Morphological and physiological analysis of cleistogamy in barley (Hordeum vulgare)

We previously reported that cleistogamy/chasmogamy (CL/CH) of barley ( Hordeum vulgare L.) is controlled by either two tightly linked genes or one gene with multiple alleles. To clarify the morphological and physiological mechanisms of barley CL, we analysed the lodicule size and auxin response of two cultivars whose CL/CH was controlled by two different genes, cly1 and Cly2 . In both cases, lodicules of the CL parent were smaller than those of the CH parent. Analyses of lodicule size and flowering phenotype of f ₁ plants and segregation analyses of the mapping population indicated that lodicule size co-segregated with the flowering phenotype. Indole-3-acetic acid (IAA) and other synthetic auxins, such as 2,4-dichlorophenoxyacetic acid, induced abnormal flowering in CH ears, in which florets remained open for a few days instead of the normal hour or so, but not in CL ears. This auxin effect also co-segregated with the flowering phenotype. Analyses of auxin-related compounds in the floret organs revealed that the anther contained high levels of IAA, whereas indole-3-carboxylic acid, a putative decarboxylated metabolite of IAA, accumulated only in lodicules of CH plants just at flowering. These results indicate that lodicule size and auxin response are pleiotropic effects of the CL gene, which may play a role in auxin response or metabolism.     

Asynchronous flowering and within-plant flowering diversity in wheat and the implications for crop resilience to heat

Background and Aims

Self-pollination dominates in wheat, with a small level of out-crossing due to flowering asynchrony and male sterility. However, the timing and synchrony of male and female flowering in wheat is a crucial determinant of seed set and may be an important factor affecting gene flow and resilience to climate change. Here, a methodology is presented for assessing the timing and synchrony of flowering in wheat, Triticum aestivum.

Methods

From the onset of flowering until the end of anthesis, the anther and stigma activity of each floret was assessed on the first five developing ears in potted plants grown under ambient conditions and originating from ‘Paragon’ or ‘Spark-Rialto’ backgrounds. At harvest maturity, seed presence, size and weight was recorded for each floret scored.

Key Results and Conclusions

The synchrony between pollen dehiscence and stigma collapse within a flower was dependent on its relative position in a spike and within a floret. Determined on the basis of synchrony within each flower, the level of pollination by pollen originating from other flowers reached approx. 30 % and did not change throughout the duration of flowering. A modelling exercise parameterized by flowering observations indicated that the temporal and spatial variability of anther activity within and between spikes may influence the relative resilience of wheat to sudden, extreme climatic events which has direct relevance to predicted future climate scenarios in the UK.     

QTL analysis of Fusarium head blight resistance using a high-density linkage map in barley

Fusarium head blight (FHB) resistance was evaluated in a set of recombinant inbred (RI) lines from a cross between Russia 6 (resistant) and H.E.S. 4 (susceptible), which had one of the widest differences of FHB resistance reactions among ca. 5,000 barley germplasm accessions in Okayama University. Field-grown spikes were sampled and inoculated by the ‘cut-spike test’. Resistance reactions on the parents and RI lines were scored by eleven grades, from resistant (0) to susceptible (10). Quantitative trait loci (QTL) analysis detected three QTL: two located on the long arm of chromosome 2H, and another on the short arm of chromosome 5H. A QTL located on chromosome 2H was coincident with the vrs1 locus, which governs inflorescence row type. The other QTL on chromosome 2H was positioned in the vicinity of cleistogamy locus (cly1 or Cly2) that determines inflorescence opening/closing. Resistant gene analog (RGA) and expressed sequence tag (EST) markers with homology for disease resistance genes were integrated into the high-density linkage map. Most of these markers were not localized near the identified resistance QTL, except for one RGA marker (FXLRRfor_XLRRrev170) localized in the vicinity of the cly1/Cly2 locus. Five AFLP markers localized in the vicinity of the identified QTL were sequenced to convert them into sequence tagged site (STS) markers. Genotyping of each RI line using two AFLP–STS markers and the vrs1 locus indicated that the RI lines with three Russia 6 QTL alleles exhibited the same level of high FHB resistance reactions as Russia 6. In contrast, RI lines with three susceptible alleles showed reactions close to H.E.S. 4. Therefore, the markers closely linked to the QTL can be efficiently used for the selection of resistance.     

