Comparative de novo transcriptome analysis of barley varieties with different malting qualities.

Barley is one of the most important crops in the world. Barley is used as both food and feed and is important for malt production. Demands for malting quality differ among countries and customs. Malting quality is a complex characteristic involving barley genetics, the environmental conditions during barley growth, and the technological parameters of the malting process. In this study, the hypothesis was that there were no differences between two groups of barley varieties with different but defined malting qualities, which was tested using RNA sequencing during selected stages of malting. In total, 919 differentially transcribed genes between the two barley groups were identified and annotated. Differentially expressed genes (DEGs) were primarily assigned to gene ontology (GO) terms of oxidation-reduction process - oxidoreductase activity, response to stress, carbohydrate metabolic process, and proteolysis - hydrolase activity, and metal ion binding. Genes connected with the plasma membrane and its integral components also play important roles in malting quality. DEG profiles of selected genes in the three malting stages indicate a complex character of malting quality. Many single-nucleotide polymorphisms (SNPs) and insertions and deletions (indels) were identified. SNPs and indels with the best quality were used for primer design. After optimization and validation, five molecular markers were developed for use in barley breeding.

The dehydration stress of couch grass is associated with its lipid metabolism, the induction of transporters and the re-programming of development coordinated by ABA.

BACKGROUND: The wild relatives of crop species represent a potentially valuable source of novel genetic variation, particularly in the context of improving the crop's level of tolerance to abiotic stress. The mechanistic basis of these tolerances remains largely unexplored. Here, the focus was to characterize the transcriptomic response of the nodes (meristematic tissue) of couch grass (a relative of barley) to dehydration stress, and to compare it to that of the barley crown formed by both a drought tolerant and a drought sensitive barley cultivar. RESULTS: Many of the genes up-regulated in the nodes by the stress were homologs of genes known to be mediated by abscisic acid during the response to drought, or were linked to either development or lipid metabolism. Transporters also featured prominently, as did genes acting on root architecture. The resilience of the couch grass node arise from both their capacity to develop an altered, more effective root architecture, but also from their formation of a lipid barrier on their outer surface and their ability to modify both their lipid metabolism and transporter activity when challenged by dehydration stress. CONCLUSIONS: Our analysis revealed the nature of dehydration stress response in couch grass. We suggested the tolerance is associated with lipid metabolism, the induction of transporters and the re-programming of development coordinated by ABA. We also proved the applicability of barley microarray for couch grass stress-response analysis.

Changes in polyphenol compounds and barley laccase expression during the malting process.

BACKGROUND: Polyphenols and phenolic acid are able to slow down or prevent oxidation processes and are therefore thought to have important effects in malting and brewing. Laccase catalyses the oxidation of a wide variety of substrates, including polyphenols. The aim of this paper was to determine the changes in polyphenol compounds and the relative expression of the HvLac1 gene during malting. RESULTS: The dominant phenolic acid was ferulic acid. The amount of ferulic acid increased, whereas the amount of vanillic acid decreased during malting. The highest levels of expression of the HvLac1 gene were observed during the third air rest period in varieties with the 'Haruna Nijo' (HN) allele, as recommended for the production of beer with the protected geographical indication (PGI) 'Ceske pivo' (Czech beer), whereas the highest expression was observed in the first day of germination in varieties with the 'Morex' (M) allele. However, the profiles of HvLac1 gene expression in varieties with alternative alleles during malting were similar, and the level of polyphenol compounds throughout malting was different. CONCLUSION: The polyphenol contents in barley increased several-fold during malting, and the degree of increase differed with variety. The expression of HvLac1 transcript was similar in every barley variety.

Garlic (A. sativum L.) alliinase gene family polymorphism reflects bolting types and cysteine sulphoxides content.

