A guide to barley mutants.

BACKGROUND: Mutants have had a fundamental impact upon scientific and applied genetics. They have paved the way for the molecular and genomic era, and most of today's crop plants are derived from breeding programs involving mutagenic treatments. RESULTS: Barley (Hordeum vulgare L.) is one of the most widely grown cereals in the world and has a long history as a crop plant. Barley breeding started more than 100 years ago and large breeding programs have collected and generated a wide range of natural and induced mutants, which often were deposited in genebanks around the world. In recent years, an increased interest in genetic diversity has brought many historic mutants into focus because the collections are regarded as valuable resources for understanding the genetic control of barley biology and barley breeding. The increased interest has been fueled also by recent advances in genomic research, which provided new tools and possibilities to analyze and reveal the genetic diversity of mutant collections. CONCLUSION: Since detailed knowledge about phenotypic characters of the mutants is the key to success of genetic and genomic studies, we here provide a comprehensive description of mostly morphological barley mutants. The review is closely linked to the International Database for Barley Genes and Barley Genetic Stocks ( bgs.nordgen.org ) where further details and additional images of each mutant described in this review can be found.

Genome-Wide Association Study of Salt Tolerance-Related Traits during Germination and Seedling Development in an Intermedium-Spike Barley Collection.

Increased salinity is one of the major consequences of climatic change affecting global crop production. The early stages in the barley (Hordeum vulgare L.) life cycle are considered the most critical phases due to their contributions to final crop yield. Particularly, the germination and seedling development are sensitive to numerous environmental stresses, especially soil salinity. In this study, we aimed to identify SNP markers linked with germination and seedling development at 150 mM NaCl as a salinity treatment. We performed a genome-wide association study (GWAS) using a panel of 208 intermedium-spike barley (H. vulgare convar. intermedium (Körn.) Mansf.) accessions and their genotype data (i.e., 10,323 SNPs) using the genome reference sequence of "Morex". The phenotypic results showed that the 150 mM NaCl salinity treatment significantly reduced all recorded germination and seedling-related traits compared to the control treatment. Furthermore, six accessions (HOR 11747, HOR 11718, HOR 11640, HOR 11256, HOR 11275 and HOR 11291) were identified as the most salinity tolerant from the intermedium-spike barley collection. GWAS analysis indicated that a total of 38 highly significantly associated SNP markers under control and/or salinity traits were identified. Of these, two SNP markers on chromosome (chr) 1H, two on chr 3H, and one on chr 4H were significantly linked to seedling fresh and dry weight under salinity stress treatment. In addition, two SNP markers on chr 7H were also significantly associated with seedling fresh and dry weight but under control condition. Under salinity stress, one SNP marker on chr 1H, 5H and 7H were detected for more than one phenotypic trait. We found that in most of the accessions exhibiting the highest salinity tolerance, most of the salinity-related QTLs were presented. These results form the basis for detailed studies, leading to improved salt tolerance breeding programs in barley.

Analyses of MADS-box Genes Suggest HvMADS56 to Regulate Lateral Spikelet Development in Barley.

MADS-box transcription factors are crucial regulators of inflorescence and flower development in plants. Therefore, the recent interest in this family has received much attention in plant breeding programs due to their impact on plant development and inflorescence architecture. The aim of this study was to investigate the role of HvMADS-box genes in lateral spikelet development in barley (Hordeum vulgare L.). A set of 30 spike-contrasting barley lines were phenotypically and genotypically investigated under controlled conditions. We detected clear variations in the spike and spikelet development during the developmental stages among the tested lines. The lateral florets in the deficiens and semi-deficiens lines were more reduced than in two-rowed cultivars except cv. Kristina. Interestingly, cv. Kristina, int-h.43 and int-i.39 exhibited the same behavior as def.5, def.6, semi-def.1, semi-def.8 regarding development and showed reduced lateral florets size. In HOR1555, HOR7191 and HOR7041, the lateral florets continued their development, eventually setting seeds. In contrast, lateral florets in two-rowed barley stopped differentiating after the awn primordia stage giving rise to lateral floret sterility. At harvest, the lines tested showed large variation for all central and lateral spikelet-related traits. Phylogenetic analysis showed that more than half of the 108 MADS-box genes identified are highly conserved and are expressed in different barley tissues. Re-sequence analysis of a subset of these genes showed clear polymorphism in either SNPs or in/del. Variation in HvMADS56 correlated with altered lateral spikelet morphology. This suggests that HvMADS56 plays an important role in lateral spikelet development in barley.

