Transformation of rice with the Arabidopsis floral regulator LEAFY causes early heading

Onset of flowering, or heading date, is an important agronomic trait of cereal crops such as rice and early-heading varieties are required for certain regions in which rice is cultivated. Since the floral control gene LEAFY from Arabidopsis can dramatically accelerate flowering in dictoyledonous plants, the usefulness of LEAFY for manipulating heading date in rice has been tested. Constitutive expression of LEAFY from the cauliflower mosaic virus 35S promoter caused early flowering in transgenic rice, with a heading date that was 26–34 days earlier than that of wild-type plants. Early flowering was accompanied by a small yield penalty and some panicle abnormality. These observations suggest that floral regulatory genes from Arabidopsis are useful tools for heading date improvement in cereal crops.     

Association of barley photoperiod and vernalization genes with QTLs for flowering time and agronomic traits in a BC2DH population and a set of wild barley introgression lines

The control of flowering time has important impacts on crop yield. The variation in response to day length (photoperiod) and low temperature (vernalization) has been selected in barley to provide adaptation to different environments and farming practices. As a further step towards unraveling the genetic mechanisms underlying flowering time control in barley, we investigated the allelic variation of ten known or putative photoperiod and vernalization pathway genes between two genotypes, the spring barley elite cultivar ‘Scarlett’ (Hordeum vulgare ssp. vulgare) and the wild barley accession ‘ISR42-8’ (Hordeum vulgare ssp. spontaneum). The genes studied are Ppd-H1, VRN-H1, VRN-H2, VRN-H3, HvCO1, HvCO2, HvGI, HvFT2, HvFT3 and HvFT4. ‘Scarlett’ and ‘ISR42-8’ are the parents of the BC₂DH advanced backcross population S42 and a set of wild barley introgression lines (S42ILs). The latter are derived from S42 after backcrossing and marker-assisted selection. The genotypes and phenotypes in S42 and S42ILs were utilized to determine the genetic map location of the candidate genes and to test if these genes may exert quantitative trait locus (QTL) effects on flowering time, yield and yield-related traits in the two populations studied. By sequencing the characteristic regions of the genes and genotyping with diagnostic markers, the contrasting allelic constitutions of four known flowering regulation genes were identified as ppd-H1, Vrn-H1, vrn-H2 and vrn-H3 in ‘Scarlett’ and as Ppd-H1, vrn-H1, Vrn-H2 and a novel allele of VRN-H3 in ‘ISR42-8’. All candidate genes could be placed on a barley simple sequence repeat (SSR) map. Seven candidate genes (Ppd-H1, VRN-H2, VRN-H3, HvGI, HvFT2, HvFT3 and HvFT4) were associated with flowering time QTLs in population S42. Four exotic alleles (Ppd-H1, Vrn-H2, vrn-H3 and HvCO1) possibly exhibited significant effects on flowering time in S42ILs. In both populations, the QTL showing the strongest effect corresponded to Ppd-H1. Here, the exotic allele was associated with a reduction of number of days until flowering by 8.0 and 12.7%, respectively. Our data suggest that Ppd-H1, Vrn-H2 and Vrn-H3 may also exert pleiotropic effects on yield and yield-related traits.     

Expression and functional analysis of <Emphasis Type="Italic">NUCLEAR FACTOR</Emphasis>-<Emphasis Type="Italic">Y</Emphasis>, subunit B genes in barley

NUCLEAR FACTOR-Y, subunit B (NF-YB) comprises a multigene family in plants and has been shown to play important roles in growth, development, and response to environmental stress. In this study, five NF-YBs containing the full-length coding region were obtained from barley (Hordeum vulgare) through database sequence analysis, cloning, and sequencing. Sequence alignment and phylogenetic analysis showed that HvNF-YB3 and HvNF-YB1 were clustered with NF-YB2 and NF-YB3 in Arabidopsis, suggesting these NF-YBs are evolutionary and functionally related. To test this hypothesis, HvNF-YB3 and HvNF-YB1 were overexpressed in Arabidopsis. Overexpression of HvNF-YB1 greatly promoted early flowering in Arabidopsis, supporting that HvNF-YB1may have conserved gene function in flowering time control as NF-YB2 and NF-YB3 in Arabidopsis. Overexpression of HvNF-YB3 in Arabidopsis had no effect on flowering time. An analysis of barley single-nucleotide polymorphism (SNP) data, however, revealed a significant association between an HvNF-YB3 SNP and heading date. While it is unknown whether HvNF-YB3 directly contributes to heading date regulation, the results implied that HvNF-YB3 may also have conserved function in flowering time (heading date in barley) control. Further studies are needed to directly verify these gene functions in barley. Barley NF-YBs showed different expression patterns associated with tissue types, developmental stages, and response to different stress treatments, suggesting that barley NF-YBs may be involved in other physiological processes.     

