Influence of CNV on transcript levels of HvCBF genes at Fr-H2 locus revealed by resequencing in resistant barley cv. 'Nure' and expression analysis.

Resequencing in resistant cultivar 'Nure' and structural comparison with the same region of susceptible 'Morex' was performed in order to gain a better insight into barley Frost-resistance-H2 locus. Accurate annotation showed copy number variation (CNV) in the proximal part of the locus. In 'Nure', two exact copies of the HvCBF4-HvCBF2A region and one of the HvCBF4-HvCBF2B segment were observed, while in 'Morex' the corresponding region harboured a single HvCBF4-HvCBF2A (22 kb) segment. Abundance and diversity of repetitive element classes, gene function gain/losses, regulatory motifs and SNPs in gene sequences were identified. An expression study of key HvCBFs with/without CNV on selected genotypes contrasting for frost resistance and estimated HvCBF4-HvCBF2B copy number (2-10 copies) was also performed. Under light stimulus at warm temperature (23 °C), CNV of HvCBF2A and HvCBF4 correlated with their expression levels and reported frost resistance of genotypes; moreover, expression levels of HvCBF2A and HvCBF14 were strongly correlated (r = 0.908, p < 0.01). On the other hand, frost resistance correlated to HvCBF14 expression (r = 0.871, p < 0.01) only after cold induction (6°C) in the dark. A complex interplay of HvCBFs expression levels under different light/temperature stimuli is discussed in light of CNV and presence/number of regulatory elements that integrate different signal transduction pathways.

Determination of Free Soluble Phenolic Compounds in Grains of Ancient Wheat Varieties ( Triticum sp. pl.) by Liquid Chromatography-Tandem Mass Spectrometry.

A method of liquid chromatography coupled with tandem mass spectrometry was developed and validated for the determination of free soluble phenolic compounds in eight ancient varieties of wheat ( Triticum sp. pl.): Autonomia, Gentil rosso, Inallettabile, Leone aristato, Mentana, Poulard di Ciano, Risciola, and Terminillo. Trace compounds such as two conjugated flavones, vitexin (17.13-34.32 µg/kg) and isovitexin (9.76-30.01 µg/kg), were also determined. Poulard di Ciano, presumably an autochthonous wheat of the Reggio Emilia province (northern Italy), showed a peculiar quali/quantitative phenolic profile (7097.03 µg/kg sum of total phenolic compounds and 1.97 sum of hydroxycinnamic acids to sum of hydroxybenzoic acids ratio) along with a tetraploid genome. Terminillo, Risciola, Gentil rosso, Mentana, and Leone aristato showed hexaploid genomes and high concentrations of phenolic compounds (ranging from 6796.12 to 7605.78 µg/kg), also in comparison with two modern varieties of bread wheat, Bolero and Blasco. The targeted metabolomic approach proved to be effective to determine some secondary metabolites of wheat. The richness in phenolic compounds combined with high rusticity and adaptability to marginal soils showed by ancient wheat varieties make them suitable for sustainable agricultural and organic cultivation.