Barley Viridis-k links an evolutionarily conserved C-type ferredoxin to chlorophyll biosynthesis.

Ferredoxins are single-electron carrier proteins involved in various cellular reactions. In chloroplasts, the most abundant ferredoxin accepts electrons from photosystem I and shuttles electrons via ferredoxin NADP+ oxidoreductase to generate NADPH or directly to ferredoxin dependent enzymes. In addition, plants contain other isoforms of ferredoxins. Two of these, named FdC1 and FdC2 in Arabidopsis thaliana, have C-terminal extensions and functions that are poorly understood. Here we identified disruption of the orthologous FdC2 gene in barley (Hordeum vulgare L.) mutants at the Viridis-k locus; these mutants are deficient in the aerobic cyclase reaction of chlorophyll biosynthesis. The magnesium-protoporphyrin IX monomethyl ester cyclase is one of the least characterized enzymes of the chlorophyll biosynthetic pathway and its electron donor has long been sought. Agroinfiltrations showed that the viridis-k phenotype could be complemented in vivo by Viridis-k but not by canonical ferredoxin. VirK could drive the cyclase reaction in vitro and analysis of cyclase mutants showed that in vivo accumulation of VirK is dependent on cyclase enzyme levels. The chlorophyll deficient phenotype of viridis-k mutants suggests that VirK plays an essential role in chlorophyll biosynthesis that cannot be replaced by other ferredoxins, thus assigning a specific function to this isoform of C-type ferredoxins.

Dissecting the Genetic Basis of Lateral and Central Spikelet Development and Grain Traits in Intermedium-Spike Barley (Hordeum vulgare Convar. Intermedium).

Barley (Hordeum vulgare L.) is one of the major grain crops worldwide and considered as a model plant for temperate cereals. One of the barley row-type groups, named intermedium-barley, was used in our previous study where we reported that other genetic loci rather than vrs1 and Int-c could play a role in lateral spikelet development and even in setting grains. To continue this work, we used phenotypic and genotypic data of 254 intermedium-spike barley accessions aimed at dissecting the genetic basis of development and grain traits of lateral and central spikelet using genome wide association (GWAS) analysis. After genotypic data filtering, 8,653 single-nucleotide polymorphism (SNPs) were used for GWAS analysis. A total of 169 significant associations were identified and we focused only on the subset of associations that exceeded the p < 10-4 threshold. Thirty-three highly significant marker-trait-associations (MTAs), represented in 28 different SNPs on all seven chromosomes for the central and/or lateral spikelet traits; such as kernel length, width, area, weight, unfilled spikelet and 1000-kernel weight, were detected. Highly significant associated markers were anchored physically using barley genome sequencing to identify candidate genes to either contain the SNPs or the closest gene to the SNP position. The results showed that 12 MTAs were specific for lateral spikelet traits, nine MTAs were specific for central spikelet traits and seven MTAs for both central and lateral traits. All together, the GWAS and candidate gene results support our hypothesis that lateral spikelet development could be regulated by loci different from those regulating central spikelet development.

COMPOSITUM 1 contributes to the architectural simplification of barley inflorescence via meristem identity signals.

Grasses have varying inflorescence shapes; however, little is known about the genetic mechanisms specifying such shapes among tribes. Here, we identify the grass-specific TCP transcription factor COMPOSITUM 1 (COM1) expressing in inflorescence meristematic boundaries of different grasses. COM1 specifies branch-inhibition in barley (Triticeae) versus branch-formation in non-Triticeae grasses. Analyses of cell size, cell walls and transcripts reveal barley COM1 regulates cell growth, thereby affecting cell wall properties and signaling specifically in meristematic boundaries to establish identity of adjacent meristems. COM1 acts upstream of the boundary gene Liguleless1 and confers meristem identity partially independent of the COM2 pathway. Furthermore, COM1 is subject to purifying natural selection, thereby contributing to specification of the spike inflorescence shape. This meristem identity pathway has conceptual implications for both inflorescence evolution and molecular breeding in Triticeae.

