasonable hypothesis is that most of the accessions represent interspecific hybrids sharing the same species as maternal parent (i.e., Mandevilla sanderi).

Genotyping Analysis by RAD-Seq Reads Is Useful to Assess the Genetic Identity and Relationships of Breeding Lines in Lavender Species Aimed at Managing Plant Variety Protection.

Lavender species are widely distributed in their wild forms around the Mediterranean Basin and they are also cultivated worldwide as improved and registered clonal varieties. The economic interest of the species belonging to the Lavandula genus is determined by their use as ornamental plants and important source of essential oils that are destinated to the production of cosmetics, pharmaceuticals and foodstuffs. Because of the increasing number of cases of illegal commercialization of selected varieties, the protection of plant breeders' rights has become of main relevance for the recognition of breeding companies' royalties. With this aim, genomic tools based on molecular markers have been demonstrated to be very reliable and transferable among laboratories, and also much more informative than morphological descriptors. With the rising of the next-generation sequencing (NGS) technologies, several genotyping-by-sequencing approaches are now available. This study deals with a deep characterization of 15 varietal clones, belonging to two distinct Lavandula species, by means of restriction-site associated DNA sequencing (RAD-Seq). We demonstrated that this technology screens single nucleotide variants that enable to assess the genetic identity of individual accessions, to reconstruct genetic relationships among related breeding lines, to group them into genetically distinguishable main subclusters, and to assign their molecular lineages to distinct ancestors. Moreover, a number of polymorphic sites were identified within genes putatively involved in biosynthetic pathways related to both tissue pigmentation and terpene production, useful for breeding and/or protecting newly registered varieties. Overall, the results highlighted the presence of pure ancestries and interspecific hybrids for the analyzed Lavandula species, and demonstrated that RAD-Seq analysis is very informative and highly reliable for characterizing Lavandula clones and managing plant variety protection.

Developing and Testing Molecular Markers in Cannabis sativa (Hemp) for Their Use in Variety and Dioecy Assessments.

Cannabis sativa (2n = 2x = 20) is a popular species belonging to the Cannabaceae family. Despite its use for medical, recreational, and industrial purposes as well as its long history, the genetic research on this species is in its infancy due to the legal implications and the prohibition campaigns. The recent legalization of Cannabis in many countries along with the use of genomics boosted the approaches aimed at marker-assisted selection, germplasm management, genetic discrimination, and authentication of cultivars. Nonetheless, the exploitation of molecular markers for the development of commercial varieties through marker-assisted breeding schemes is still rare. The present study aimed to develop an informative panel of simple sequence repeat markers to be used for the genotyping of high breeding value C. sativa lines. Starting from 41 nuclear SSR designated by in silico analyses, we selected 20 highly polymorphic and discriminant loci that were tested in 104 individuals belonging to 11 distinct hemp varieties. The selected markers were successful in accessing homozygosity, genetic uniformity, and genetic variation within and among varieties. Population structure analysis identified eight genetic groups, clustering individuals based on sexual behaviors (dioecious and monoecious) and geographical origins. Overall, this study provides important tools for the genetic characterization, authentication, conservation of biodiversity, genetic improvement and traceability of this increasingly important plant species.

Molecular Hallmarks, Agronomic Performances and Seed Nutraceutical Properties to Exploit Neglected Genetic Resources of Common Beans Grown by Organic Farming in Two Contrasting Environments.

Common bean (Phaseolus vulgaris L.) is an essential source of food proteins and an important component of sustainable agriculture systems around the world. Thus, conserving and exploiting the genetic materials of this crop species play an important role in achieving global food safety and security through the preservation of functional and serependic opportunities afforded by plant species diversity. Our research aimed to collect and perform agronomic, morpho-phenological, molecular-genetic, and nutraceutical characterizations of common bean accessions, including lowland and mountain Venetian niche landraces (ancient farmer populations) and Italian elite lineages (old breeder selections). Molecular characterization with SSR and SNP markers grouped these accessions into two well-separated clusters that were linked to the original Andean and Mesoamerican gene pools, which was consistent with the outputs of ancestral analysis. Genetic diversity in the two main clusters was not distributed equally the Andean gene pool was found to be much more uniform than the Mesoamerican pool. Additional subdivision resulted in subclusters, supporting the existence of six varietal groups. Accessions were selected according to preliminary investigations and historical records and cultivated in two contrasting Venetian environments: sea-level and mountain territories. We found that the environment significantly affected some nutraceutical properties of the seeds, mainly protein and starch contents. The antioxidant capacity was found significantly greater at sea level for climbing accessions and in the mountains for dwarf accessions. The seed yield at sea level was halved than mountain due to a seeds reduction in weight, volume, size and density. At sea level, bean landraces tended to have extended flowering periods and shorter fresh pod periods. The seed yield was positively correlated with the length of the period during which plants had fresh pods and negatively correlated with the length of the flowering period. Thus, the agronomic performance of these genetic resources showed their strong connection and adaptation to mountainous environments. On the whole, the genetic-molecular information put together for these univocal bean entries was combined with overall results from plant and seed analyses to select and transform the best accessions into commercial varieties (i.e., pure lines) suitable for wider cultivation.

