Project description:Sorghum is a recalcitrant crop for Agrobacterium-mediated genetic transformation. Several parameters related to Agrobacterium-mediated transformation were tested to optimize sorghum transformation frequencies. In this study, we evaluated pretreatment of sorghum variety Tx430 immature embryos using Agrobacterium strain GV2260. Pretreatment of immature embryos with heat (43°C) treatment for 15 or 21?min, and centrifugation resulted in a transformation efficiency of up to 1.9% of immature embryos treated. Although further optimization to enhance transformation efficiency is required, this study contributes to the genetic validation of genes of interest and molecular breeding in sorghum plants.
Project description:Background:Sorghum (Sorghum bicolor L.) is one of the world's most important cereal crops grown for multiple applications and has been identified as a potential biofuel crop. Despite several decades of study, sorghum has been widely considered as a recalcitrant major crop for transformation due to accumulation of phenolic compounds, lack of model genotypes, low regeneration frequency and loss of regeneration potential through sub-cultures. Among different explants used for genetic transformation of sorghum, immature embryos are ideal over other explants. However, the continuous supply of quality immature embryos for transformation is labour intensive and expensive. In addition, transformation efficiencies are also influenced by environmental conditions (light and temperature). Despite these challenges, immature embryos remain the predominant choice because of their success rate and also due to non-availability of other dependable explants without compromising the transformation efficiency. Results:We report here a robust genetic transformation method for sorghum (Tx430) using differentiating embryogenic calli (DEC) with nodular structures induced from immature embryos and maintained for more than a year without losing regeneration potential on modified MS media. The addition of lipoic acid (LA) to callus induction media along with optimized growth regulators increased callus induction frequency from 61.3 ± 3.2 to 79 ± 6.5% from immature embryos (1.5-2.0 mm in length) isolated 12-15 days after pollination. Similarly, the regeneration efficiency and the number of shoots from DEC tissue was enhanced by LA. The optimized regeneration system in combination with particle bombardment resulted in an average transformation efficiency (TE) of 27.2 or 46.6% based on the selection strategy, 25% to twofold higher TE than published reports in Tx430. Up to 100% putative transgenic shoots were positive for npt-II by PCR and 48% of events had < 3 copies of transgenes as determined by digital droplet PCR. Reproducibility of this method was demonstrated by generating ~ 800 transgenic plants using 10 different gene constructs. Conclusions:This protocol demonstrates significant improvements in both efficiency and ease of use over existing sorghum transformation methods using PDS, also enables quick hypothesis testing in the production of various high value products in sorghum.
Project description:This study utilized next generation sequencing technology (RNA-Seq and BS-Seq) to examine the transcriptome and methylome of various tissues within sorghum plants with the ultimate goal of improving the Sorghum bicolor annotation We examined the mRNA of various Sorghum bicolor (BTx623) tissues (flowers, vegitative and floral meristems, embryos, roots and shoots) and bisulfite treated DNA from two root samples
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: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
Project description:Sorghum bicolor, a photosynthetically efficient C4 grass, represents an important source of grain, forage, fermentable sugars, and cellulosic fibers that can be utilized in myriad applications ranging from bioenergy to bioindustrial feedstocks. Sorghum's efficient fixation of carbon per unit time per unit area per unit input has led to its classification as a preferred biomass crop highlighted by its designation as an advanced biofuel by the U.S. Department of Energy. Due to its extensive genetic diversity and worldwide colonization, sorghum has considerable diversity for a range of phenotypes influencing productivity, composition, and sink/source dynamics. To dissect the genetic basis of these key traits, we present a sorghum carbon-partitioning nested association mapping (NAM) population generated by crossing 11 diverse founder lines with Grassl as the single recurrent female. By exploiting existing variation among cellulosic, forage, sweet, and grain sorghum carbon partitioning regimes, the sorghum carbon-partitioning NAM population will allow the identification of important biomass-associated traits, elucidate the genetic architecture underlying carbon partitioning and improve our understanding of the genetic determinants affecting unique phenotypes within Poaceae. We contrast this NAM population with an existing grain population generated using Tx430 as the recurrent female. Genotypic data are assessed for quality by examining variant density, nucleotide diversity, linkage decay, and are validated using pericarp and testa phenotypes to map known genes affecting these phenotypes. We release the 11-family NAM population along with corresponding genomic data for use in genetic, genomic, and agronomic studies with a focus on carbon-partitioning regimes.
