The C-terminal motif of SiAGO1b is required for the regulation of growth, development and stress responses in foxtail millet (\u3ci\u3eSetaria italica\u3c/i\u3e (L.) P. Beauv)

Foxtail millet (Setaria italica (L.) P. Beauv), which belongs to the Panicoideae tribe of the Poaceae, is an important grain crop widely grown in Northern China and India. It is currently developing into a novel model species for functional genomics of the Panicoideae as a result of its fully available reference genome sequence, small diploid genome (2n=18, ~510 Mb), short life cycle, small stature and prolific seed production. Argonaute 1 (AGO1), belonging to the argonaute (AGO) protein family, recruits small RNAs and regulates plant growth and development. Here, we characterized an AGO1 mutant (siago1b) in foxtail millet, which was induced by ethyl methanesulfonate treatment. The mutant exhibited pleiotropic developmental defects, including dwarfing stem, narrow and rolled leaves, smaller panicles and lower rates of seed setting. Map-based cloning analysis demonstrated that these phenotypic variations were attributed to a C–A transversion, and a 7-bp deletion in the C-terminus of the SiAGO1b gene in siago1b. Yeast two-hybrid assays and BiFC experiments revealed that the mutated region was an essential functional motif for the interaction between SiAGO1b and SiHYL1. Furthermore, 1598 differentially expressed genes were detected via RNAseq- based comparison of SiAGO1b and wild-type plants, which revealed that SiAGO1b mutation influenced multiple biological processes, including energy metabolism, cell growth, programmed death and abiotic stress responses in foxtail millet. This study may provide a better understanding of the mechanisms by which SiAGO1b regulates the growth and development of crops

The C-terminal motif of SiAGO1b is required for the regulation of growth, development and stress responses in foxtail millet (\u3ci\u3eSetaria italica\u3c/i\u3e (L.) P. Beauv)

Foxtail millet (Setaria italica (L.) P. Beauv), which belongs to the Panicoideae tribe of the Poaceae, is an important grain crop widely grown in Northern China and India. It is currently developing into a novel model species for functional genomics of the Panicoideae as a result of its fully available reference genome sequence, small diploid genome (2n=18, ~510 Mb), short life cycle, small stature and prolific seed production. Argonaute 1 (AGO1), belonging to the argonaute (AGO) protein family, recruits small RNAs and regulates plant growth and development. Here, we characterized an AGO1 mutant (siago1b) in foxtail millet, which was induced by ethyl methanesulfonate treatment. The mutant exhibited pleiotropic developmental defects, including dwarfing stem, narrow and rolled leaves, smaller panicles and lower rates of seed setting. Map-based cloning analysis demonstrated that these phenotypic variations were attributed to a C–A transversion, and a 7-bp deletion in the C-terminus of the SiAGO1b gene in siago1b. Yeast two-hybrid assays and BiFC experiments revealed that the mutated region was an essential functional motif for the interaction between SiAGO1b and SiHYL1. Furthermore, 1598 differentially expressed genes were detected via RNAseq- based comparison of SiAGO1b and wild-type plants, which revealed that SiAGO1b mutation influenced multiple biological processes, including energy metabolism, cell growth, programmed death and abiotic stress responses in foxtail millet. This study may provide a better understanding of the mechanisms by which SiAGO1b regulates the growth and development of crops

Screening of Mutants Related to the C4 Photosynthetic Kranz Structure in Foxtail Millet

C4 plants exhibit significantly higher photosynthetic, water and nutrient use efficiency compared with C3 plants. Kranz anatomy is associated with many C4 plants in which bundle sheath cells surround the veins and are themselves surrounded by mesophyll cells. This specialized Kranz anatomy is elucidated as an important contributor to C4 photosynthetic activities in C4 plant. Characterizing the molecular basis of Kranz structure formation has become a key objective for studies of C4 photosynthesis. However, severe mutants that specifically disrupt Kranz anatomy have not been identified. In this study, we detected 549 stable ethyl methane sulfonate-induced foxtail millet (cultivar Yugu1) mutants related to leaf development and photosynthesis among 2,709 mutants screened (M3/M4 generation). The identified mutants included 52 that had abnormal leaf veins (with abnormal starch accumulation based on iodine staining). Each of the 52 mutants was characterized through an analysis of leaf morphology, and through microscopic observations of leaf tissue sections embedded in resin and paraffin. In total, 14 mutants were identified with abnormal Kranz structures exemplified by small bundle sheath cell size. Additional phenotypes of the mutants included poorly differentiated mesophyll and bundle sheath cells, increased vein density and the absence of chloroplasts in the bundle sheath cells. Kranz structure mutations were accompanied by varying leaf thickness, implying these mutations induced complex effects. We identified mutations related to Kranz structure development in this trial, which may be useful for the mapping and cloning of genes responsible for mediating Kranz structure development

Genome-Wide DNA Polymorphism Analysis and Molecular Marker Development for the Setaria italica Variety “SSR41” and Positional Cloning of the Setaria White Leaf Sheath Gene SiWLS1

