Genetic Improvement of Camelina sativa (L.) Crantz: Opportunities and Challenges.

In recent years, a renewed interest in novel crops has been developing due to the environmental issues associated with the sustainability of agricultural practices. In particular, a cover crop, Camelina sativa (L.) Crantz, belonging to the Brassicaceae family, is attracting the scientific community's interest for several desirable features. It is related to the model species Arabidopsis thaliana, and its oil extracted from the seeds can be used either for food and feed, or for industrial uses such as biofuel production. From an agronomic point of view, it can grow in marginal lands with little or no inputs, and is practically resistant to the most important pathogens of Brassicaceae. Although cultivated in the past, particularly in northern Europe and Italy, in the last century, it was abandoned. For this reason, little breeding work has been conducted to improve this plant, also because of the low genetic variability present in this hexaploid species. In this review, we summarize the main works on this crop, focused on genetic improvement with three main objectives: yield, seed oil content and quality, and reduction in glucosinolates content in the seed, which are the main anti-nutritional substances present in camelina. We also report the latest advances in utilising classical plant breeding, transgenic approaches, and CRISPR-Cas9 genome-editing.

Distinct gene networks modulate floral induction of autonomous maize and photoperiod-dependent teosinte.

Temperate maize was domesticated from its tropical ancestor, teosinte. Whereas temperate maize is an autonomous day-neutral plant, teosinte is an obligate short-day plant that requires uninterrupted long nights to induce flowering. Leaf-derived florigenic signals trigger reproductive growth in both teosinte and temperate maize. To study the genetic mechanisms underlying floral inductive pathways in maize and teosinte, mRNA and small RNA genome-wide expression analyses were conducted on leaf tissue from plants that were induced or not induced to flower. Transcriptome profiles reveal common differentially expressed genes during floral induction, but a comparison of candidate flowering time genes indicates that photoperiod and autonomous pathways act independently. Expression differences in teosinte are consistent with the current paradigm for photoperiod-induced flowering, where changes in circadian clock output trigger florigen production. Conversely, differentially expressed genes in temperate maize link carbon partitioning and flowering, but also show altered expression of circadian clock genes that are distinct from those altered upon photoperiodic induction in teosinte. Altered miRNA399 levels in both teosinte and maize suggest a novel common connection between flowering and phosphorus perception. These findings provide insights into the molecular mechanisms underlying a strengthened autonomous pathway that enabled maize growth throughout temperate regions.

Variability in Seed Traits in a Collection of Cannabis sativa L. Genotypes.

The seed of Cannabis sativa L. is an expanding source of proteins and oil for both humans and animals. In this study, the proximate composition of a collection of hemp cultivars and accessions of different geographical origins grown under the same conditions for 1 year was analyzed in order to identify potential accessions to improve hemp cultivars. Fatty acids, tocopherols, and antinutritional components, as well as concentrations of crude protein and oil were quantified. The seed oil concentrations varied between 285 and 360 g kg(-1) dry seed (DS), while crude protein ranged between 316 and 356 g kg(-1) dry matter (DM). The seed oil was mainly composed of unsaturated fatty acids and, as expected, the dominant fatty acids were linoleic and α-linolenic acid. A high variability among the cultivars and accessions was also detected for polyphenolic content which ranged from 5.88 to 10.63 g kg(-1) DM, cv. Felina was the richest, whereas cv. Finola had the lowest polyphenolic content. Regarding antinutritional compounds in seed, a high variability was detected among all genotypes analyzed and phytic acid was particularly abundant (ranging between 43 and 75 g kg(-1) DM). In conclusion, our results reveal noticeable differences among hemp seed genotypes for antinutritional components, oil and protein content. Collectively, this study suggests that the hemp seed is an interesting product in terms of protein, oil and antioxidant molecules but a reduction of phytic acid would be desirable for both humans and monogastric animals. The high variability detected among the different genotypes indicates that an improvement of hemp seed might be possible by conventional and/or molecular breeding.

Molecular characterization of edestin gene family in Cannabis sativa L.

