RNA-Seq of Sorghum bicolor 9d seedlings in response to osmotic stress and abscisic acid
ABSTRACT: This study utilized next generation sequencing technology (RNA-Seq) to examine the transcriptome of sorghum plants challenged with osmotic stress and exogenous abscisic acid (ABA) to elucidate those genes and gene networks that contribute to sorghum's tolerance to water-limiting environments with a long-term aim of developing strategies to improve plant productivity under drought. We examined the mRNA of 9 day old Sorghum bicolor (BTx623) from 2 tissue types (roots and shoots) for 2 treatments (20 uM ABA and 20% PEG) with corresponding controls (0.2M NaOH and H2O) for 27 hrs prior to harvesting, each done in triplicate biological replicates - resulting in 24 unique runs
Project description:This study utilized next generation sequencing technology (RNA-Seq) to examine the transcriptome of sorghum plants challenged with osmotic stress and exogenous abscisic acid (ABA) to elucidate those genes and gene networks that contribute to sorghum's tolerance to water-limiting environments with a long-term aim of developing strategies to improve plant productivity under drought. We examined the mRNA of 9 day old Sorghum bicolor (BTx623) from 2 tissue types (roots and shoots) for 2 treatments (20 uM ABA and 20% PEG) with corresponding controls (0.2M NaOH and H2O) for 27 hrs prior to harvesting, each done in triplicate biological replicates - resulting in 24 unique runs
Project description:BACKGROUND: Higher plants exhibit remarkable phenotypic plasticity allowing them to adapt to an extensive range of environmental conditions. Sorghum is a cereal crop that exhibits exceptional tolerance to adverse conditions, in particular, water-limiting environments. This study utilized next generation sequencing (NGS) technology to examine the transcriptome of sorghum plants challenged with osmotic stress and exogenous abscisic acid (ABA) in order to elucidate genes and gene networks that contribute to sorghum's tolerance to water-limiting environments with a long-term aim of developing strategies to improve plant productivity under drought. RESULTS: RNA-Seq results revealed transcriptional activity of 28,335 unique genes from sorghum root and shoot tissues subjected to polyethylene glycol (PEG)-induced osmotic stress or exogenous ABA. Differential gene expression analyses in response to osmotic stress and ABA revealed a strong interplay among various metabolic pathways including abscisic acid and 13-lipoxygenase, salicylic acid, jasmonic acid, and plant defense pathways. Transcription factor analysis indicated that groups of genes may be co-regulated by similar regulatory sequences to which the expressed transcription factors bind. We successfully exploited the data presented here in conjunction with published transcriptome analyses for rice, maize, and Arabidopsis to discover more than 50 differentially expressed, drought-responsive gene orthologs for which no function had been previously ascribed. CONCLUSIONS: The present study provides an initial assemblage of sorghum genes and gene networks regulated by osmotic stress and hormonal treatment. We are providing an RNA-Seq data set and an initial collection of transcription factors, which offer a preliminary look into the cascade of global gene expression patterns that arise in a drought tolerant crop subjected to abiotic stress. These resources will allow scientists to query gene expression and functional annotation in response to drought.
Project description:Sorghum (Sorghum bicolor Moench, L.) plant accumulates copious layers of epi-cuticular wax (EW) on its aerial surfaces, to a greater extent than most other crops. EW provides a vapor barrier that reduces water loss, and is therefore considered to be a major determinant of sorghum's drought tolerance. However, little is known about the genes responsible for wax accumulation in sorghum. We isolated two allelic mutants, bloomless40-1 (bm40-1) and bm40-2, from a mutant library constructed from ethyl methane sulfonate (EMS) treated seeds of an inbred, BTx623. Both mutants were nearly devoid of the EW layer. Each bm mutant was crossed to the un-mutated BTx623 to generated F2 populations that segregated for the bm phenotype. Genomic DNA from 20 bm F2 plants from each population was bulked for whole genome sequencing. A single gene, Sobic.001G228100, encoding a GDSL-like lipase/acylhydrolase, had unique homozygous mutations in each bulked F2 population. Mutant bm40-1 harbored a missense mutation in the gene, whereas bm40-2 had a splice donor site mutation. Our findings thus provide strong evidence that mutation in this GDSL-like lipase gene causes the bm phenotype, and further demonstrate that this approach of sequencing two independent allelic mutant populations is an efficient method for identifying causal mutations. Combined with allelic mutants, MutMap provides powerful method to identify all causal genes for the large collection of bm mutants in sorghum, which will provide insight into how sorghum plants accumulate such abundant EW on their aerial surface. This knowledge may facilitate the development of tools for engineering drought-tolerant crops with reduced water loss.
Project description:Sorghum (Sorghum bicolor) is the fifth most important cereal crop in the world. It is an annual C4 crop due to its high biomass and wide usage, and has a strong resistance to stress. Obviously, there are many benefits of planting sorghum on marginal soils such as saline-alkali land. Although it is known that abscisic acid (ABA) is involved in plant abiotic stress responses, there are few reports on sorghum. Here, we obtained RNA-seq data, which showed gene expression at the genome-wide level under saline-alkali stress. The genes related to ABA biosynthesis, catabolism, and signaling were identified and analyzed. Meanwhile, their amino acid sequences were intermingled with rice genes to form several distinct orthologous and paralogous groups. ABA-related differentially expressed genes under saline-alkali stress were identified, and family members involved in ABA signaling were hypothesized based on the expression levels and homologous genes in rice. Furthermore, the ABA signaling pathway in Sorghum bicolor was understood better by interaction analysis. These findings present a comprehensive overview of the genes regulating ABA biosynthesis, catabolism, and signaling in Sorghum bicolor under saline-alkali stress, and provide a foundation for future research regarding their biological roles in sorghum stress tolerance.