Aerobic Barley Mg-protoporphyrin IX Monomethyl Ester Cyclase is Powered by Electrons from Ferredoxin.

Chlorophyll is the light-harvesting molecule central to the process of photosynthesis. Chlorophyll is synthesized through 15 enzymatic steps. Most of the reactions have been characterized using recombinant proteins. One exception is the formation of the isocyclic E-ring characteristic of chlorophylls. This reaction is catalyzed by the Mg-protoporphyrin IX monomethyl ester cyclase encoded by Xantha-l in barley (Hordeum vulgare L.). The Xantha-l gene product (XanL) is a membrane-bound diiron monooxygenase, which requires additional soluble and membrane-bound components for its activity. XanL has so far been impossible to produce as an active recombinant protein for in vitro assays, which is required for deeper biochemical and structural analyses. In the present work, we performed cyclase assays with soluble and membrane-bound fractions of barley etioplasts. Addition of antibodies raised against ferredoxin or ferredoxin-NADPH oxidoreductase (FNR) inhibited assays, strongly suggesting that reducing electrons for the cyclase reaction involves ferredoxin and FNR. We further developed a completely recombinant cyclase assay. Expression of active XanL required co-expression with an additional protein, Ycf54. In vitro cyclase activity was obtained with recombinant XanL in combination with ferredoxin and FNR. Our experiment demonstrates that the cyclase is a ferredoxin-dependent enzyme. Ferredoxin is part of the photosynthetic electron-transport chain, which suggests that the cyclase reaction might be connected to photosynthesis under light conditions.

Heterologous Expression of the Barley (Hordeum vulgare L.) Xantha-f, -g and -h Genes that Encode Magnesium Chelatase Subunits.

Biosynthesis of chlorophyll involves several enzymatic reactions of which many are shared with the heme biosynthesis pathway. Magnesium chelatase is the first specific enzyme in the chlorophyll pathway. It catalyzes the formation of Mg-protoporphyrin IX from the insertion of Mg2+ into protoporphyrin IX. The enzyme consists of three subunits encoded by three genes. The three genes are named Xantha-h, Xantha-g and Xantha-f in barley (Hordeum vulgare L.). The products of the genes have a molecular weight of 38, 78 and 148 kDa, respectively, as mature proteins in the chloroplast. Most studies on magnesium chelatase enzymes have been performed using recombinant proteins of Rhodobacter capsulatus, Synechocystis sp. PCC6803 and Thermosynechococcus elongatus, which are photosynthetic bacteria. In the present study we established a recombinant expression system for barley magnesium chelatase with the long-term goal to obtain structural information of this enigmatic enzyme complex from a higher plant. The genes Xantha-h, -g and -f were cloned in plasmid pET15b, which allowed the production of the three subunits as His-tagged proteins in Escherichia coli BL21(DE3)pLysS. The purified subunits stimulated magnesium chelatase activity of barley plastid extracts and produced activity in assays with only recombinant proteins. In preparation for future structural analyses of the barley magnesium chelatase, stability tests were performed on the subunits and activity assays were screened to find an optimal buffer system and pH.

Correction to: Genetic mapping of the labile (lab) gene: a recessive locus causing irregular spikelet fertility in labile-barley (Hordeum vulgare convar. labile).

While continuing our quest towards the identification of the labile (lab) locus in barley, we discovered that the previously assigned map location on the long arm of chromosome 5H was wrong.

Spike developmental stages and ABA role in spikelet primordia abortion contribute to the final yield in barley (Hordeum vulgare L.).