Genome-wide DNA polymorphism analysis and molecular marker development are important for forward genetics research and DNA marker-assisted breeding. As an ideal model system for Panicoideae grasses and an important minor crop in East Asia, foxtail millet (Setaria italica) has a high-quality reference genome as well as large mutant libraries based on the “Yugu1” variety. However, there is still a lack of genetic and mutation mapping tools available for forward genetics research on S. italica. Here, we screened another S. italica genotype, “SSR41”, which is morphologically similar to, and readily cross-pollinates with, “Yugu1”. High-throughput resequencing of “SSR41” identified 1,102,064 reliable single nucleotide polymorphisms (SNPs) and 196,782 insertions/deletions (InDels) between the two genotypes, indicating that these two genotypes have high genetic diversity. Of the 8,361 high-quality InDels longer than 20 bp that were developed as molecular markers, 180 were validated with 91.5% accuracy. We used “SSR41” and these developed molecular markers to map the white leaf sheath gene SiWLS1. Further analyses showed that SiWLS1 encodes a chloroplastlocalized protein that is involved in the regulation of chloroplast development in bundle sheath cells in the leaf sheath in S. italica and is related to sensitivity to heavy metals. Our study provides the methodology and an important resource for forward genetics research on Setaria

SiYGL2 Is Involved in the Regulation of Leaf Senescence and Photosystem II Efficiency in Setaria italica (L.) P. Beauv.

A yellow-green leaf mutant was isolated from EMS-mutagenized lines of Setaria italica variety Yugu1. Map-based cloning revealed the mutant gene is a homolog of Arabidopsis thaliana AtEGY1. EGY1 (ethylene-dependent gravitropism-deficient and yellow-green 1) is an ATP-independent metalloprotease (MP) that is required for chloroplast development, photosystem protein accumulation, hypocotyl gravitropism, leaf senescence, and ABA signal response in A. thaliana. However, the function of EGY1 in monocotyledonous C4 plants has not yet been described. The siygl2 mutant is phenotypically characterized by chlorotic organs, premature senescence, and damaged PS II function. Sequence comparisons of the AtEGY1 and SiYGL2 proteins reveals the potential for SiYGL2 to encode a partially functional protein. Phenotypic characterization and gene expression analysis suggested that SiYGL2 participates in the regulation of chlorophyll content, leaf senescence progression, and PS II function. Additionally, our research will contribute to further characterization of the mechanisms regulating leaf senescence and photosynthesis in S. italica, and in C4 plants in general

Novel genomes and genome constitutions identified by GISH and 5S rDNA and knotted1 genomic sequences in the genus Setaria

Background: The Setaria genus is increasingly of interest to researchers, as its two species, S. viridis and S. italica, are being developed as models for understanding C4 photosynthesis and plant functional genomics. The genome constitution of Setaria species has been studied in the diploid species S. viridis, S. adhaerans and S. grisebachii, where three genomes A, B and C were identified respectively. Two allotetraploid species, S. verticillata and S. faberi, were found to have AABB genomes, and one autotetraploid species, S. queenslandica, with an AAAA genome, has also been identified. The genomes and genome constitutions of most other species remain unknown, even though it was thought there are approximately 125 species in the genus distributed world-wide.Results: GISH was performed to detect the genome constitutions of Eurasia species of S. glauca, S. plicata, and S. arenaria, with the known A, B and C genomes as probes. No or very poor hybridization signal was detected indicating that their genomes are different from those already described. GISH was also performed reciprocally between S. glauca, S. plicata, and S. arenaria genomes, but no hybridization signals between each other were found. The two sets of chromosomes of S. lachnea both hybridized strong signals with only the known C genome of S. grisebachii. Chromosomes of Qing 9, an accession formerly considered as S. viridis, hybridized strong signal only to B genome of S. adherans. Phylogenetic trees constructed with 5S rDNA and knotted1 markers, clearly classify the samples in this study into six clusters, matching the GISH results, and suggesting that the F genome of S. arenaria is basal in the genus.Conclusions: Three novel genomes in the Setaria genus were identified and designated as genome D (S. glauca), E (S. plicata) and F (S. arenaria) respectively. The genome constitution of tetraploid S. lachnea is putatively CCC’C’. Qing 9 is a B genome species indigenous to China and is hypothesized to be a newly identified species. The difference in genome constitution and origin of S. verticillata and S. faberi is also discussed. The new genomes and the genome constitutions of Setaria species identified in this report provide useful information for Setaria germplasm management, foxtail millet breeding, grass evolution and the development of S. viridis and S. italica as a new model for functional genomics.Peer reviewedBotan

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie

geneHapR: an R package for gene haplotypic statistics and visualization

Abstract Background Together with application of next-generation sequencing technologies and increased accumulation of genomic variation data in different organism species, an opportunity for effectively identification of superior alleles of functional genes to facilitate marker-assisted selection is emerging, and the clarification of haplotypes of functional genes is becoming an essential target in recent study works. Results In this paper, we describe an R package ‘geneHapR’ developed for haplotypes identification, statistics and visualization analysis of candidate genes. This package could integrate genotype data, genomic annotating information and phenotypic variation data to clarify genotype variations, evolutionary-ship, and morphological effects among haplotypes through variants visualization, network construction and phenotypic comparison. ‘geneHapR’ also provides functions for Linkage Disequilibrium block analysis and visualizing of haplotypes geo-distribution. Conclusions The R package ‘geneHapR’ provided an easy-to-use tool for haplotype identification, statistic and visualization for candidate gene and will provide useful clues for gene functional dissection and molecular-assistant pyramiding of beneficial alleles of functional locus in future breeding programs