BACKGROUND: Alliinase is an important enzyme occurring in Allium species that converts precursors of sulfuric compounds, cysteine sulfoxides into a biologically active substance termed allicin. Allicin facilitates garlic defense against pests and produces health-promoting compounds. Alliinase is encoded by members of a multigene family that has not yet been sufficiently characterized, namely with regard to the copy numbers occurring within the genome and the polymorphisms among the family members. RESULTS: We cloned 45 full-length alliinase amplicons of cultivar (cv.) Jovan. Sequence analyses revealed nine different sequence variants (SVs), confirming the multilocus nature of this gene family. Several mutations in exons, mainly occurring in the first exon coding for vacuolar signal peptide, were found. These results enabled us to identify sequences with putatively modified vacuole-targeting abilities. We found additional sequence variants using partial amplicons. We estimated that the minimum number of gene copies in the diploid genome of the investigated cultivar was fourteen. We obtained similar results for another three cultivars, which differed in bolting type and place of origin. The further identification of high degree of polymorphisms in the intron regions allowed us to develop a specific polymerase chain reaction assay capable to capture intron length polymorphism (ILP). This assay was used to screen 131 additional accessions. Polymorphic data were used for cluster analysis, which separated the bolting and non-bolting garlic types and those with high cysteine-sulfoxide contents in a similar way as AFLP analysis in previous study. These newly developed markers can be further applied for the selection of desirable garlic genotypes. CONCLUSIONS: Detailed analysis of sequences confirmed multigenic nature of garlic alliinase. Intron and exon polymorphism analysis generated similar results as whole genome variability assessed previously by AFLP. Detected polymorphism is thus also associated with cysteine-sulphoxide content in individual genotypes. ILP markers capable to detect intron polymorphisms were newly developed. Developed markers could be applied in garlic breeding. Higher genetic variability found in bolting genotypes may indicates longer period of their sexual propagation in comparison with nonbolting genotypes.

Validation of the ß-amy1 transcription profiling assay and selection of reference genes suited for a RT-qPCR assay in developing barley caryopsis.

Reverse transcription coupled with real-time quantitative PCR (RT-qPCR) is a frequently used method for gene expression profiling. Reference genes (RGs) are commonly employed to normalize gene expression data. A limited information exist on the gene expression and profiling in developing barley caryopsis. Expression stability was assessed by measuring the cycle threshold (Ct) range and applying both the GeNorm (pair-wise comparison of geometric means) and Normfinder (model-based approach) principles for the calculation. Here, we have identified a set of four RGs suitable for studying gene expression in the developing barley caryopsis. These encode the proteins GAPDH, HSP90, HSP70 and ubiquitin. We found a correlation between the frequency of occurrence of a transcript in silico and its suitability as an RG. This set of RGs was tested by comparing the normalized level of ß-amylase (ß-amy1) transcript with directly measured quantities of the BMY1 gene product in the developing barley caryopsis. This panel of genes could be used for other gene expression studies, as well as to optimize ß-amy1 analysis for study of the impact of ß-amy1 expression upon barley end-use quality.

Characterization and mapping of a putative laccase-like multicopper oxidase gene in the barley (Hordeum vulgare L.).

Laccases constitute a multi-gene family of multi-copper glycoproteins. The barley laccase-like multicopper oxidase (LMCO) gene structure, the DNA sequence polymorphism and putative protein have not yet been described. As part of the study of LMCO in cereals, we have characterized the genomic structure of the putative LMCO gene HvLac1 from the barley variety 'Morex' and mapped HvLac1 on chromosome 4H. The genomic sequence of the HvLac1 gene is 2646 bp long and covers 100% of the coding region. It contains four exons and three introns. In this study, we have described the HvLac1 gene nucleotide polymorphisms (In/Del) in 134 barley varieties. Initial characterization of the barley and rice LMCO and the phylogeny analysis indicate that a monocot LMCO family is composed of five members. There are two high pI isoforms of putative HvLac1 protein derived from two in frame translation start codons with 602aa or 592aa residues. Isoforms differ in their predicted subcellular localization and both isoforms are characterized on C-terminus by the presence of the KDEL-like motif, which contributes to the accumulation of soluble proteins in the endoplasmic reticulum. Our results suggest that this unique feature of HvLac1 could be important for their role in physiological processes.