Project description:Sorghum (Sorghum bicolor), also known as great millet, is one of the most popular cultivated grass species in the world. Sorghum is frequently consumed as food for humans and animals as well as used for ethanol production. In this study, we conducted de novo transcriptome assembly for sorghum variety Taejin by next-generation sequencing, obtaining 8.748 GB of raw data. The raw data in this study can be available in NCBI SRA database with accession number of SRX1715644. Using the Trinity program, we identified 222,161 transcripts from sorghum variety Taejin. We further predicted coding regions within the assembled transcripts by the TransDecoder program, resulting in a total of 148,531 proteins. We carried out BLASTP against the Swiss-Prot protein sequence database to annotate the functions of the identified proteins. To our knowledge, this is the first transcriptome data for a sorghum variety derived from Korea, and it can be usefully applied to the generation of genetic markers.
Project description:Despite a "ploidy barrier," interspecific crosses to wild and/or cultivated sorghum (Sorghum bicolor, 2n = 2x = 20) may have aided the spread across six continents of Sorghum halepense, also exemplifying risks of "transgene escape" from crops that could make weeds more difficult to control. Genetic maps of two BC1F1 populations derived from crosses of S. bicolor (sorghum) and S. halepense with totals of 722 and 795 single nucleotide polymorphism (SNP) markers span 37 and 35 linkage groups, with 2-6 for each of the 10 basic sorghum chromosomes due to fragments covering different chromosomal portions or independent segregation from different S. halepense homologs. Segregation distortion favored S. halepense alleles on chromosomes 2 (1.06-4.68 Mb, near a fertility restoration gene), 7 (1.20-6.16 Mb), 8 (1.81-5.33 Mb, associated with gene conversion), and 9 (47.5-50.1 Mb); and S. bicolor alleles on chromosome 6 (0-40 Mb), which contains both a large heterochromatin block and the Ma1 gene. Regions of the S. halepense genome that are recalcitrant to gene flow from sorghum might be exploited as part a multi-component system to reduce the likelihood of spread of transgenes or other modified genes. Its SNP profile suggests that chromosome segments from its respective progenitors S. bicolor and Sorghum propinquum have extensively recombined in S. halepense. This study reveals genomic regions that might discourage crop-to-weed gene escape, and provides a foundation for marker-trait association analysis to determine the genetic control of traits contributing to weediness, invasiveness, and perenniality of S. halepense.
Project description:Long-read sequencing technologies have greatly facilitated assemblies of large eukaryotic genomes. In this paper, Oxford Nanopore sequences generated on a MinION sequencer are combined with Bionano Genomics Direct Label and Stain (DLS) optical maps to generate a chromosome-scale de novo assembly of the repeat-rich Sorghum bicolor Tx430 genome. The final assembly consists of 29 scaffolds, encompassing in most cases entire chromosome arms. It has a scaffold N50 of 33.28 Mbps and covers 90% of the expected genome length. A sequence accuracy of 99.85% is obtained after aligning the assembly against Illumina Tx430 data and 99.6% of the 34,211 public gene models align to the assembly. Comparisons of Tx430 and BTx623 DLS maps against the public BTx623 v3.0.1 genome assembly suggest substantial discrepancies whose origin remains to be determined. In summary, this study demonstrates that informative assemblies of complex plant genomes can be generated by combining nanopore sequencing with DLS optical maps.
Project description:Parallel Analysis of RNA Ends (PARE) sequencing reads were generated to validate putative microRNAs and identify cleavage sites in Sorghum bicolor and Setaria viridis. Overall design: For Sorghum bicolor, a variety of conditions were used to generate total RNA, including leaf and three stages of anther development. For Setaria viridis, single replicates of leaf, panicle, and two stages of spikelets were sampled.