Mutation at the circadian clock gene EARLY MATURITY 8 adapts domesticated barley (Hordeum vulgare) to short growing seasons.

The circadian clock is an autonomous oscillator that produces endogenous biological rhythms with a period of about 24 h. This clock allows organisms to coordinate their metabolism and development with predicted daily and seasonal changes of the environment. In plants, circadian rhythms contribute to both evolutionary fitness and agricultural productivity. Nevertheless, we show that commercial barley varieties bred for short growing seasons by use of early maturity 8 (eam8) mutations, also termed mat-a, are severely compromised in clock gene expression and clock outputs. We identified EAM8 as a barley ortholog of the Arabidopsis thaliana circadian clock regulator EARLY FLOWERING3 (ELF3) and demonstrate that eam8 accelerates the transition from vegetative to reproductive growth and inflorescence development. We propose that eam8 was selected as barley cultivation moved to high-latitude short-season environments in Europe because it allowed rapid flowering in genetic backgrounds that contained a previously selected late-flowering mutation of the photoperiod response gene Ppd-H1. We show that eam8 mutants have increased expression of the floral activator HvFT1, which is independent of allelic variation at Ppd-H1. The selection of independent eam8 mutations shows that this strategy facilitates short growth-season adaptation and expansion of the geographic range of barley, despite the pronounced clock defect.

The impact of photoperiod insensitive Ppd-1a mutations on the photoperiod pathway across the three genomes of hexaploid wheat (Triticum aestivum).

Flowering time is a trait that has been extensively altered during wheat domestication, enabling it to be highly productive in diverse environments and providing a rich source of variation for studying adaptation mechanisms. Hexaploid wheat is ancestrally a long-day plant, but many environments require varieties with photoperiod insensitivity (PI) that can flower in short days. PI results from mutations in the Ppd-1 gene on the A, B or D genomes, with individual mutations conferring different degrees of earliness. The basis of this is poorly understood. Using a common genetic background, the effects of A, B and D genome PI mutations on genes of the circadian clock and photoperiod pathway were studied using genome-specific expression assays. Ppd-1 PI mutations did not affect the clock or immediate clock outputs, but affected TaCO1 and TaFT1, with a reduction in TaCO1 expression as TaFT1 expression increased. Therefore, although Ppd-1 is related to PRR genes of the Arabidopsis circadian clock, Ppd-1 affects flowering by an alternative route, most likely by upregulating TaFT1 with a feedback effect that reduces TaCO1 expression. Individual genes in the circadian clock and photoperiod pathway were predominantly expressed from one genome, and there was no genome specificity in Ppd-1 action. Lines combining PI mutations on two or three genomes had enhanced earliness with higher levels, but not earlier induction, of TaFT1, showing that there is a direct quantitative relationship between Ppd-1 mutations, TaFT1 expression and flowering.

The effect of day-neutral mutations in barley and wheat on the interaction between photoperiod and vernalization.

Vernalization-2 (Vrn-2) is the major flowering repressor in temperate cereals. It is only expressed under long days in wild-type plants. We used two day-neutral (photoperiod insensitive) mutations that allow rapid flowering in short or long days to investigate the day length control of Vrn-2. The barley (Hordeum vulgare) early maturity8 (eam8) mutation affects the barley ELF3 gene. eam8 mutants disrupt the circadian clock resulting in elevated expression of Ppd-H1 and the floral activator HvFT1 under short or long days. When eam8 was crossed into a genetic background with a vernalization requirement Vrn-2 was expressed under all photoperiods and the early flowering phenotype was partially repressed in unvernalized (UV) plants, likely due to competition between the constitutively active photoperiod pathway and the repressing effect of Vrn-2. We also investigated the wheat (Triticum aestivum) Ppd-D1a mutation. This differs from eam8 in causing elevated levels of Ppd-1 and TaFT1 expression without affecting the circadian clock. We used genotypes that differed in "short-day vernalization". Short days were effective in promoting flowering in individuals wild type at Ppd-D1, but not in individuals that carry the Ppd-D1a mutation. The latter showed Vrn-2 expression in short days. In summary, eam8 and Ppd-D1a mimic long days in terms of photoperiod response, causing Vrn-2 to become aberrantly expressed (in short days). As Ppd-D1a does not affect the circadian clock, this also shows that clock regulation of Vrn-2 operates indirectly through one or more downstream genes, one of which may be Ppd-1.