Project description:This experiment contains the subset of data corresponding to sorghum RNA-Seq data from experiment E-GEOD-50464 (http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-50464/), which goal is to examine the transcriptome of various Sorghum bicolor (BTx623) tissues: flowers, vegetative and floral meristems, embryos, roots and shoots. Thus, we expanded the existing transcriptome atlas for sorghum by conducting RNA-Seq analysis on meristematic tissues, florets, and embryos, and these data sets have been used to improve on the existing community structural annotations.
Project description:Target leaf spot (TLS) of sorghum, a foliar disease caused by the necrotrophic fungus Bipolaris cookei (also known as Bipolaris sorghicola), can affect grain yield in sorghum by causing premature drying of leaves and defoliation. Two sorghum recombinant inbred line (RIL) populations, BTx623/BTx642 and BTx623/SC155-14E, were assessed for TLS resistance in replicated trials. Using least square mean trait data, four TLS resistance QTL were identified, two in each population. Of these, three were previously unidentified while a major QTL on chromosome 5 in the BTx623/BTx642 RIL population corresponded to the previously identified TLS resistance gene ds1. A set of sorghum lines were assessed for production of reactive oxygen species induced by treatment with the microbe-associated molecular pattern (MAMP) flg22 (a derivative of flagellin). Flg22-induced ROS production varied between lines in a consistent fashion. One QTL associated with variation in the flg22 response was detected in the RIL populations. No evidence was found to link variation in the MAMP response to variation in TLS resistance.
Project description:Simple sequence repeat (SSR) markers with a high degree of polymorphism contribute to the molecular dissection of agriculturally important traits in sorghum (Sorghum bicolor (L.) Moench). We designed 5599 non-redundant SSR markers, including regions flanking the SSRs, in whole-genome shotgun sequences of sorghum line ATx623. (AT/TA)n repeats constituted 26.1% of all SSRs, followed by (AG/TC)n at 20.5%, (AC/TG)n at 13.7% and (CG/GC)n at 11.8%. The chromosomal locations of 5012 SSR markers were determined by comparing the locations identified by means of electronic PCR with the predicted positions of 34 008 gene loci. Most SSR markers had a similar distribution to the gene loci. Among 970 markers validated by fragment analysis, 67.8% (658 of 970) markers successfully provided PCR amplification in sorghum line BTx623, with a mean polymorphism rate of 45.1% (297 of 658) for all SSR loci in combinations of 11 sorghum lines and one sudangrass (Sorghum sudanense (Piper) Stapf) line. The product of 5012 and 0.678 suggests that approximately 3400 SSR markers could be used to detect SSR polymorphisms and that more than 1500 (45.1% of 3400) markers could reveal SSR polymorphisms in combinations of Sorghum lines.
Project description:Sorghum (Sorghum bicolor L.) ranks as the fifth most widely planted cereal in the world and is used for food as well as a biomass plant for ethanol production. Use of the TX430 non-tannin sorghum variety has enhanced Agrobacterium-mediated sorghum transformation. These protocols could not be applied, however, to other tannin producing sorghum varieties such as the BTx623 model cultivar for sorghum with full genome information of sorghum. Here we report an improved protocol for Agrobacterium-mediated genetic transformation of tannin-producing sorghum variety BTx623. We successfully developed modification of root regeneration condition for generation of transgenic plant of BTx623. We inoculated immature embryos with Agrobacterium tumefaciens strain EHA105 harboring pMDC32-35S-GFP to generate transgenic plants. In the root regeneration step, we found that regeneration from transformed calli was affected by tannin. For root regeneration, shoots that appeared were not transferred to agar plate, but instead transferred to vermiculite in a plastic pod. Direct planting of regenerated shoots into vermiculite prevented the toxic effect of tannin. Root regeneration efficiency from calli emerged shoots in vermiculite was 78.57%. Presence of sGFP transgene in the genome of transgenic plants was confirmed by PCR and sGFP expression was confirmed in transgenic plants. This improved protocol of Agrobacterium-mediated transformation for tannin-producing sorghum BTx623 could be a useful tool for functional genomics using this plant.
Project description:<h4>Background</h4>Sorghum bicolor is the fifth most commonly grown cereal worldwide and is remarkable for its drought and abiotic stress tolerance. For these reasons and the large size of biomass varieties, it has been proposed as a bioenergy crop. However, little is known about the genes underlying sorghum's abiotic stress tolerance and biomass yield.<h4>Results</h4>To uncover the genetic basis of drought tolerance in sorghum at a genome-wide level, we undertook a high-density phenomics genome wide association study (GWAS) in which 648 diverse sorghum lines were phenotyped at two locations in California once per week by drone over the course of a growing season. Biomass, height, and leaf area were measured by drone for individual field plots, subjected to two drought treatments and a well-watered control. The resulting dataset of ~?171,000 phenotypic data-points was analyzed along with 183,989 genotype by sequence markers to reveal 213 high-quality, replicated, and conserved GWAS associations.<h4>Conclusions</h4>The genomic intervals defined by the associations include many strong candidate genes, including those encoding heat shock proteins, antifreeze proteins, and other domains recognized as important to plant stress responses. The markers identified by our study can be used for marker assisted selection for drought tolerance and biomass. In addition, our results are a significant step toward identifying specific sorghum genes controlling drought tolerance and biomass yield.
Project description:This study used with RNA-Seq to examine the tissue specific expression data within sorghum plants for improving the Sorghum bicolor gene annotation. We examined the RNA from tissues (spikelet, seed and stem) in Sorghum bicolor (BTx623).Total RNAs form each tissues were extracted using SDS/phenol method followed by LiCl purification