DNA polymorphisms and haplotype patterns of transcription factors involved in barley endosperm development are associated with key agronomic traits

Background  

Association mapping is receiving considerable attention in plant genetics for its potential to fine map quantitative trait loci (QTL), validate candidate genes, and identify alleles of interest. In the present study association mapping in barley (Hordeum vulgare L.) is investigated by associating DNA polymorphisms with variation in grain quality traits, plant height, and flowering time to gain further understanding of gene functions involved in the control of these traits. We focused on the four loci BLZ1, BLZ2, BPBF and HvGAMYB that play a role in the regulation of B-hordein expression, the major fraction of the barley storage protein. The association was tested in a collection of 224 spring barley accessions using a two-stage mixed model approach.     

Control of floral transition in the bioenergy crop switchgrass

Switchgrass (Panicum virgatum L.), a perennial warm season bunchgrass native to North America, has been a target in the U.S. as a renewable bioenergy crop because of its ability to produce moderate to high biomass yield on marginal soils. Delaying flowering can increase vegetative biomass production by allowing prolonged growth before switching to the reproductive phase. Despite the identification of flowering time as a biomass trait in switchgrass, the molecular regulatory factors involved in controlling floral transition are poorly understood. Here we identified PvFT1, PvAPL1‐3 and PvSL1, 2 as key flowering regulators required from floral transition initiation to development of floral organs. PvFT1 expression in leaves is developmentally regulated peaking at the time of floral transition, and diurnally regulated with peak at approximately 2 h into the dark period. Ectopic expression of PvFT1 in Arabidopsis, Brachypodium and switchgrass led to extremely early flowering, and activation of FT downstream target genes, confirming that it is a strong activator of flowering in switchgrass. Ectopic expression of PvAPL1‐3 and PvSL1, 2 in Arabidopsis also activated early flowering with distinct floral organ phenotypes. Our results suggest that switchgrass has conserved flowering pathway regulators similar to Arabidopsis and rice.     

The gibberellic-acid insensitive dwarfing gene<Emphasis Type="Italic"> sdw3</Emphasis> of barley is located on chromosome 2HS in a region that shows high colinearity with rice chromosome 7L

In this study, comparative high resolution genetic mapping of the GA-insensitive dwarfing gene sdw3 of barley revealed highly conserved macrosynteny of the target region on barley chromosome 2HS with rice chromosome 7L. A rice contig covering the sdw3-orthologous region was identified and subsequently exploited for marker saturation of the target interval in barley. This was achieved by (1) mapping of rice markers from the orthologous region of the rice genetic map, (2) mapping of rice ESTs that had been physically localized on the rice contig, or (3) mapping of barley ESTs that show strong sequence similarity to coding sequences present in the rice contig. Finally, the sdw3 gene was mapped to an interval of 0.55 cM in barley, corresponding to a physical distance of about 252 kb in rice, after employing orthologous EST-derived rice markers. Three putative ORFs were identified in this interval in rice, which exhibited significant sequence similarity to known signal regulator genes from different species. These ORFs can serve as starting points for the map-based isolation of the sdw3 gene from barley.Communicated by R. Hagemann     

Identification and molecular characterization of a Brachypodium distachyon GIGANTEA gene: functional conservation in monocot and dicot plants