Meta-QTL analysis of the genetic control of ear emergence in elite European winter wheat germplasm.

Variation in ear emergence time is critical for the adaptation of wheat (Triticum aestivum L.) to specific environments. The aim of this study was to identify genes controlling ear emergence time in elite European winter wheat germplasm. Four doubled haploid populations derived from the crosses: Avalon x Cadenza, Savannah x Rialto, Spark x Rialto, and Charger x Badger were selected which represent diversity in European winter wheat breeding programmes. Ear emergence time was recorded as the time from 1st May to heading in replicated field trials in the UK, France and Germany. Genetic maps based on simple sequence repeat (SSR) and Diversity Arrays Technology (DArT) markers were constructed for each population. One hundred and twenty-seven significant QTL were identified in the four populations. These effects were condensed into 19 meta-QTL projected onto a consensus SSR map of wheat. These effects are located on chromosomes 1B (2 meta-QTL), 1D, 2A (2 meta-QTL), 3A, 3B (2 meta-QTL), 4B, 4D, 5A (2 meta-QTL), 5B, 6A, 6B 7A (2 meta-QTL), 7B and 7D. The identification of environmentally robust earliness per se effects will facilitate the fine tuning of ear emergence in predictive wheat breeding programmes.

Specificity and clinical performance of two commercial TRACP 5b immunoassays.

Two forms of tartrate-resistant acid phosphatase (TRACP) circulate in human blood, TRACP 5a derived from inflammatory macrophages and TRACP 5b derived from osteoclasts. Serum TRACP 5b is a clinically useful marker of osteoclast number and bone resorption. We have studied TRACP 5b specificity of two commercially available immunoassays that are stated to be TRACP 5b specific, the BoneTRAP assay and the MetraTRAP5b assay, and investigated their clinical performance for monitoring the efficacy of alendronate treatment. Both assays bound TRACP 5b equally and had similar cross-reactivity to TRACP 5a. The mean decrease in the alendronate group was higher with the MetraTRAP5b assay, but the clinical performance of the two assays for monitoring alendronate treatment was equal due to higher variability of the MetraTRAP5b assay. We conclude that the BoneTRAP assay and the MetraTRAP5b assay have similar specificity for TRACP 5b, and similar clinical performance for monitoring alendronate treatment.

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.

Association mapping of partitioning loci in barley.

BACKGROUND: Association mapping, initially developed in human disease genetics, is now being applied to plant species. The model species Arabidopsis provided some of the first examples of association mapping in plants, identifying previously cloned flowering time genes, despite high population sub-structure. More recently, association genetics has been applied to barley, where breeding activity has resulted in a high degree of population sub-structure. A major genotypic division within barley is that between winter- and spring-sown varieties, which differ in their requirement for vernalization to promote subsequent flowering. To date, all attempts to validate association genetics in barley by identifying major flowering time loci that control vernalization requirement (VRN-H1 and VRN-H2) have failed. Here, we validate the use of association genetics in barley by identifying VRN-H1 and VRN-H2, despite their prominent role in determining population sub-structure. RESULTS: By taking barley as a typical inbreeding crop, and seasonal growth habit as a major partitioning phenotype, we develop an association mapping approach which successfully identifies VRN-H1 and VRN-H2, the underlying loci largely responsible for this agronomic division. We find a combination of Structured Association followed by Genomic Control to correct for population structure and inflation of the test statistic, resolved significant associations only with VRN-H1 and the VRN-H2 candidate genes, as well as two genes closely linked to VRN-H1 (HvCSFs1 and HvPHYC). CONCLUSION: We show that, after employing appropriate statistical methods to correct for population sub-structure, the genome-wide partitioning effect of allelic status at VRN-H1 and VRN-H2 does not result in the high levels of spurious association expected to occur in highly structured samples. Furthermore, we demonstrate that both VRN-H1 and the candidate VRN-H2 genes can be identified using association mapping. Discrimination between intragenic VRN-H1 markers was achieved, indicating that candidate causative polymorphisms may be discerned and prioritised within a larger set of positive associations. This proof of concept study demonstrates the feasibility of association mapping in barley, even within highly structured populations. A major advantage of this method is that it does not require large numbers of genome-wide markers, and is therefore suitable for fine mapping and candidate gene evaluation, especially in species for which large numbers of genetic markers are either unavailable or too costly.

