Benzoxazinoids (BX) are major secondary metabolites of gramineous plants that play an important role in disease resistance and allelopathy. They also have many other unique properties including anti-bacterial and anti-fungal activity, and the ability to reduce alfa–amylase activity. The biosynthesis and modification of BX are controlled by the genes Bx1 ÷ Bx10, GT and glu, and the majority of these Bx genes have been mapped in maize, wheat and rye. However, the genetic basis of BX biosynthesis remains largely uncharacterized apart from some data from maize and wheat. The aim of this study was to isolate, sequence and characterize five genes (ScBx1, ScBx2, ScBx3, ScBx4 and ScBx5) encoding enzymes involved in the synthesis of DIBOA, an important defense compound of rye. Using a modified 3D procedure of BAC library screening, seven BAC clones containing all of the ScBx genes were isolated and sequenced. Bioinformatic analyses of the resulting contigs were used to examine the structure and other features of these genes, including their promoters, introns and 3’UTRs. Comparative analysis showed that the ScBx genes are similar to those of other Poaceae species, especially to the TaBx genes. The polymorphisms present both in the coding sequences and non-coding regions of ScBx in relation to other Bx genes are predicted to have an impact on the expression, structure and properties of the encoded proteins.Electronic supplementary materialThe online version of this article (doi:10.1007/s13353-015-0271-z) contains supplementary material, which is available to authorized users.

The first step of the benzoxazinoid (BX) synthesis pathway is catalyzed by an enzyme with indole-3-glycerol phosphate lyase activity encoded by 3 genes, Bx1, TSA and Igl. A gene highly homologous to maize and wheat Bx1 has been identified in rye. The goal of the study was to analyze the gene and to experimentally verify its role in the rye BX biosynthesis pathway as a rye ortholog of the Bx1 gene. Expression of the gene showed peak values 3 days after imbibition (dai) and at 21 dai it was undetectable. Changes of the BX content in leaves were highly correlated with the expression pattern until 21 dai. In plants older than 21 dai despite the undetectable expression of the analyzed gene there was still low accumulation of BXs. Function of the gene was verified by correlating its native expression and virus-induced silencing with BX accumulation. Barley stripe mosaic virus (BSMV)-based vectors were used to induce transcriptional (TGS) and posttranscriptional (PTGS) silencing of the analyzed gene. Both strategies (PTGS and TGS) significantly reduced the transcript level of the analyzed gene, and this was highly correlated with lowered BX content. Inoculation with virus-based vectors specifically induced expression of the analyzed gene, indicating up-regulation by biotic stressors. This is the first report of using the BSMV-based system for functional analysis of rye gene. The findings prove that the analyzed gene is a rye ortholog of the Bx1 gene. Its expression is developmentally regulated and is strongly induced by biotic stress. Stable accumulation of BXs in plants older than 21 dai associated with undetectable expression of ScBx1 indicates that the function of the ScBx1 in the BX biosynthesis is redundant with another gene. We anticipate that the unknown gene is a putative ortholog of the Igl, which still remains to be identified in rye.

Seed aging is a complex biological process and its fundamentals and mechanisms have not yet been fully recognized. This is a key issue faced by research teams involved in the collection and storage of plant genetic resources in gene banks every day. Transcriptomic changes associated with seed aging in the dry state have barely been studied. The aim of the study was to develop an efficient protocol for construction of RNA-Seq libraries from long-term stored seeds with very low viability and low RNA integrity number (RIN). Here, barley seeds that have almost completely lost their viability as a result of long-term storage were used. As a control, fully viable seeds obtained in the course of field regeneration were used. The effectiveness of protocols dedicated to RNA samples with high and low RIN values was compared. The experiment concluded that library construction from low viable or long-term stored seeds with degraded RNA (RIN < 3) should be carried out with extraordinary attention due to the possibility of uneven degradation of different RNA fractions.

Landraces are considered a key element of agrobiodiversity because of their high variability and adaptation to local environmental conditions, but at the same time, they represent a breeding potential hidden in gene banks that has not yet been fully appreciated and utilized. Here, we present a genome-wide DArTseq analysis of the diversity of 116 spring barley landraces preserved in the collection of the Polish gene bank. Genetic analysis revealed considerable variation in this collection and several distinct groups related to the landraces’ country of origin and the grain type were identified. The genetic distinctness of hulless accessions may provide a basis for pro-quality breeding aimed at functional food production. However, the variable level of accession heterogeneity can be a significant obstacle. A solution to this problem is the establishment of special collections composed of pure lines that are accessible to breeders. Regions lacking genetic diversity have also been identified on 1H and 4H chromosomes. A small region of reduced heterogeneity was also present in the hulless forms in the vicinity of the nud gene that determines the hulless grain type. However, the SNPs present in this area may also be important in selection for traits related to grain weight and size because their QTLs were found there. This may support breeding of hulless forms of spring barley which may have applications in the production of high-quality foods with health-promoting values.

GLYCOGEN SYNTHASE KINASE3/Shaggy-like kinases (GSKs) represent a highly conserved group of proteins found in all eukaryotes. In plants they are encoded by multigene families and integrate signaling of brassinosteroids, auxin and abscisic acid in wide range of physiological and developmental processes with a strong impact on plant responses to environmental and biotic factors. Based on comprehensively studied structures of 10 Arabidopsis thaliana GSK genes and encoded proteins we report identification and phylogenetic reconstruction of 7 transcriptionally active GSK genes in barley. We re-evaluated annotation of the GSK genes in the current barley genome (Hv_IBSC_PGSB_v2) and provided data that a single gene annotated in the previous barley genome ensemble should be retained in the current one. The novel structure of another GSK, predicted in Hv_IBSC_PGSB_v2 to encode both GSK and amine oxidase domains, was proposed and experimentally confirmed based on the syntenic region in Brachypodium distachyon. The genes were assigned to 4 groups based on their encoded amino acid sequences and protein kinase domains. The analysis confirmed high level of conservation of functional protein domains and motifs among plant GSKs and the identified barley orthologs. Each of the seven identified HvGSK genes was expressed indicating semi-constitutive regulation in all tested organs and developmental stages. Regulation patterns of GSKs from the indicated groups showed a shift in organ-preferential expression in A. thaliana and barley illustrating diversification of biological roles of individual HvGSKs in different plant species.