Developmental phase change and flowering transition are emerging as potential targets for biomass agriculture in recent years. The GIGANTEA (GI) gene is one of the central regulators that direct flowering promotion and phase transition. In this work, we isolated a GI gene orthologue from the small annual grass Brachypodium distachyon inbred line Bd21 (Brachypodium), which is perceived as a potential model monocot for studies on bioenergy grass species. A partial GI gene sequence was identified from a Brachypodium expressed sequence tag library, and a full-size gene (BdGI) was amplified from a Brachypodium cDNA library using specific primer sets designed through analysis of monocot GI gene sequences. The BdGI gene was up-regulated by light and cold. A circadian rhythm set by light–dark transition also regulated the expression of the BdGI gene. The deduced amino acid sequence of the BdGI protein shares higher than 70% of sequence identity with the GI proteins in monocots and Arabidopsis. In addition, the BdGI protein is constitutively targeted to the nucleus and physically interacts with the ZEITLUPE (ZTL) and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) proteins, like the Arabidopsis GI protein. Interestingly, heterologous expression of the BdGI gene in a GI-deficient Arabidopsis mutant rescued efficiently the late flowering phenotype. Together, our data indicate that the role of the GI gene in flowering induction is conserved in Arabidopsis and Brachypodium. It is envisioned that the GI genes of bioenergy grasses as well as Brachypodium could be manipulated to improve biomass by engineering developmental timing of phase transitions.     

The FLOWERING LOCUS T-like gene family in barley (Hordeum vulgare)

The FLOWERING LOCUS T (FT) gene plays a central role in integrating flowering signals in Arabidopsis because its expression is regulated antagonistically by the photoperiod and vernalization pathways. FT belongs to a family of six genes characterized by a phosphatidylethanolamine-binding protein (PEBP) domain. In rice (Oryza sativa), 19 PEBP genes were previously described, 13 of which are FT-like genes. Five FT-like genes were found in barley (Hordeum vulgare). HvFT1, HvFT2, HvFT3, and HvFT4 were highly homologous to OsFTL2 (the Hd3a QTL), OsFTL1, OsFTL10, and OsFTL12, respectively, and this relationship was supported by comparative mapping. No rice equivalent was found for HvFT5. HvFT1 was highly expressed under long-day (inductive) conditions at the time of the morphological switch of the shoot apex from vegetative to reproductive growth. HvFT2 and HvFT4 were expressed later in development. HvFT1 was therefore identified as the main barley FT-like gene involved in the switch to flowering. Mapping of HvFT genes suggests that they provide important sources of flowering-time variation in barley. HvFTI was a candidate for VRN-H3, a dominant mutation giving precocious flowering, while HvFT3 was a candidate for Ppd-H2, a major QTL affecting flowering time in short days.     

Analysis of a post-translational steroid induction system for GIGANTEA in Arabidopsis

Background  

To investigate the link between the flowering time gene GIGANTEA (GI) and downstream genes, an inducible GI system was developed in Arabidopsis thaliana L. Heynh. Transgenic Arabidopsis plant lines were generated with a steroid-inducible post-translational control system for GI. The gene expression construct consisted of the coding region of the GI protein fused to that of the ligand binding domain of the rat glucocorticoid receptor (GR). This fusion gene was expressed from the constitutive cauliflower mosaic virus 35S promoter and was introduced into plants carrying the gi-2 mutation. Application of the steroid dexamethasone (DEX) was expected to result in activation of the GI-GR protein and its relocation from the cytoplasm to the nucleus.     

Expression of heterologous phytochromes A, B or C in transgenic tobacco plants alters vegetative development and flowering time

