Genetic Loci Underlying Awn Morphology in Barley

Barley awns are highly active in photosynthesis and account for 30–50% of grain weight in barley. They are diverse in length, ranging from long to awnless, and in shape from straight to hooded or crooked. Their diversity and importance have intrigued geneticists for several decades. A large collection of awnness mutants are available—over a dozen of them have been mapped on chromosomes and a few recently cloned. Different awnness genes interact with each other to produce diverse awn phenotypes. With the availability of the sequenced barley genome and application of new mapping and gene cloning strategies, it will now be possible to identify and clone more awnness genes. A better understanding of the genetic basis of awn diversity will greatly facilitate development of new barley cultivars with improved yield, adaptability and sustainability.

Genome-wide identification and expression profile under abiotic stress of the barley non-specific lipid transfer protein gene family and its Qingke Orthologues

Background Plant non-specific lipid transfer proteins (nsLTPs), a group of small, basic ubiquitous proteins to participate in lipid transfer, cuticle formation and stress response, are involved in the regulation of plant growth and development. To date, although the nsLTP gene family of barley (Hordeum vulgare L.) has been preliminarily identified, it is still unclear in the recently completed genome database of barley and Qingke, and its transcriptional profiling under abiotic stress has not been elucidated as well. Results We identified 40 barley nsLTP (HvLTP) genes through a strict screening strategy based on the latest barley genome and 35 Qingke nsLTP (HtLTP) orthologues using blastp, and these LTP genes were divided into four types (1, 2, D and G). At the same time, a comprehensive analysis of the physical and chemical characteristics, homology alignment, conserved motifs, gene structure and evolution of HvLTPs and HtLTPs further supported their similar nsLTP characteristics and classification. The genomic location of HvLTPs and HtLTPs showed that these genes were unevenly distributed, and obvious HvLTP and HtLTP gene clusters were found on the 7 chromosomes including six pairs of tandem repeats and one pair of segment repeats in the barley genome, indicating that these genes may be co-evolutionary and co-regulated. A spatial expression analysis showed that most HvLTPs and HtLTPs had different tissue-specific expression patterns. Moreover, the upstream cis-element analysis of HvLTPs and HtLTPs showed that there were many different stress-related transcriptional regulatory elements, and the expression pattern of HvLTPs and HtLTPs under abiotic stress also indicated that numerous HvLTP and HtLTP genes were related to the abiotic stress response. Taken together, these results may be due to the differences in promoters rather than by genes themselves resulting in different expression patterns under abiotic stress. Conclusion Due to a stringent screening and comprehensive analysis of the nsLTP gene family in barley and Qingke and its expression profile under abiotic stress, this study can be considered a useful source for the future studies of nsLTP genes in either barley or Qingke or for comparisons of different plant species.

Genome-Wide Analysis of NLR Disease Resistance Genes in an Updated Reference Genome of Barley

Barley is one of the top 10 crop plants in the world. During its whole lifespan, barley is frequently infected by various pathogens. In this study, we performed genome-wide analysis of the largest group of plant disease resistance (R) genes, the nucleotide binding site–leucine-rich repeat receptor (NLR) gene, in an updated barley genome. A total of 468 NLR genes were identified from the improved barley genome, including one RNL subclass and 467 CNL subclass genes. Proteins of 43 barley CNL genes were shown to contain 25 different integrated domains, including WRKY and BED. The NLR gene number identified in this study is much larger than previously reported results in earlier versions of barley genomes, and only slightly fewer than that in the diploid wheat Triticum urartu. Barley Chromosome 7 contains the largest number of 112 NLR genes, which equals to seven times of the number of NLR genes on Chromosome 4. The majority of NLR genes (68%) are located in multigene clusters. Phylogenetic analysis revealed that at least 18 ancestral CNL lineages were presented in the common ancestor of barley, T. urartu and Arabidopsis thaliana. Among them fifteen lineages expanded to 533 sub-lineages prior to the divergence of barley and T. urartu. The barley genome inherited 356 of these sub-lineages and duplicated to the 467 CNL genes detected in this study. Overall, our study provides an updated profile of barley NLR genes, which should serve as a fundamental resource for functional gene mining and molecular breeding of barley.

Genome editing of barley

Although barley is of great importance for the brewing and animal feed industries and is regarded as a model for small grain cereals, only a few results on targeted gene modification using CRISPR/Cas endonuclease technology have been published to date. In this chapter, the frontiers and achievements of the currently used techniques in barley genome modification will be shown and discussed.