Floret Initiation and Development in Spring Wheat (Triticum aestivum L.)

The number and developmental stages of florets were determinedin each spikelet of the spike in the main shoots of spring wheat.Samples were taken frequently from plants grown in a phytotronand in a nitrogen application field-test. Ten stages of development,from floret initiation until anthesis, were recognized and described. Inter-spikelet variation in the total number of initiated floretswas rather small. However, the number of florets at advancedstages of development, as well as the number of grains, washighest in the central spikelets in which florets initiatedfirst. Floret initiation did not proceed beyond spike emergence,whereafter the distal florets and the spikelet apex degenerated.Grain-set was restricted to florets which had developed at leastto the stage of visible anther lobes at spike emergence. Thenumber of these florets was increased significantly by nitrogenapplication. Wheat, Triticum aestivum L., spikelet, floret, grain set, nitrogen     

Detection of Fusarium head blight resistance QTLs using five populations of top-cross progeny derived from two-row × two-row crosses in barley

Fusarium head blight (FHB) resistance was evaluated in five recombinant inbred (RI) populations. The RI populations consisted of top-cross progeny derived from a diallel set of crosses. Each of five two-row barley lines differing in response to FHB were crossed with ‘Harbin 2-row’. FHB severity was scored on an 11-point scale, where resistant = 0 and susceptible = 10, based on the ‘cut-spike test’. Disease data were obtained for each population for 2 or 3 years. Linkage maps comprised of expressed sequence tag (EST) markers were developed for each population and used for quantitative trait locus (QTL) detection. Thirty two QTLs were detected using all data sets (individual populations and years). Thirteen QTLs were detected using averages across years; 10 of these were consistent across the individual year and average data sets. These QTLs clustered at 14 regions, with clusters on all chromosomes. At 11 of these clusters, Harbin 2-row contributed FHB resistance alleles. No QTLs were detected near the row type (vrs1) locus in any of the five RI populations, suggesting that the FHB resistance QTL in this region reported in two-row × six-row crosses may be pleiotropic effect of vrs1. QTL were coincident with the flowering type locus (cly1/Cly2) on chromosome 2H in every population. Some QTL × QTL interactions were significant, but these were smaller than QTL main effects. Considering the pleiotropic effect of spike morphology on FHB resistance, future FHB resistance mapping efforts in barley should focus on cross combinations in which alleles at vrs1 are not segregating. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification and mapping of cleistogamy genes in barley

Cleistogamy is a closed type of flowering with ensured self-pollination and an important trait to study evolutionary development in flower organs, reproduction systems, gene flow, and disease control. Still, very limited information is available about the genetic control and regulatory mechanism of this trait in barley. In this work, from the eight crosses between cleistogamous and chasmogamous accessions, five crosses generated chasmogamous F₁ plants and their F₂ plants segregated as 3 chasmogamous:1 cleistogamous, whereas three crosses generated cleistogamous F₁ plants, and their F₂ plants segregated as 1 chasmogamous:3 cleistogamous. Although a single gene was responsible for the control of cleistogamy in these two groups of crosses, the direction of dominance was opposite, suggesting two genes, cly1 and Cly2, for the genetic control of cleistogamy in barley. Epistatic type of gene interaction between the two loci was detected. In the analysis of 99 recombinant inbred lines of Azumamugi × Kanto Nakate Gold and doubled haploid lines of Harrington × Mikamo Golden, where in both crosses F₁ was chasmogamous, the cly1 locus has been mapped on chromosome 2HL. Using the analysis of the F₂ population of Misato Golden and Satsuki Nijo where F₁ was cleistogamous, the Cly2 locus was mapped in the same region of chromosome 2HL. Because the cly1 and Cly2 loci were mapped in the same region in these three different mapping populations, it was concluded that the expression of cleistogamy is under the control of two tightly linked genes or different alleles of the same gene.     