In this study, oat phytochrome A (phyA), Arabidopsis phytochrome B (phyB) or Arabidopsis phytochrome C (phyC) were expressed in both day-neutral and photo-period-sensitive (short-day) tobacco (Nicotiana tabacum cv. Hicks). Introgression of the Maryland Mammoth (MM) gene into cv Hicks was used to confer short-day photo-periodic sensitivity. Expression of oat phyA led to characteristic hypersensitivity of hypocotyls to red light (R) and far-red light (FR) and an overall dwarfing of the mature plant. Expression of Arabidopsis phyB enhanced the sensitivity of hypocotyls to R and caused even more marked dwarfing of the mature plant. In contrast, the expression of Arabidopsis phyC had no detectable consequences for the photocontrol of hypocotyl elongation. However, phyC expression did lead to a R-dependent increase in cotyledon expansion in de-etiolating seedlings and to a significant increase in leaf area in mature plants. This provides the first experimental evidence that phyC is biologically active. The flowering time of cv Hicks plants grown under 8 h photoperiods was virtually unaffected by a 30 min white light (W) night break given 8 h into the dark period. In contrast, cv Hicks MM plants responded to a night break with a delay in flowering. Expression of phyA or phyB led to a night break-dependent delay in flowering in cv Hicks plants. For cv Hicks MM plants, the expression of any of phyA, phyB or phyC caused a marked enhancement of the flower-delaying effect of a night break. These observations indicate that transgenic phyA, phyB or phyC can interact with the endogenous mechanisms controlling flowering time in tobacco.     

Modelling the genetic architecture of flowering time control in barley through nested association mapping

Background

Barley, globally the fourth most important cereal, provides food and beverages for humans and feed for animal husbandry. Maximizing grain yield under varying climate conditions largely depends on the optimal timing of flowering. Therefore, regulation of flowering time is of extraordinary importance to meet future food and feed demands. We developed the first barley nested association mapping (NAM) population, HEB-25, by crossing 25 wild barleys with one elite barley cultivar, and used it to dissect the genetic architecture of flowering time.

Results

Upon cultivation of 1,420 lines in multi-field trials and applying a genome-wide association study, eight major quantitative trait loci (QTL) were identified as main determinants to control flowering time in barley. These QTL accounted for 64% of the cross-validated proportion of explained genotypic variance (p_G). The strongest single QTL effect corresponded to the known photoperiod response gene Ppd-H1. After sequencing the causative part of Ppd-H1, we differentiated twelve haplotypes in HEB-25, whereof the strongest exotic haplotype accelerated flowering time by 11 days compared to the elite barley haplotype. Applying a whole genome prediction model including main effects and epistatic interactions allowed predicting flowering time with an unmatched accuracy of 77% of cross-validated p_G.

Conclusions

The elaborated causal models represent a fundamental step to explain flowering time in barley. In addition, our study confirms that the exotic biodiversity present in HEB-25 is a valuable toolbox to dissect the genetic architecture of important agronomic traits and to replenish the elite barley breeding pool with favorable, trait-improving exotic alleles.

     

Characterizing the pathotype structure of barley powdery mildew and effectiveness of resistance genes to this pathogen in Kazakhstan

Background

Alike to Reduced height-1 (Rht-1) genes in wheat and the semi dwarfing (sd-1) gene in rice, the sdw1/denso locus involved in the metabolism of the GA, was designated as the ‘Green Revolution’ gene in barley. The recent molecular characterization of the candidate gene HvGA20ox2 for sdw1/denso locus allows to estimate the impact of the functional polymorphism of this gene on the variation of agronomically important traits in barley.

Results

We investigated the effect of the 7-bp deletion in exon 1 of HvGA20ox2 gene (sdw1.d mutation) on the variation of yield-related and malting quality traits in the population of DHLs derived from cross of medium tall barley Morex and semi-dwarf barley Barke. Segregation of plant height, flowering time, thousand grain weight, grain protein content and grain starch was evaluated in two diverse environments separated from one another by 15° of latitude. The 7-bp deletion in HvGA20ox2 gene reduced plant height by approximately 13 cm and delayed flowering time by 3–5 days in the barley segregating DHLs population independently on environmental cue. On other hand, the sdw1.d mutation did not affect significantly either grain quality traits (protein and starch content) or thousand grain weight.

Conclusions

The beneficial effect of the sdw1.d allele could be associated in barley with lodging resistance and extended period of vegetative growth allowing to accumulate additional biomass that supports higher yield in certain environments. However, no direct effect of the sdw1.d mutation on thousand grain weight or grain quality traits in barley was detected.

     

Quantitative trait loci controlling vernalisation requirement, heading time and number of panicles in meadow fescue (Festuca pratensis Huds.)