Measurement of vitamin D status: background, clinical use, and methodologies.

Vitamin D and its metabolites are crucial to the overall health and well-being of humans and animals, having important functions in calcium homeostasis and bone metabolism. Exposure of the skin to sunlight may provide adequate levels of vitamin D; however, there are numerous reports of vitamin D insufficiency or deficiency. 25-hydroxyvitamin D (calcidiol, 25(OH)D) is regarded as the best measurement of overall vitamin D status. 1,25-dihydroxyvitamin D (calcitriol, 1,25(OH)2D) is the most biologically active vitamin D metabolite. 25(OH)D has higher affinity for vitamin D binding protein (VDBP) than 1,25-dihydroxyvitamin D; whereas, 1,25-dihydroxyvitamin D has higher affinity for the vitamin D receptor (VDR) than 25-hydroxyvitamin D. HPLC and immunoassays allow the determination of vitamin D status, as measured by 25(OH)D, and 1,25(OH)2D. Recently it has been shown that the vitamin D requirements have been underestimated and that vitamin D2 is much less potent than vitamin D3. Future studies will determine the amount of vitamin D3 necessary for optimal health and well-being.

Comparative mapping of the barley Ppd-H1 photoperiod response gene region, which lies close to a junction between two rice linkage segments.

Comparative mapping of cereals has shown that chromosomes of barley, wheat, and maize can be described in terms of rice "linkage segments." However, little is known about marker order in the junctions between linkage blocks or whether this will impair comparative analysis of major genes that lie in such regions. We used genetic and physical mapping to investigate the relationship between the distal part of rice chromosome 7L, which contains the Hd2 heading date gene, and the region of barley chromosome 2HS containing the Ppd-H1 photoperiod response gene, which lies near the junction between rice 7 and rice 4 linkage segments. RFLP markers were mapped in maize to identify regions that might contain Hd2 or Ppd-H1 orthologs. Rice provided useful markers for the Ppd-H1 region but comparative mapping was complicated by loss of colinearity and sequence duplications that predated the divergence of rice, maize, and barley. The sequences of cDNA markers were used to search for homologs in the Arabidopsis genome. Homologous sequences were found for 13 out of 16 markers but they were dispersed in Arabidopsis and did not identify any candidate equivalent region. The implications of the results for comparative trait mapping in junction regions are discussed.

Barley Hv CIRCADIAN CLOCK ASSOCIATED 1 and Hv PHOTOPERIOD H1 Are Circadian Regulators That Can Affect Circadian Rhythms in Arabidopsis.

