Elena Ponzoni

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 β‐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 β‐tubulin genes can successfully detect ILPs among different bean varieties.

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.

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.

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 β-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.

From milk to diet: feed recognition for milk authenticity.

The presence of plastidial DNA fragments of plant origin in animal milk samples has been confirmed. An experimental plan was arranged with 4 groups of goats, each provided with a different monophytic diet: 3 fresh forages (oats, ryegrass, and X-triticosecale) and one 2-wk-old silage (X-triticosecale). Feed-derived rubisco (ribulose bisphosphate carboxylase, rbcL) DNA fragments were detected in 100% of the analyzed goat milk samples, and the nucleotide sequence of the PCR-amplified fragments was found to be 100% identical to the corresponding fragments amplified from the plant species consumed in the diet. Two additional chloroplast-based molecular markers were used to set up an assay for distinctiveness, conveniently based on a simple PCR. In one case, differences in single nucleotides occurring within the gene encoding for plant maturase K (matK) were exploited. In the other, plant species recognition was based on the difference in the length of the intron present within the transfer RNA leucine (trnL) gene. The presence of plastidial plant DNA, ascertained by the PCR-based amplification of the rbcL fragment, was also assessed in raw cow milk samples collected directly from stock farms or taken from milk sold on the commercial market. In this case, the nucleotide sequence of the amplified DNA fragments reflected the multiple forages present in the diet fed to the animals.

Genome-wide identification and organization of seed storage protein genes of Cannabis sativa

Hemp (Cannabis sativa L.) seeds have been recognized as a nutritional protein source for humans and animals. In this study, gene families encoding precursor polypeptides of three storage protein classes, including six 11S edestin, two 2S albumin and one 7S vicilin-like genes were identified and characterized from an inbred line of hemp. All edestins showed typical 11S globulin features but based on the amino acid composition, they were grouped in three edestin types (type1, -2 and -3). Genes encoding edestin type1 and -3 were very close to each other in a DNA fragment of 16 071 bp, whereas the two isoforms of edestin type2 were linked on a different DNA fragment of 8 232 bp and arranged in a tailto- tail fashion. All edestin types were very rich in arginine and glutamic acid, but edestin type3 was the richest in cysteine and methionine. Regarding the 2S albumin (Cs2S) two genes were identified in a fragment of 13 738 bp in a tail-to-head array. Finally, only one 7S-vicilin like gene (Cs7S) that exhibited typical 7S vicilin features, such as the presence of two cupin domains and several N-glycosylation sites, was isolated. Southern blot hybridization is in agreement with the number of genes isolated, and real-time qPCR analysis revealed that all genes are expressed in the seed. The highest expression was observed for edestin type1 (CsEde1) and Cs2S, whereas the lowest expression was detected for Cs7S. The results of this study provide a complete overview of the genes encoding hemp storage proteins and significantly advance our knowledge on the organization of these gene families.