Non-destructive staging of barley reproductive development for molecular analysis based upon external morphology

A prerequisite to study the molecular genetic pathways of pollen and anther development is an accurate staging system for reproductive development. However in barley, floret formation occurs mainly within the pseudostem, which makes the observation of floret development and access to the floret particularly difficult without dissecting the plant. Thus selecting stages for molecular analysis cannot be done non-destructively. A staging method has therefore been developed for barley in order to define the relationship between readily detectable growth points and reproductive development, to provide a clear key to enable accurate selection of reproductive material. Initial staging followed the traditional Zadoks decimal system, with minor adaptations to stages 31-34 and stage 37. The later stages, from 37 onward, were replaced by growth staging based upon the last flag elongation (LFE) and the position occupied by the spike within the pseudostem. Spike size could be readily predicted by using the staging system incorporating Zadoks stages 31-37, supplemented with substages and by LFE staging to improve accuracy. The different spike sizes, as well as the LFE stages, showed a clear relationship to events occurring within the anther, as confirmed by light microscopy of the anthers. The defined relationship between spike size and development to anther development now makes possible the accurate prediction of anther and pollen progression by external staging. This, therefore, provides a mechanism for non-destructive selection of material for analysis that is critical for the molecular characterization of genes in anther and pollen development.     

GA-Responsive Dwarfing Gene Rht12 Affects the Developmental and Agronomic Traits in Common Bread Wheat

Opportunities exist for replacing reduced height (Rht) genes Rht-B1b and Rht-D1b with alternative dwarfing genes, such as the gibberellin-responsive gene Rht12, for bread wheat improvement. However, a comprehensive understanding of the effects and mode of action of Rht12 is lacking. In the present study, the effects of Rht12 were characterized by analyzing its effects on seeding vigour, seedling roots, leaf and stem morphology, spike development and carbohydrate assimilation and distribution. This was carried out in the four genotypes of F_2:3 lines derived from a cross between Ningchun45 and Karcagi (12) in two experiments of autumn sowing and spring sowing. Rht12 significantly decreased stem length (43%∼48% for peduncle) and leaf length (25%∼30% for flag leaf) while the thickness of the internode walls and width of the leaves were increased. Though the final plant stature was shortened (40%) by Rht12, the seedling vigour, especially coleoptile length and root traits at the seedling stage, were not affected adversely. Rht12 elongated the duration of the spike development phase, improved the proportion of spike dry weight at anthesis and significantly increased floret fertility (14%) in the autumn sowing experiment. However, Rht12 delayed anthesis date by around 5 days and even the dominant Vrn-B1 allele could not compensate this negative effect. Additionally, grain size was reduced with the ability to support spike development after anthesis decreased in Rht12 lines. Finally, grain yield was similar between the dwarf and tall lines in the autumn sowing experiment. Thus, Rht12 could substantially reduce plant height without altering seeding vigour and significantly increase spikelet fertility in the favourable autumn sowing environment. The successful utilization of Rht12 in breeding programs will require careful selection since it might delay ear emergence. Nonetheless, the potential exists for wheat improvement by using Rht12.     

OsJAR1 is required for JA-regulated floret opening and anther dehiscence in rice

Jasmonates are important phytohormones regulating reproductive development. We used two recessive rice Tos17 alleles of OsJAR1, osjar1-2 and osjar1-3, to study the biological function of jasmonates in rice anthesis. The florets of both osjar1 alleles stayed open during anthesis because the lodicules, which control flower opening in rice, were not withering on time. Furthermore, dehiscence of the anthers filled with viable pollen, was impaired, resulting in lower fertility. In situ hybridization and promoter GUS transgenic analysis confirmed OsJAR1 expression in these floral tissues. Flower opening induced by exogenous applied methyl jasmonate was impaired in osjar1 plants and was restored in a complementation experiment with transgenics expressing a wild type copy of OsJAR1 controlled by a rice actin promoter. Biochemical analysis showed that OsJAR1 encoded an enzyme conjugating jasmonic acid (JA) to at least Ile, Leu, Met, Phe, Trp and Val and both osjar1 alleles had substantial reduction in content of JA-Ile, JA-Leu and JA-Val in florets. We conclude that OsJAR1 is a JA-amino acid synthetase that is required for optimal flower opening and closing and anther dehiscence in rice.     