NGS-based barcoding with mini-COI gene target is useful for pet food market surveys aimed at mislabelling detection.

Pet food industry has grown considerably in the last few years and it is expected to continue with this rate. Despite the economic impact of this sector and the consumer concerns for the increasing number of food and feed adulteration cases, few studies have been published on mislabelling in pet foods. We therefore investigated the capability of a next generation sequencing-based mini-barcoding approach to identify animal species in pet food products. In a preliminary analysis, a 127 bp fragment of the COI gene was tested on both individual specimens and ad hoc mixed fresh samples used as testers, to evaluate its discrimination power and primers effectiveness. Eighteen pet food products of different price categories and forms available on the market (i.e. kibbles, bites, pâté and strips) were analysed through an NGS approach in biological replicates. At least one of the species listed in the ingredients was not detected in half of the products, while seven products showed supplementary species in addition to those stated on the label. Due to the accuracy, sensitivity and specificity demonstrated, this method can be proposed as food genetic traceability system to evaluate both the feed and food quality timely along the supply chain.

Potentials and Challenges of Genomics for Breeding Cannabis Cultivars.

Cannabis (Cannabis sativa L.) is an influential yet controversial agricultural plant with a very long and prominent history of recreational, medicinal, and industrial usages. Given the importance of this species, we deepened some of the main challenges-along with potential solutions-behind the breeding of new cannabis cultivars. One of the main issues that should be fixed before starting new breeding programs is the uncertain taxonomic classification of the two main taxa (e.g., indica and sativa) of the Cannabis genus. We tried therefore to examine this topic from a molecular perspective through the use of DNA barcoding. Our findings seem to support a unique species system (C. sativa) based on two subspecies: C. sativa subsp. sativa and C. sativa subsp. indica. The second key issue in a breeding program is related to the dioecy behavior of this species and to the comprehension of those molecular mechanisms underlying flower development, the main cannabis product. Given the role of MADS box genes in flower identity, we analyzed and reorganized all the genomic and transcriptomic data available for homeotic genes, trying to decipher the applicability of the ABCDE model in Cannabis. Finally, reviewing the limits of the conventional breeding methods traditionally applied for developing new varieties, we proposed a new breeding scheme for the constitution of F1 hybrids, without ignoring the indisputable contribution offered by genomics. In this sense, in parallel, we resumed the main advances in the genomic field of this species and, ascertained the lack of a robust set of SNP markers, provided a discriminant and polymorphic panel of SSR markers as a valuable tool for future marker assisted breeding programs.

Genetic Structure of Cultivated Varieties of Radicchio (Cichorium intybus L.): A Comparison between F1 Hybrids and Synthetics.

Cichorium intybus L., well known in Italy with the common name "Radicchio", is an important leafy vegetable that is prevalently reproduced by allogamy due to very efficient barriers of self-incompatibility. Marker-assisted breeding is widely used by seed firms to develop new hybrid varieties that manifest genetic distinctiveness, uniformity and stability. A total of 29 mapped microsatellite markers were used for genotyping 504 samples of the Red of Chioggia biotype: First, two synthetics, four F1 hybrids and two derived F2 populations were compared to assess the distinctiveness of their gene pool and structure; then, the uniformity and stability of 3 years of production of a commercial F1 variety were also investigated. Genetic similarity and diversity statistics as well as the genetic structure of populations were analysed, including allele and genotype frequencies. The mean estimates and ranges of genetic similarity enabled the molecular discrimination of OP synthetics from F1 varieties and their F2 progenies and the determination of individual plant memberships. Moreover, the genetic structure of F1 hybrids produced in 3 years unexpectedly revealed two main clusters that discriminate the first 2 years from the 3rd, mainly because of the presence of uncommon specific alleles and different allele frequencies. Overall, this molecular information will enable breeders to determine the genetic distinctness, uniformity and stability of commercial and experimental varieties, as well as their genetic relationships and relatedness. Hence, this work provides a useful tool for achieving the molecular characterisation and genetic identification of different radicchio populations.