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

Population genetics of foxtail millet and its wild ancestor

<p>Abstract</p> <p>Background</p> <p>Foxtail millet (<it>Setaria italica </it>(L.) P. Beauv.), one of the most ancient domesticated crops, is becoming a model system for studying biofuel crops and comparative genomics in the grasses. However, knowledge on the level of genetic diversity and linkage disequilibrium (LD) is very limited in this crop and its wild ancestor, green foxtail (<it>Setaria viridis </it>(L.) P. Beauv.). Such information would help us to understand the domestication process of cultivated species and will allow further research in these species, including association mapping and identification of agricultural significant genes involved in domestication.</p> <p>Results</p> <p>In this study, we surveyed DNA sequence for nine loci across 50 accessions of cultivated foxtail millet and 34 of its wild progenitor. We found a low level of genetic diversity in wild green foxtail (θ = 0.0059), θ means Watterson's estimator of θ. Despite of a 55% loss of its wild diversity, foxtail millet still harbored a considerable level of diversity (θ = 0.0027) when compared to rice and sorghum (θ = 0.0024 and 0.0034, respectively). The level of LD in the domesticated foxtail millet extends to 1 kb, while it decayed rapidly to a negligible level within 150 bp in wild green foxtail. Using coalescent simulation, we estimated the bottleneck severity at k = 0.6095 when ρ/θ = 1. These results indicated that the domestication bottleneck of foxtail millet was more severe than that of maize but slightly less pronounced than that of rice.</p> <p>Conclusions</p> <p>The results in this study establish a general framework for the domestication history of foxtail millet. The low level of genetic diversity and the increased level of LD in foxtail millet are mainly caused by a population bottleneck, although gene flow from foxtail millet to green foxtail is another factor that may have shaped the pattern of genetic diversity of these two related gene pools. The knowledge provided in this study will benefit future population based studies in foxtail millet.</p

A Larger Root System Is Coupled With Contrasting Expression Patterns of Phosphate and Nitrate Transporters in Foxtail Millet [Setaria italica (L.) Beauv.] Under Phosphate Limitation

Foxtail millet [Setaria italica (L.) Beauv.], a widely cultivated food and fodder crop, develops a smaller root system while enlarges the root diameter facilitating nutrient transport under nitrogen limitation. How foxtail millet responds to phosphate limitation (LP) remains unaddressed. LP seedlings of the sequenced variety Yugu1 had significantly lower P concentrations in both shoots and roots and displayed higher levels of anthocyanin accumulation in leaves, indicating that the seedlings suffered from P limitation under hydroponic culture. One obvious and adaptive phenotype of LP plants was the larger root system mostly as the result of stimulation of lateral root proliferation in terms of the number, density, and length. Preferential biomass accumulation in the root under LP ensured carbon provision for root expansion and resulted in significant increases in the total and specific root length, which substantially extended the absorptive surface of P in the growth medium. Elevation of auxin and gibberellin concentrations might serve as an internal boost underpinning root architectural re-patterning under LP. Not just morphological adaptation, up-regulation of expression of SiPHT1;1 and SiPHT1;4 in roots and that of SiPHT1;2 in roots and shoots preconditioned adaptive enhancement of P uptake and translocation under LP. Interestingly, internal nitrogen surpluses occurred as indicated by dramatic increases in free amino acids in LP shoots and roots and higher concentrations of nitrogen in roots. Such nitrogen surplus ‘signals’ tended to switch down expression of nitrate transporters SiNRT2.1 and SiNAR2.1 in the root and that of SiNRT1.11 and SiNRT1.12 in the shoot to reduce nitrate mobilization toward or within the shoot. Together, our work provided new insights into adaption of a critical cereal crop to LP and its innate connection with nitrogen nutrition

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

Horizontal Transfer of a Plant Transposon

The majority of well-documented cases of horizontal transfer between higher eukaryotes involve the movement of transposable elements between animals. Surprisingly, although plant genomes often contain vast numbers of these mobile genetic elements, no evidence of horizontal transfer of a nuclear-encoded transposon between plant species has been detected to date. The most mutagenic known plant transposable element system is the Mutator system in maize. Mu-like elements (MULEs) are widespread among plants, and previous analysis has suggested that the distribution of various subgroups of MULEs is patchy, consistent with horizontal transfer. We have sequenced portions of MULE transposons from a number of species of the genus Setaria and compared them to each other and to publicly available databases. A subset of these elements is remarkably similar to a small family of MULEs in rice. A comparison of noncoding and synonymous sequences revealed that the observed similarity is not due to selection at the amino acid level. Given the amount of time separating Setaria and rice, the degree of similarity between these elements excludes the possibility of simple vertical transmission of this class of MULEs. This is the first well-documented example of horizontal transfer of any nuclear-encoded genes between higher plants

Phytolith Analysis for Differentiating between Foxtail Millet (Setaria italica) and Green Foxtail (Setaria viridis)

Foxtail millet (Setaria italica) is one of the oldest domesticated cereal crops in Eurasia, but identifying foxtail millets, especially in charred grains, and differentiating it from its wild ancestor, green foxtail (Setaria viridis), in the archaeobotanical remains, is still problematic. Phytolithic analysis provides a meaningful method for identifying this important crop. In this paper, the silicon structure patterns in the glumes, lemmas, and paleas from inflorescence bracts in 16 modern plants of foxtail millet and green foxtail from China and Europe are examined using light microscopy with phase-contrast and a microscopic interferometer. Our research shows that the silicon structure of ΩIII from upper lemmas and paleas in foxtail millet and green foxtail can be correspondingly divided into two groups. The size of ΩIII type phytolith of foxtail millet is bigger than that from green foxtail. Discriminant function analysis reveals that 78.4% of data on foxtail millet and 76.9% of data on green foxtail are correctly classified. This means certain morphotypes of phytoliths are relatively reliable tools for distinguishing foxtail millet from green foxtail. Our results also revealed that the husk phytolith morphologies of foxtail millets from China and Eastern Europe are markedly different from those from Western Europe. Our research gives a meaningful method of separating foxtail millet and green foxtail. The implications of these findings for understanding the history of foxtail millet domestication and cultivation in ancient civilizations are significant