Changes in Respiration and Cyanide Sensitivity of the Barley Floret during Development and Maturation

The 6-week period of development and maturation of the barley (Hordeum vulgare L.) floret from anthesis to harvest is characterized by two phases: an early phase of rapid increase in respiration rate and dry weight, and a late phase during which respiration decreased rapidly whereas dry weight remained unchanged. Consumption of O₂ by the embryo changed little during the entire developmental period, whereas O₂ uptake by the endosperm and the lemma and palea decreased significantly during the late phase.     

Molecular and comparative mapping of genes governing spike compactness from wild emmer wheat

The development and morphology of the wheat spike is important because the spike is where reproduction occurs and it holds the grains until harvest. Therefore, genes that influence spike morphology are of interest from both theoretical and practical stand points. When substituted for the native chromosome 2A in the tetraploid Langdon (LDN) durum wheat background, the Triticum turgidum ssp. dicoccoides chromosome 2A from accession IsraelA confers a short, compact spike with fewer spikelets per spike compared to LDN. Molecular mapping and quantitative trait loci (QTL) analysis of these traits in a homozygous recombinant population derived from LDN × the chromosome 2A substitution line (LDNIsA-2A) indicated that the number of spikelets per spike and spike length were controlled by linked, but different, loci on the long arm of 2A. A QTL explaining most of the variation for spike compactness coincided with the QTL for spike length. Comparative mapping indicated that the QTL for number of spikelets per spike overlapped with a previously mapped QTL for Fusarium head blight susceptibility. The genes governing spike length and compactness were not orthologous to either sog or C, genes known to confer compact spikes in diploid and hexaploid wheat, respectively. Mapping and sequence analysis indicated that the gene governing spike length and compactness derived from wild emmer could be an ortholog of the barley Cly1/Zeo gene, which research indicates is an AP2-like gene pleiotropically affecting cleistogamy, flowering time, and rachis internode length. This work provides researchers with knowledge of new genetic loci and associated markers that may be useful for manipulating spike morphology in durum wheat.     

Effects of the deficiens allele on ear development and yield in barley

Lines partially-isogenic for the deficiens (V^tV^t and two-rowed (VV) barley ear types were developed in the genetic background of cv. Kym, for comparisons of ear development and yield components. The suppression of lateral floret growth by the V^t allele during pre-anthesis ear development occurred at a relatively late stage and only occasionally was median floret survival enhanced in the deficiens line. The final size of lateral florets in both deficiens and normal ear types appeared to be largely governed by assimilate supply. At anthesis, the lateral florets comprised about 10% of the dry matter of normal ears, whereas in the deficiens ears the median florets were correspondingly heavier. After anthesis, grain growth in the deficiens line was slower than in the normal line, although final grain weight was not affected. In another experiment the V^t allele occasionally increased the number of grains per ear, but consistently decreased the weight per grain. In drilled plot trials, the mean yield of the deficiens line was 8% lower than the normal. Excision of lateral florets at anthesis showed that photosynthesis in these organs did not contribute to the yield advantage of the normal ear type. It is suggested that the inferior performance of the deficiens type was attributable to the greater physical constriction of endosperm expansion imposed by the development of thicker, more rigid, palea and lemma tissues.     

Wheat floret survival as related to pre-anthesis spike growth

Further improvements to wheat yield potential will be essential to meet future food demand. As yield is related to the number of fertile florets and grains, an understanding of the basis of their generation is instrumental to raising yield. Based on (i) a strong positive association between the number of fertile florets or grains and spike dry weight at anthesis; and (ii) the finding that floret death occurs when spikes grow at maximum rate, it was always assumed that floret survival depends on the growth of the spike. However, this assumption was recently questioned, suggesting that assimilates diverted to the spike do not determine the number of florets and grains and that the onset of floret death may instead be a developmental process that is not associated with spike growth. In this study, the relationships between the fate of floret primordia and spike growth from six independent experiments that included different growing conditions (greenhouse/field experiments, growing seasons, photoperiod/shading treatments during the floret primordia phase) and diverse cultivar types (winter/spring, semi-dwarf/standard-height, photoperiod sensitive/insensitive) were re-analysed together. Onset of floret death was associated with the beginning of spike growth at the maximum rate in c. 80% of the cases analysed; and the rate of floret death (the main determinant of floret survival) showed a negative quantitative relationship with spike weight at anthesis. As floret death and survival were shown to be linked to pre-anthesis spike growth, the strategy of focusing on traits associated with pre-anthesis spike growth when breeding to increase wheat yield potential further is valuable.     

