Project description:<h4>Background</h4>Quantitative real-time polymerase chain reaction (RT-qPCR) is the key platform for the quantitative analysis of gene expression in a wide range of experimental systems and conditions. However, the accuracy and reproducibility of gene expression quantification via RT-qPCR is entirely dependent on the identification of reliable reference genes for data normalisation. Green foxtail (<i>Setaria viridis</i>) has recently been proposed as a potential experimental model for the study of C<sub>4</sub> photosynthesis and is closely related to many economically important crop species of the Panicoideae subfamily of grasses, including <i>Zea mays</i> (maize), <i>Sorghum bicolor</i> (sorghum) and <i>Sacchurum officinarum</i> (sugarcane). <i>Setaria viridis</i> (Accession 10) possesses a number of key traits as an experimental model, namely; (i) a small sized, sequenced and well annotated genome; (ii) short stature and generation time; (iii) prolific seed production, and; (iv) is amendable to <i>Agrobacterium tumefaciens</i>-mediated transformation. There is currently however, a lack of reference gene expression information for <i>Setaria viridis</i> (<i>S. viridis</i>). We therefore aimed to identify a cohort of suitable <i>S. viridis</i> reference genes for accurate and reliable normalisation of <i>S. viridis</i> RT-qPCR expression data.<h4>Results</h4>Eleven putative candidate reference genes were identified and examined across thirteen different <i>S. viridis</i> tissues. Of these, the geNorm and NormFinder analysis software identified <i>SERINE</i>/<i>THERONINE</i>-<i>PROTEIN PHOSPHATASE 2A</i> (<i>PP2A</i>), <i>5</i>'-<i>ADENYLYLSULFATE REDUCTASE 6</i> (<i>ASPR6</i>) and <i>DUAL SPECIFICITY PHOSPHATASE</i> (<i>DUSP</i>) as the most suitable combination of reference genes for the accurate and reliable normalisation of <i>S. viridis</i> RT-qPCR expression data. To demonstrate the suitability of the three selected reference genes, <i>PP2A</i>, <i>ASPR6</i> and <i>DUSP</i>, were used to normalise the expression of <i>CINNAMYL ALCOHOL DEHYDROGENASE</i> (<i>CAD</i>) genes across the same tissues.<h4>Conclusions</h4>This approach readily demonstrated the suitably of the three selected reference genes for the accurate and reliable normalisation of <i>S. viridis</i> RT-qPCR expression data. Further, the work reported here forms a highly useful platform for future gene expression quantification in <i>S. viridis</i> and can also be potentially directly translatable to other closely related and agronomically important C<sub>4</sub> crop species.
Project description:Setaria viridis has emerged as a model species for the larger C4 grasses. Here the cellulose synthase (CesA) superfamily has been defined, with an emphasis on the amounts and distribution of (1,3;1,4)-?-glucan, a cell wall polysaccharide that is characteristic of the grasses and is of considerable value for human health.Orthologous relationship of the CesA and Poales-specific cellulose synthase-like (Csl) genes among Setaria italica (Si), Sorghum bicolor (Sb), Oryza sativa (Os), Brachypodium distachyon (Bradi) and Hordeum vulgare (Hv) were compared using bioinformatics analysis. Transcription profiling of Csl gene families, which are involved in (1,3;1,4)-?-glucan synthesis, was performed using real-time quantitative PCR (Q-PCR). The amount of (1,3;1,4)-?-glucan was measured using a modified Megazyme assay. The fine structures of the (1,3;1,4)-?-glucan, as denoted by the ratio of cellotriosyl to cellotetraosyl residues (DP3:DP4 ratio) was assessed by chromatography (HPLC and HPAEC-PAD). The distribution and deposition of the MLG was examined using the specific antibody BG-1 and captured using fluorescence and transmission electron microscopy (TEM).The cellulose synthase gene superfamily contains 13 CesA and 35 Csl genes in Setaria. Transcript profiling of CslF, CslH and CslJ gene families across a vegetative tissue series indicated that SvCslF6 transcripts were the most abundant relative to all other Csl transcripts. The amounts of (1,3;1,4)-?-glucan in Setaria vegetative tissues ranged from 0.2% to 2.9% w/w with much smaller amounts in developing grain (0.003% to 0.013% w/w). In general, the amount of (1,3;1,4)-?-glucan was greater in younger than in older tissues. The DP3:DP4 ratios varied between tissue types and across developmental stages, and ranged from 2.4 to 3.0:1. The DP3:DP4 ratios in developing grain ranged from 2.5 to 2.8:1. Micrographs revealing the distribution of (1,3;1,4)-?-glucan in walls of different cell types and the data were consistent with the quantitative (1,3;1,4)-?-glucan assays.The characteristics of the cellulose synthase gene superfamily and the accumulation and distribution of (1,3;1,4)-?-glucans in Setaria are similar to those in other C4 grasses, including sorghum. This suggests that Setaria is a suitable model plant for cell wall polysaccharide biology in C4 grasses.