BACKGROUND: Salinity is a significant environmental stress factor limiting crops productivity. Barley (Hordeum vulgare L.) has a natural tolerance to salinity stress, making it an interesting study object in stress biology research. In the present study, for the first time the effect of salinity stress on barley inflorescence developmental stages was investigated. Five spring barley genotypes irrigated with saline water (12.5 ds/m NaCl) were compared to controls treated with normal tap water. We measured abscisic acid (ABA) concentrations in the apical, central and basal sections of the immature inflorescence at green anther (GA) stage. The role of ABA in spikelet primordia development, atrophy and abortion and final yield was evaluated. RESULTS: A time course experiment starting from double ridge until green anther (GA) stages revealed that salinity reduced the length of spike developmental stages in all genotypes causing shortened of the plant life cycle. The shortened plant life cycle negatively affected plant height and number of tillers/plant. Salinity also affected spikelet primordia development. In both control and salinity treated plants apical spikelet abortion started in late awn primordium (AP) stage. However, under salinity treatment, significantly more spikelets were aborted, thus directly affecting plant yield potential. ABA, which plays a role in the spikelet/floret abortion process, was markedly elevated in the base and apex of salt treated spikes correlating with an increased spikelet abortion in these regions. CONCLUSIONS: Overall, salinity treatment reduced all plant and yield-related parameters investigated and turned some of the correlations among them from positive to negative or vice versa. Investigations of ABA role in floral development and phase duration of barley spike showed that, ABA regulates the spikelet/floret abortion process affecting the yield potential under salinity and control conditions.

Crosstalk among hormones in barley spike contributes to the yield.

KEY MESSAGE: The hormonal ratios along the barley spike regulate the development, atrophy and abortion of the spikelets and could be the mechanism by which the barley spike adapts its yield potential. Barley (Hordeum vulgare L.) is one of the oldest cereal crops known to be cultivated since about 10,000 years. The inflorescence of cultivated barley is an indeterminate spike that produces three single-flowered spikelets at each rachis node which make it unique among the grasses. The yield production in barley is predominantly controlled by very important parameters such as number of tillers and number of spikelets per spike. These two parameters are negatively correlated. Therefore, studying the biological and genetics of the spikelet development during the spike developmental stages is essential for breeding programs. Here we summarize our current understanding of the crosstalk between hormones such as auxin, cytokinin, gibberellin and abscisic acid along the spike and what is their role in regulating spike and spikelet development in barley. We conclude that the hormonal ratios at the apical, central, and basal sections of the spike not only regulate the spike developmental stages, but also the development, atrophy, and abortion of the spikelets. This hormonal dependent modification of the grain number along the spike could be the mechanism by which the barley spike adapts its yield potential.

Natural variation and genetic make-up of leaf blade area in spring barley.

KEY MESSAGE: GWAS analysis for leaf blade area (LA) revealed intriguing genomic regions associated with putatively novel QTL and known plant stature-related phytohormone and sugar-related genes. Despite long-standing studies in the morpho-physiological characters of leaf blade area (LA) in cereal crops, advanced genetic studies to explore its natural variation are lacking. The importance of modifying LA in improving cereal grain yield and the genes controlling leaf traits have been well studied in rice but not in temperate cereals. To better understand the natural genetic variation of LA at four developmental stages, main culm LA was measured from 215 worldwide spring barleys including 92 photoperiod-sensitive accessions [PHOTOPERIOD RESPONSE LOCUS 1 (Ppd-H1)] and 123 accessions with reduced photoperiod sensitivity (ppd-H1) locus under controlled greenhouse conditions (long-day; 16/8 h; ~ 20/~ 16 °C day/night). The LA of Ppd-H1-carrying accessions was always smaller than in ppd-H1-carrying accessions. We found that nine SNPs from the Ppd-H1 gene were present in the collection of which marker 9 (M9; G/T in the CCT-domain) showed the most significant and consistent effect on LA at all studied developmental stages. Genome-wide association scans (GWAS) showed that the accessions carrying the ppd-H1 allele T/M9 (late heading) possessed more genetic variation in LA than the Ppd-H1 group carrying G/M9 (early heading). Several QTL with major effects on LA variation were found close to plant stature-related heading time, phytohormone- and sugar-related genes. The results provide evidence that natural variation of LA is an important source for improving grain yield, adaptation and canopy architecture of temperate cereals.

VRS2 regulates hormone-mediated inflorescence patterning in barley.