Globulins are the predominant class of seed storage proteins in a wide variety of plants. In many plant species globulins are present in several isoforms encoded by gene families. The major seed storage protein of Cannabis sativa L. is the globulin edestin, widely known for its nutritional potential. In this work, we report the isolation of seven cDNAs encoding for edestin from the C. sativa variety Carmagnola. Southern blot hybridization is in agreement with the number of identified edestin genes. All seven sequences showed the characteristic globulin features, but they result to be divergent members/forms of two edestin types. According to their sequence similarity four forms named CsEde1A, CsEde1B, CsEde1C, CsEde1D have been assigned to the edestin type 1 and the three forms CsEde2A, CsEde2B, CsEde2C to the edestin type 2. Analysis of the coding sequences revealed a high percentage of similarity (98-99%) among the different forms belonging to the same type, which decreased significantly to approximately 64% between the forms belonging to different types. Quantitative RT-PCR analysis revealed that both edestin types are expressed in developing hemp seeds and the amount of CsEde1 was 4.44 ± 0.10 higher than CsEde2. Both edestin types exhibited a high percentage of arginine (11-12%), but CsEde2 resulted particularly rich in methionine residues (2.36%) respect to CsEde1 (0.82%). The amino acid composition determined in CsEde1 and CsEde2 types suggests that these seed proteins can be used to improve the nutritional quality of plant food-stuffs.

Traceback identification of plant components in commercial compound feed through an oligonucleotide microarray based on tubulin intron polymorphism.

According to EU Regulations, all components of commercial compound feed need to be declared on the label. Effective protection against fraud requires severe controls based on accurate analytical methods to ascertain what is declared by the producers. The aim of this work was to develop an oligonucleotide microarray for the molecular recognition of multiple plant components in commercial feeds. We tested the potential of the highly polymorphic first intron sequences from members of the plant ß-tubulin gene family as a target for plant DNA identification. 23 oligonucleotide capture probes, targeting species-specific intron sequences, were assembled within a low density microarray for the identification of 10 plant species, selected from among those most commonly used in cattle feed formulation. The ability of the array to detect specific components in complex flour blends and in compound feed was evaluated.

A multiplex, bead-based array for profiling plant-derived components in complex food matrixes.

Authentication of processed food ingredients is becoming an important issue for customers, and some DNA-based analytical methods have been developed, especially for animal products. As food products typically contain several different ingredients, a current challenge is to increase the multiplexing capacity of DNA-based methods, to develop "all-in-one" assays. Oligonucleotide-coupled, bead-based suspension arrays are sensitive and reproducible multiplex analytical tools. We applied the Multi-Analyte Profile (xMAP™) technology to develop an assay able to concurrently detect five different plant components in mixed flours and in processed feed and food. Capture probes were targeted to species-specific DNA polymorphisms present within the first intron of plant ß-tubulin genes, which can be amplified by the tubulin-based polymorphism-amplification method (TBP-PCR). The workflow is very simple and straightforward, consisting of a PCR amplification step with universal primers, followed by the direct hybridization assay. Results are highly reproducible. For each single plant species, the absolute detection limit was as low as one target DNA copy. In complex mixtures of flours derived from seeds or from commercial dry "pasta," relative limits of detection ranged, in weight, from 2% for soybean to less than 0.5% for wheat. The specificity of the capture probes and the high sensitivity of the method allowed the successful determination of the analytical composition of three feeds as well as eleven food samples, such as snacks, biscuits, and pasta. The multiplexing ability of the assay (up to 100 different analytes) provides scalability and flexibility, in response to specific needs.

Traceability of plant diet contents in raw cow milk samples.

The use of molecular marker in the dairy sector is gaining large acceptance as a reliable diagnostic approach for food authenticity and traceability. Using a PCR approach, the rbcL marker, a chloroplast-based gene, was selected to amplify plant DNA fragments in raw cow milk samples collected from stock farms or bought on the Italian market. rbcL-specific DNA fragments could be found in total milk, as well as in the skimmed and the cream fractions. When the PCR amplified fragments were sent to sequence, the nucleotide composition of the chromatogram reflected the multiple contents of the polyphytic diet.

Plant tubulin intronics.

Introns of plant tubulin genes are useful molecular tools to study IME (Intron Mediated Enhancement of gene expression) and to define plant genetic and evolutionary relationships through ILP (Intron Length Polymorphism). Here we show that the intron present within the 5'UTR sequence of some rice beta-tubulin genes can sustain IME in rice transgenic plants and that degenerated oligonucleotide mixtures designed to amplify the first and the second intron present within the coding sequence of plant beta-tubulin genes can successfully detect ILPs among different bean varieties.