Project description:Setaria viridis (green foxtail) has been identified as a potential experimental model system to genetically and molecularly characterise the C4 monocotyledonous grasses due to its small physical size, short generation time and prolific seed production, together with a sequenced and annotated genome. Setaria viridis is the wild ancestor of the cropping species, foxtail millet (Setaria italica), with both Setaria species sharing a close evolutionary relationship with the agronomically important species, maize, sorghum, and sugarcane, as well as the bioenergy feedstocks, switchgrass, and Miscanthus. However, an efficient and reproducible transformation protocol is required to further advance the use of S. viridis to study the molecular genetics of C4 monocotyledonous grasses. An efficient and reproducible protocol was established for Agrobacterium tumefaciens-mediated transformation of S. viridis (Accession A10) regenerable callus material derived from mature seeds, a protocol that returned an average transformation efficiency of 6.3%. The efficiency of this protocol was the result of the: (i) use of mature embryo derived callus material; (ii) age of the seed used to induce callus formation; (iii) composition of the callus induction media, including the addition of the ethylene inhibitor, silver nitrate; (iv) use of a co-cultivation approach, and; (v) concentration of the selective agent. Our protocol furthers the use of S. viridis as an experimental model system to study the molecular genetics of C4 monocotyledonous grasses for the potential future development of improved C4 cropping species.
Project description:The CRISPR/Cas9 system has been used for genome editing in several organisms, including higher plants. This system induces site-specific mutations in the genome based on the nucleotide sequence of engineered guide RNAs. The complex genomes of C4 grasses makes genome editing a challenge in key grass crops like maize (<i>Zea mays</i>), sorghum (<i>Sorghum bicolor</i>), <i>Brachiaria</i> spp., switchgrass (<i>Panicum virgatum</i>), and sugarcane (<i>Saccharum</i> spp.). <i>Setaria viridis</i> is a diploid C4 grass widely used as a model for these C4 crop plants. Here, an optimized CRISPR/Cas9 binary vector that exploits the non-homologous end joining (NHEJ) system was used to knockout a <i>green fluorescent protein</i> (<i>gfp</i>) transgene in <i>S. viridis</i> accession A10.1. Transformation of embryogenic callus by <i>A. tumefaciens</i> generated ten glufosinate-ammonium resistant transgenic events. In the T0 generation, 60% of the events were biallelic mutants in the <i>gfp</i> transgene with no detectable accumulation of GFP protein and without insertions or deletions in predicted off-target sites. The <i>gfp</i> mutations generated by CRISPR/Cas9 were stable and displayed Mendelian segregation in the T1 generation. Altogether, the system described here is a highly efficient genome editing system for <i>S. viridis</i>, an important model plant for functional genomics studies in C4 grasses. Also, this system is a potential tool for improvement of agronomic traits in C4 crop plants with complex genomes.