Circadian clocks regulate many aspects of plant physiology and development that contribute to essential agronomic traits. Circadian clocks contain transcriptional feedback loops that are thought to generate circadian timing. There is considerable similarity in the genes that comprise the transcriptional and translational feedback loops of the circadian clock in the plant Kingdom. Functional characterisation of circadian clock genes has been restricted to a few model species. Here we provide a functional characterisation of the Hordeum vulgare (barley) circadian clock genes Hv circadian clock associated 1 (HvCCA1) and Hv photoperiodh1, which are respectively most similar to Arabidopsis thaliana circadian clock associated 1 (AtCCA1) and pseudo response regulator 7 (AtPRR7). This provides insight into the circadian regulation of one of the major crop species of Northern Europe. Through a combination of physiological assays of circadian rhythms in barley and heterologous expression in wild type and mutant strains of A. thaliana we demonstrate that HvCCA1 has a conserved function to AtCCA1. We find that Hv photoperiod H1 has AtPRR7-like functionality in A. thaliana and that the effects of the Hv photoperiod h1 mutation on photoperiodism and circadian rhythms are genetically separable.

Mutant alleles of Photoperiod-1 in wheat (Triticum aestivum L.) that confer a late flowering phenotype in long days.

Flowering time in wheat and barley is known to be modified by mutations in the Photoperiod-1 (Ppd-1) gene. Semi-dominant Ppd-1a mutations conferring an early flowering phenotype are well documented in wheat but gene sequencing has also identified candidate loss of function mutations for Ppd-A1 and Ppd-D1. By analogy to the recessive ppd-H1 mutation in barley, loss of function mutations in wheat are predicted to delay flowering under long day conditions. To test this experimentally, introgression lines were developed in the spring wheat variety 'Paragon'. Plants lacking a Ppd-B1 gene were identified from a gamma irradiated 'Paragon' population. These were crossed with the other introgression lines to generate plants with candidate loss of function mutations on one, two or three genomes. Lines lacking Ppd-B1 flowered 10 to 15 days later than controls under long days. Candidate loss of function Ppd-A1 alleles delayed flowering by 1 to 5 days while candidate loss of function Ppd-D1 alleles did not affect flowering time. Loss of Ppd-A1 gave an enhanced effect, and loss of Ppd-D1 became detectable in lines where Ppd-B1 was absent, indicating effects may be buffered by functional Ppd-1 alleles on other genomes. Expression analysis revealed that delayed flowering was associated with reduced expression of the TaFT1 gene and increased expression of TaCO1. A survey of the GEDIFLUX wheat collection grown in the UK and North Western Europe between the 1940s and 1980s and the A.E. Watkins global collection of landraces from the 1920s and 1930s showed that the identified candidate loss of function mutations for Ppd-D1 were common and widespread, while the identified candidate Ppd-A1 loss of function mutation was rare in countries around the Mediterranean and in the Far East but was common in North Western Europe. This may reflect a possible benefit of the latter in northern locations.

Copy number variation affecting the Photoperiod-B1 and Vernalization-A1 genes is associated with altered flowering time in wheat (Triticum aestivum).

The timing of flowering during the year is an important adaptive character affecting reproductive success in plants and is critical to crop yield. Flowering time has been extensively manipulated in crops such as wheat (Triticum aestivum L.) during domestication, and this enables them to grow productively in a wide range of environments. Several major genes controlling flowering time have been identified in wheat with mutant alleles having sequence changes such as insertions, deletions or point mutations. We investigated genetic variants in commercial varieties of wheat that regulate flowering by altering photoperiod response (Ppd-B1 alleles) or vernalization requirement (Vrn-A1 alleles) and for which no candidate mutation was found within the gene sequence. Genetic and genomic approaches showed that in both cases alleles conferring altered flowering time had an increased copy number of the gene and altered gene expression. Alleles with an increased copy number of Ppd-B1 confer an early flowering day neutral phenotype and have arisen independently at least twice. Plants with an increased copy number of Vrn-A1 have an increased requirement for vernalization so that longer periods of cold are required to potentiate flowering. The results suggest that copy number variation (CNV) plays a significant role in wheat adaptation.

A generic operational strategy to qualify translational safety biomarkers.