BarleyVarDB: a database of barley genomic variation

Barley (Hordeum vulgare L.) is one of the first domesticated grain crops and represents the fourth most important cereal source for human and animal consumption. BarleyVarDB is a database of barley genomic variation. It can be publicly accessible through the website at http://146.118.64.11/BarleyVar. This database mainly provides three sets of information. First, there are 57 754 224 single nuclear polymorphisms (SNPs) and 3 600 663 insertions or deletions (InDels) included in BarleyVarDB, which were identified from high-coverage whole genome sequencing of 21 barley germplasm, including 8 wild barley accessions from 3 barley evolutionary original centers and 13 barley landraces from different continents. Second, it uses the latest barley genome reference and its annotation information publicly accessible, which has been achieved by the International Barley Genome Sequencing Consortium (IBSC). Third, 522 212 whole genome-wide microsatellites/simple sequence repeats (SSRs) were also included in this database, which were identified in the reference barley pseudo-molecular genome sequence. Additionally, several useful web-based applications are provided including JBrowse, BLAST and Primer3. Users can design PCR primers to asses polymorphic variants deposited in this database and use a user-friendly interface for accessing the barley reference genome. We envisage that the BarleyVarDB will benefit the barley genetic research community by providing access to all publicly available barley genomic variation information and barley reference genome as well as providing them with an ultra-high density of SNP and InDel markers for molecular breeding and identification of functional genes with important agronomic traits in barley. Database URL: http://146.118.64.11/BarleyVar

Genome-Wide Exploration of Thaumatin-like Protein (TLP) Gene Family in Cereals

Background: TLP genes are the members of a conserved pathogenesis-related protein 5 (PR-5) gene family. They play role in abiotic stress response, hormone signaling, cell death, cold tolerance, enzyme inactivation, fruit maturation and seed germination. In this study, we characterized the TLP gene family in barley with specific emphasis on germination and malting. Results: We identified 19 TLP genes from the reference genome of Hordeum vulgare L. cv. Morex and 37, 28 and 35 TLP genes from the Oryza sativa, Brachypodium distachyon and Sorghum bicolor genome respectively. Comparative phylogenetic analysis and thaumatin domain organization of TLPs using the conserved region classified the TLP family into nine groups. Data revealed that localized gene duplications contributed to the expansion of the TLP gene family in cereals with diverse exon/intron structures. In the barley genome, most HvTLPs were localized on chromosome 5H. The differential spatiotemporal expression pattern of HvTLP genes in barley indicate that TLPs have been expressed predominantly in the embryo, developing grains, root and shoot tissues. Additionally, transcript abundance of HvTLP genes was measured between 16 hrs. to 96 hrs. of grain germination. Differential expression of HvTLP14, HvTLP17 and HvTLP18 in the malting variety (Morex), as compared to the feed variety (Steptoe) at different stages of seed germination indicates their possible role in malting. Conclusion: Barley genome contains higher number (19) of TLP genes as previously thought (8). This study provides a description of the TLP gene family in barley and their differential expression between 16-96 hrs. of germination. The results indicate their possible involvement in the malting process. Keywords: Cereals, Barley, Thaumatin like-proteins, Phylogenetics, Expression analysis

Duplicated flavonoid 3’-hydroxylase and flavonoid 3’, 5’-hydroxylase genes in barley genome

Background Anthocyanin compounds playing multiple biological functions can be synthesized in different parts of barley (Hordeum vulgare L.) plant. The diversity of anthocyanin molecules is related with branching the pathway to alternative ways in which dihydroflavonols may be modified either with the help of flavonoid 3′-hydroxylase (F3′H) or flavonoid 3′,5′-hydroxylase (F3′5′H)—the cytochrome P450-dependent monooxygenases. The F3′H and F3′5′H gene families are among the least studied anthocyanin biosynthesis structural genes in barley. The aim of this study was to identify and characterise duplicated copies of the F3′H and F3′5′H genes in the barley genome. Results Four copies of the F3′5′H gene (on chromosomes 4HL, 6HL, 6HS and 7HS) and two copies of the F3′H gene (on chromosomes 1HL and 6HS) were identified in barley genome. These copies have either one or two introns. Amino acid sequences analysis demonstrated the presence of the flavonoid hydroxylase-featured conserved motifs in all copies of the F3′H and F3′5′H genes with the exception of F3′5′H-3 carrying a loss-of-function mutation in a conservative cytochrome P450 domain. It was shown that the divergence between F3′H and F3′5′H genes occurred 129 million years ago (MYA) before the emergence of monocot and dicot plant species. The F3′H copy approximately occurred 80 MYA; the appearance of F3′5′H copies occurred 8, 36 and 91 MYA. qRT-PCR analysis revealed the tissue-specific activity for some copies of the studied genes. The F3′H-1 gene was transcribed in aleurone layer, lemma and pericarp (with an increased level in the coloured pericarp), whereas the F3′H-2 gene was expressed in stems only. The F3′5′H-1 gene was expressed only in the aleurone layer, and in a coloured aleurone its expression was 30-fold higher. The transcriptional activity of F3′5′H-2 was detected in different tissues with significantly higher level in uncoloured genotype in contrast to coloured ones. The F3′5′H-3 gene expressed neither in stems nor in aleurone layer, lemma and pericarp. The F3′5′H-4 gene copy was weakly expressed in all tissues analysed. Conclusion F3′H and F3′5′H-coding genes involved in anthocyanin synthesis in H. vulgare were identified and characterised, from which the copies designated F3′H-1, F3′H-2, F3′5′H-1 and F3′5′H-2 demonstrated tissue-specific expression patterns. Information on these modulators of the anthocyanin biosynthesis pathway can be used in future for manipulation with synthesis of diverse anthocyanin compounds in different parts of barley plant. Finding both the copies with tissue-specific expression and a copy undergoing pseudogenization demonstrated rapid evolutionary events tightly related with functional specialization of the duplicated members of the cytochrome P450-dependent monooxygenases gene families.