Haplotype variability and identification of new functional alleles at the Rdg2a leaf stripe resistance gene locus

The barley Rdg2a locus confers resistance to the leaf stripe pathogen Pyrenophora graminea and, in the barley genotype Thibaut, it is composed of a gene family with three highly similar paralogs. Only one member of the gene family (called as Rdg2a) encoding for a CC-NB-LRR protein is able to confer resistance to the leaf stripe isolate Dg2. To study the genome evolution and diversity at the Rdg2a locus, sequences spanning the Rdg2a gene were compared in two barley cultivars, Thibaut and Morex, respectively, resistant and susceptible to leaf stripe. An overall high level of sequence conservation interrupted by several rearrangements that included three main deletions was observed in the Morex contig. The main deletion of 13,692 bp was most likely derived from unequal crossing over between Rdg2a paralogs leading to the generation of a chimeric Morex rdg2a gene which was not associated to detectable level of resistance toward leaf stripe. PCR-based analyses of genic and intergenic regions at the Rdg2a locus in 29 H. vulgare lines and one H. vulgare ssp. spontaneum accession indicated large haplotype variability in the cultivated barley gene pool suggesting rapid and recent divergence at this locus. Barley genotypes showing the same haplotype as Thibaut at the Rdg2a locus were selected for a Rdg2a allele mining through allele re-sequencing and two lines with polymorphic nucleotides leading to amino acid changes in the CC-NB and LRR encoding domains, respectively, were identified. Analysis of nucleotide diversity of the Rdg2a alleles revealed that the polymorphic sites were subjected to positive selection. Moreover, strong positively selected sites were located in the LRR encoding domain suggesting that both positive selection and divergence at homologous loci are possibly representing the molecular mechanism for the generation of high diversity at the Rdg2a locus in the barley gene pool.     

Yersinochloa gen. nov. (Gramineae: Bambusoideae‐Bambusineae) endemic to the Lam Vien Plateau,southern Vietnam

A clambering bamboo endemic to the Lam Vien plateau, southern Vietnam and representing a new monotypic endemic genus, Yersinochloa H. N. Nguyen & V. T. Tran (Gramineae–Bambusoideae–Bambusinae), is described and illustrated. It has an indeterminate inflorescence, terminate leafy branches, pseudo‐spikelets with only one perfect floret, unkeeled palea, three lodicules, six stamens with free filaments, anther apices with tiny spines, glabrous ovary with a long style, 3 plumose stigmas, and an oblong caryopsis with a relatively thin pericarp.     

Lodicule Function and Filament Extension in the Grasses: Potassium Ion Movement and Tissue Specialization

Flowering in the grasses has been the subject of a great deal of previous research, yet much remains to be learned concerning the role of environmental and endogenous factors in controlling the actual events of anthesis. The objective of this study has been to investigate further two key processes, namely lodicule function and stamen extension.The lodicules are the two diminutive bodies lying between the lemma and the ovary base in the grass floret which, by expanding rapidly at the time of anthesis, lever away the rigid lemma allowing anthers and stigmas to emerge. Expansion results from the swelling of a cushion of tissue at the base of each lodicule. We show that this is driven by the influx of water associated with the accumulation of K⁺in specialized distensible cells of the basal cushion. The stamen filaments normally elongate synchronously with the enlarging lodicules, again with the passage of K⁺into the extending cells. The walls of distensible cells of the lodicule bear lattice-like cellulosic thickenings with transverse bars in an outer layer and longitudinal bars in an inner, a basket-like disposition allowing rapid expansion while preserving the integrity of the protoplast. The microfibrils in the walls of the cylindrical filament cells are disposed helically, constraining expansion to the longitudinal axis. The principal extraneous factors initiating lodicule function in mature florets of rye are mechanical disturbance and light. In the field radiant heat is unlikely to be a dominant factor. In a customarily cleistogamous cultivar of wheat, sporadic florets open chasmogamously in intense light, both lodicules and stamen filaments enlarging. In cleistogamous flowers in the same inflorescence the lodicules fail to enlarge while the filaments extend, suggesting that the influx of osmoticum into the two sets of organs is independently controlled.