The importance of using translational safety biomarkers that can predict, detect and monitor drug-induced toxicity during human trials is becoming increasingly recognized. However, suitable processes to qualify biomarkers in clinical studies have not yet been established. There is a need to define clear scientific guidelines to link biomarkers to clinical processes and clinical endpoints. To help define the operational approach for the qualification of safety biomarkers the IMI SAFE-T consortium has established a generic qualification strategy for new translational safety biomarkers that will allow early identification, assessment and management of drug-induced injuries throughout R&D.

Comparative genomics of flowering time pathways using Brachypodium distachyon as a model for the temperate grasses.

Brachypodium distachyon (Brachypodium) is a model for the temperate grasses which include important cereals such as barley, wheat and oats. Comparison of the Brachypodium genome (accession Bd21) with those of the model dicot Arabidopsis thaliana and the tropical cereal rice (Oryza sativa) provides an opportunity to compare and contrast genetic pathways controlling important traits. We analysed the homologies of genes controlling the induction of flowering using pathways curated in Arabidopsis Reactome as a starting point. Pathways include those detecting and responding to the environmental cues of day length (photoperiod) and extended periods of low temperature (vernalization). Variation in these responses has been selected during cereal domestication, providing an interesting comparison with the wild genome of Brachypodium. Brachypodium Bd21 has well conserved homologues of circadian clock, photoperiod pathway and autonomous pathway genes defined in Arabidopsis and homologues of vernalization pathway genes defined in cereals with the exception of VRN2 which was absent. Bd21 also lacked a member of the CO family (CO3). In both cases flanking genes were conserved showing that these genes are deleted in at least this accession. Segmental duplication explains the presence of two CO-like genes in temperate cereals, of which one (Hd1) is retained in rice, and explains many differences in gene family structure between grasses and Arabidopsis. The conserved fine structure of duplications shows that they largely evolved to their present structure before the divergence of the rice and Brachypodium. Of four flowering-time genes found in rice but absent in Arabidopsis, two were found in Bd21 (Id1, OsMADS51) and two were absent (Ghd7, Ehd1). Overall, results suggest that an ancient core photoperiod pathway promoting flowering via the induction of FT has been modified by the recruitment of additional lineage specific pathways that promote or repress FT expression.

The European and American use of exploratory approaches for first-in-human studies.

Exploratory approaches for first-in-human clinical studies have evolved over the last few years and have stimulated the issuance of national regulatory guidances in some European countries as well as the United States. With the increasing implementation of these approaches and the recent preparation of a multiregional regulatory guidance (ICH M3 rev2), an exchange of experiences on the opportunities and challenges of exploratory clinical trials was desirable; thus, a workshop focusing on the use of this clinical approach was planned and conducted in Lisbon, Portugal, March 18-19, 2009 sponsored by the Portuguese Health Authority (INFARMED) and DIA. The structure of the workshop focused in three main areas. Regulatory representatives from Portugal, Belgium, Germany, the United Kingdom and the United States formally reviewed their experiences. This was followed by a discussion on issues from an ethics review perspective as well as an insight to the opportunities in the area of biologics. The industry perspective was presented by representatives from Merck, Pfizer, J&J, Novartis, Speedel, AstraZeneca, GSK, and Roche. Finally, through break out sessions, issues were identified to be addressed moving forward. It is the purpose of this paper to report on the outcome of this workshop.

Urinary clusterin, cystatin C, beta2-microglobulin and total protein as markers to detect drug-induced kidney injury.