Plant architecture has clear agronomic and economic implications for crops such as wheat and barley, as it is a critical factor for determining grain yield. Despite this, only limited molecular information is available about how grain-bearing inflorescences, called spikes, are formed and maintain their regular, distichous pattern. Here we elucidate the molecular and hormonal role of Six-rowed spike 2 (Vrs2), which encodes a SHORT INTERNODES (SHI) transcriptional regulator during barley inflorescence and shoot development. We show that Vrs2 is specifically involved in floral organ patterning and phase duration by maintaining hormonal homeostasis and gradients during normal spike development and similarly influences plant stature traits. Furthermore, we establish a link between the SHI protein family and sucrose metabolism during organ growth and development that may have implications for deeper molecular insights into inflorescence and plant architecture in crops.

The Genetic Basis of Composite Spike Form in Barley and 'Miracle-Wheat'.

Inflorescences of the tribe Triticeae, which includes wheat (Triticum sp. L.) and barley (Hordeum vulgare L.) are characterized by sessile spikelets directly borne on the main axis, thus forming a branchless spike. 'Compositum-Barley' and tetraploid 'Miracle-Wheat' (T. turgidum convar. compositum (L.f.) Filat.) display noncanonical spike-branching in which spikelets are replaced by lateral branch-like structures resembling small-sized secondary spikes. As a result of this branch formation 'Miracle-Wheat' produces significantly more grains per spike, leading to higher spike yield. In this study, we first isolated the gene underlying spike-branching in 'Compositum-Barley,' i.e., compositum 2 (com2). Moreover, we found that COM2 is orthologous to the branched head(t) (bh(t)) locus regulating spike branching in tetraploid 'Miracle-Wheat.' Both genes possess orthologs with similar functions in maize BRANCHED SILKLESS 1 (BD1) and rice FRIZZY PANICLE/BRANCHED FLORETLESS 1 (FZP/BFL1) encoding AP2/ERF transcription factors. Sequence analysis of the bh(t) locus in a collection of mutant and wild-type tetraploid wheat accessions revealed that a single amino acid substitution in the DNA-binding domain gave rise to the domestication of 'Miracle-Wheat.' mRNA in situ hybridization, microarray experiments, and independent qRT-PCR validation analyses revealed that the branch repression pathway in barley is governed through the spike architecture gene Six-rowed spike 4 regulating COM2 expression, while HvIDS1 (barley ortholog of maize INDETERMINATE SPIKELET 1) is a putative downstream target of COM2. These findings presented here provide new insights into the genetic basis of spike architecture in Triticeae, and have disclosed new targets for genetic manipulations aiming at boosting wheat's yield potential.

Genetic mapping of the labile (lab) gene: a recessive locus causing irregular spikelet fertility in labile-barley (Hordeum vulgare convar. labile).

KEY MESSAGE: The recessive labile locus mapped on chromosome 5HL causes irregular spikelet fertility and controls floret development as well as row-type in barley. The labile-barley displays a variable number of fertile spikelets at each rachis internode (0-3 fertile spikelets/rachis internode) which is intermediate between that observed in two- or six-rowed types. Previous re-sequencing of Vrs1 in 219 labile-barley (Hordeum vulgare L. convar. labile) accessions showed that all carried a six-rowed specific allele. We therefore hypothesized that this seemingly random reduction in spikelet fertility is most likely caused by the labile (lab) locus, which we aimed to phenotypically and genetically define. Here, we report a detailed phenotypic analysis of spikelet fertility in labile-barleys in comparison to two- and six-rowed genotypes using scanning electron microscopy analysis. We found that the first visible morphological deviation occurred during the stamen primordium stage, when we regularly observed the appearance of arrested central floral primordia in labile but not in two- or six-rowed barleys. At late stamen and early awn primordium stages, lateral florets in two-rowed and only some in labile-barley showed retarded development and reduction in size compared with fully fertile lateral florets in six-rowed barley. We used two F2 mapping populations to generate whole genome genetic linkage maps and ultimately locate the lab locus as a recessive Mendelian trait to a 4.5-5.8 cM interval at approximately 80 cM on chromosome 5HL. Our results will help identifying the role of the lab gene in relation to other spikelet fertility factors in barley.