Project description:Setaria viridis (green foxtail) is an important model system for improving cereal crops due to its diploid genome, ease of cultivation, and use of C4 photosynthesis. The S. viridis accession ME034V is exceptionally transformable, but the lack of a sequenced genome for this accession has limited its utility. We present a 397 Mb highly contiguous de novo assembly of ME034V using ultra-long nanopore sequencing technology (read N50 = 41kb). We estimate that this genome is largely complete based on our updated k-mer based genome size estimate of 401 Mb for S. viridis Genome annotation identified 37,908 protein-coding genes and >300k repetitive elements comprising 46% of the genome. We compared the ME034V assembly with two other previously sequenced Setaria genomes as well as to a diversity panel of 235 S. viridis accessions. We found the genome assemblies to be largely syntenic, but numerous unique polymorphic structural variants were discovered. Several ME034V deletions may be associated with recent retrotransposition of copia and gypsy LTR repeat families, as evidenced by their low genotype frequencies in the sampled population. Lastly, we performed a phylogenomic analysis to identify gene families that have expanded in Setaria, including those involved in specialized metabolism and plant defense response. The high continuity of the ME034V genome assembly validates the utility of ultra-long DNA sequencing to improve genetic resources for emerging model organisms. Structural variation present in Setaria illustrates the importance of obtaining the proper genome reference for genetic experiments. Thus, we anticipate that the ME034V genome will be of significant utility for the Setaria research community.
Project description:Acidic soils are distributed worldwide, predominantly in tropical and subtropical areas, reaching around 50% of the arable soil. This type of soil strongly reduces crop production, mainly because of the presence of aluminum, which has its solubility increased at low pH levels. A well-known physiological mechanism used by plants to cope with Al stress involves activation of membrane transporters responsible for organic acid anions secretion from the root apex to the rhizosphere, which chelate Al, preventing its absorption by roots. In sorghum, a membrane transporter gene belonging to multidrug and toxic compound extrusion (MATE) family was identified and characterized as an aluminum-activated citrate transporter gene responsible for Al tolerance in this crop. <i>Setaria viridis</i> is an emerging model for C4 species and it is an important model to validate some genes for further C4 crops transformation, such as sugarcane, maize, and wheat. In the present work, <i>Setaria viridis</i> was used as a model plant to overexpress a newly identified MATE gene from <i>Brachypodium distachyon</i> (<i>BdMATE</i>), closely related to <i>SbMATE</i>, for aluminum tolerance assays. Transgenic <i>S. viridis</i> plants overexpressing a <i>BdMATE</i> presented an improved Al tolerance phenotype, characterized by sustained root growth and exclusion of aluminum from the root apex in transgenic plants, as confirmed by hematoxylin assay. In addition, transgenic plants showed higher root citrate exudation into the rhizosphere, suggesting that Al tolerance improvement in these plants could be related to the chelation of the metal by the organic acid anion. These results suggest that <i>BdMATE</i> gene can be used to transform C4 crops of economic importance with improved aluminum tolerance.
Project description:An efficient method for crossing green foxtail (Setaria viridis) is currently lacking. S. viridis is considered to be the new model plant for the study of C4 system in monocots and so an effective crossing protocol is urgently needed. S. viridis is a small grass with C4-NADP (ME) type of photosynthesis and has the advantage of having small genome of about 515 Mb, small plant stature, short life cycle, multiple tillers, and profuse seed set, and hence is an ideal model species for research. The objectives of this project were to develop efficient methods of emasculation and pollination, and to speed up generation advancement. We assessed the response of S. viridis flowers to hot water treatment (48°C) and to different concentrations of gibberellic acid, abscisic acid, maleic hydrazide (MH), and kinetin. We found that 500 ?M of MH was effective in the emasculation of S. viridis, whilst still retaining the receptivity of the stigma to pollination. We also report effective ways to accelerate the breeding cycle of S. viridis for research through the germination of mature as well as immature seeds in optimized culture media. We believe these findings will be of great interest to researchers using Setaria.