The “BF14/16×HF2/7” mapping population of meadow fescue (Festuca pratensis Huds.) was characterised for number of panicles produced by non-vernalised plants in the field, vernalisation requirement (number of weeks at 6°C and 8 h photoperiod), as well as days to heading, number of panicles and proportion of shoots heading after a 12 weeks vernalisation treatment. Quantitative trait loci (QTLs) were identified and compared to QTLs and genes related to the induction of flowering in cereals and grasses. A region on chromosome 1F affected days to heading and the proportion of shoots heading. Chromosome 4F appeared to have several genes with a strong effect on vernalisation requirement. The strongest effects were located in the proximal end of 4F and may correspond to the earliness per se (eps) QTL eps6L.2 in barley and a heading time QTL in perennial ryegrass. A part of the meadow fescue orthologue of VRN1 was sequenced and mapped to another region of 4F that also had a strong effect on vernalisation requirement. The proximal end of chromosome 5F had QTLs for days to heading and proportion of heading shoots. Syntenic regions in wheat and barley contain eps-loci. A QTL for number of panicles in the field and a QTL for proportion of heading shoots were present on chromosome 6. A region on 7F affected the variation in number of panicles among plants without a vernalisation requirement, and is syntenic to regions in perennial ryegrass, barley and rice containing orthologues of Arabidopsis thaliana CO.     

The Control of Flowering in Wheat and Barley: What Recent Advances in Molecular Genetics Can Reveal

The combined forces of developmental biologists, studying primordiuminitiation at the stem apex, and mathematical modellers, developingsimulations of crop growth and development, have brought aboutconsiderable advances in the understanding of the control offlowering in wheat and barley. Nevertheless, there are stillmajor gaps in this understanding including: what determinesthe basic rate of development (magnitude of the phyllochronor plastochron); how temperature and photoperiod interact tobring about the transition from vegetative to reproductive development;and how flowering occurs eventually in the absence of inductiveconditions. Although geneticists have tended to measure cerealflowering in terms of ‘days from sowing or emergence toheading’, results of studies using aneuploids and molecularmarkers are compatible with the roles for photoperiod and low-temperaturevernalization established in purely-physiological or developmentalinvestigations. They have also revealed the existence of ‘earlinessperse’loci, whose detailed roles have yet to be established.Progress towards isolating and characterizing wheat and barleyloci is hampered by the poor resolution of mapping (locationto a precision of tens of thousands of base pairs). Neitherof these broad approaches promises a rapid resolution of thefactors controlling the induction of flowering. Two expandingareas of molecular genetics now provide potential for greaterunderstanding of cereal flowering. First, the extensive homoeologyamong members of the Gramineae can be employed to establishthe existence and location of genes or quantitative trait lociin rice which correspond to controlling loci in wheat or barley.Since the rice genome is 1/30th of the size of the wheat genome,the accuracy of mapping loci can be much higher, and there isgreater potential for precise location of loci using techniquessuch as chromosome walking. With the ultimate cloning of individualgenes, and the isolation of gene products, the relative rolesof the 20 loci apparently involved in the induction of floweringof wheat could be explored. However, progress in the moleculargenetics ofArabidopsis(the second area) may provide a more rapidroute to understanding the control of flowering in cereals forseveral reasons: its small genome (1/4 that of rice); the likelihoodof extensive homoeology with cereals, in spite of differencesin codon usage between monocots and dicots; the existence ofa wide range of flowering-time mutants; and the control of floralinduction by a similar range of environmental factors includingphotoperiod and low temperature. It is likely that the MCDK(Martinez-Zapater, Coupland, Dean and Koornneef, 1994. In: MeyerowitzEM, Somerville CR.Arabidopsis.New York: Cold Spring Harbor Laboratory,403–433) model, formulated to explain the genetic andenvironmental control of flowering inArabidopsis,could be employedusefully in the formulation of experimental work on floweringin wheat and barley. This paper reviews these issues, payingparticular attention to the significance of ‘earlinessperse’ loci and the ‘constitutive floral pathway’for wheat and barley.Copyright 1998 Annals of Botany Company Wheat, barley, rice,Arabidopsis,flowering, photoperiod, vernalization, genetics, development.