Earlier and more reliable detection of drug-induced kidney injury would improve clinical care and help to streamline drug-development. As the current standards to monitor renal function, such as blood urea nitrogen (BUN) or serum creatinine (SCr), are late indicators of kidney injury, we conducted ten nonclinical studies to rigorously assess the potential of four previously described nephrotoxicity markers to detect drug-induced kidney and liver injury. Whereas urinary clusterin outperformed BUN and SCr for detecting proximal tubular injury, urinary total protein, cystatin C and beta2-microglobulin showed a better diagnostic performance than BUN and SCr for detecting glomerular injury. Gene and protein expression analysis, in-situ hybridization and immunohistochemistry provide mechanistic evidence to support the use of these four markers for detecting kidney injury to guide regulatory decision making in drug development. The recognition of the qualification of these biomarkers by the EMEA and FDA will significantly enhance renal safety monitoring.

Voluntary exploratory data submissions to the US FDA and the EMA: experience and impact.

Heterogeneity in the underlying mechanisms of disease processes and inter-patient variability in drug responses are major challenges in drug development. To address these challenges, biomarker strategies based on a range of platforms, such as microarray gene-expression technologies, are increasingly being applied to elucidate these sources of variability and thereby potentially increase drug development success rates. With the aim of enhancing understanding of the regulatory significance of such biomarker data by regulators and sponsors, the US Food and Drug Administration initiated a programme in 2004 to allow sponsors to submit exploratory genomic data voluntarily, without immediate regulatory impact. In this article, a selection of case studies from the first 5 years of this programme - which is now known as the voluntary exploratory data submission programme, and also involves collaboration with the European Medicines Agency - are discussed, and general lessons are highlighted.

Towards consensus practices to qualify safety biomarkers for use in early drug development.

Application of any new biomarker to support safety-related decisions during regulated phases of drug development requires provision of a substantial data set that critically assesses analytical and biological performance of that biomarker. Such an approach enables stakeholders from industry and regulatory bodies to objectively evaluate whether superior standards of performance have been met and whether specific claims of fit-for-purpose use are supported. It is therefore important during the biomarker evaluation process that stakeholders seek agreement on which critical experiments are needed to test that a biomarker meets specific performance claims, how new biomarker and traditional comparators will be measured and how the resulting data will be merged, analyzed and interpreted.

Renal biomarker qualification submission: a dialog between the FDA-EMEA and Predictive Safety Testing Consortium.

The first formal qualification of safety biomarkers for regulatory decision making marks a milestone in the application of biomarkers to drug development. Following submission of drug toxicity studies and analyses of biomarker performance to the Food and Drug Administration (FDA) and European Medicines Agency (EMEA) by the Predictive Safety Testing Consortium's (PSTC) Nephrotoxicity Working Group, seven renal safety biomarkers have been qualified for limited use in nonclinical and clinical drug development to help guide safety assessments. This was a pilot process, and the experience gained will both facilitate better understanding of how the qualification process will probably evolve and clarify the minimal requirements necessary to evaluate the performance of biomarkers of organ injury within specific contexts.

Photoperiod insensitive Ppd-A1a mutations in tetraploid wheat (Triticum durum Desf.).

Variation in photoperiod response plays an important role in adapting crops to agricultural environments. In hexaploid wheat, mutations conferring photoperiod insensitivity (flowering after a similar time in short or long days) have been mapped on the 2B (Ppd-B1) and 2D (Ppd-D1) chromosomes in colinear positions to the 2H Ppd-H1 gene of barley. No A genome mutation is known. On the D genome, photoperiod insensitivity is likely to be caused by deletion of a regulatory region that causes misexpression of a member of the pseudo-response regulator (PRR) gene family and activation of the photoperiod pathway irrespective of day length. Photoperiod insensitivity in tetraploid (durum) wheat is less characterized. We compared pairs of near-isogenic lines that differ in photoperiod response and showed that photoperiod insensitivity is associated with two independent deletions of the A genome PRR gene that cause altered expression. This is associated with induction of the floral regulator FT. The A genome deletions and the previously described D genome deletion of hexaploid wheat remove a common region, suggesting a shared mechanism for photoperiod insensitivity. The identification of the A genome mutations will allow characterization of durum wheat germplasm and the construction of genotypes with novel combinations of photoperiod insensitive alleles.