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Targeted mutagenesis in tetraploid switchgrass (Panicum virgatum L.) using CRISPR/Cas9.

ABSTRACT: The CRISPR/Cas9 system has become a powerful tool for targeted mutagenesis. Switchgrass (Panicum virgatum L.) is a high yielding perennial grass species that has been designated as a model biomass crop by the U.S. Department of Energy. The self-infertility and high ploidy level make it difficult to study gene function or improve germplasm. To overcome these constraints, we explored the feasibility of using CRISPR/Cas9 for targeted mutagenesis in a tetraploid cultivar 'Alamo' switchgrass. We first developed a transient assay by which a non-functional green-fluorescent protein gene containing a 1-bp frameshift insertion in its 5' coding region was successfully mutated by a Cas9/sgRNA complex resulting in its restored function. Agrobacterium-mediated stable transformation of embryogenic calli derived from mature caryopses averaged a 3.0% transformation efficiency targeting the genes of teosinte branched 1(tb1)a and b and phosphoglycerate mutase (PGM). With a single construct containing two sgRNAs targeting different regions of tb1a and tb1b genes, primary transformants (T0) containing CRISPR/Cas9-induced mutations were obtained at frequencies of 95.5% (tb1a) and 11% (tb1b), respectively, with T0 mutants exhibiting increased tiller production. Meanwhile, a mutation frequency of 13.7% was obtained for the PGM gene with a CRISPR/Cas9 construct containing a single sgRNA. Among the PGM T0 mutants, six are heterozygous and one is homozygous for a 1-bp deletion in the target region with no apparent phenotypical alterations. We show that CRISPR/Cas9 system can generate targeted mutagenesis effectively and obtain targeted homozygous mutants in T0 generation in switchgrass, circumventing the need of inbreeding.

SUBMITTER: Liu Y

PROVIDER: S-EPMC5787850 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Targeted mutagenesis in tetraploid switchgrass (Panicum virgatum L.) using CRISPR/Cas9.

Liu Yang Y Merrick Paul P Zhang Zhengzhi Z Ji Chonghui C Yang Bing B Fei Shui-Zhang SZ

Plant biotechnology journal 20170801 2

The CRISPR/Cas9 system has become a powerful tool for targeted mutagenesis. Switchgrass (Panicum virgatum L.) is a high yielding perennial grass species that has been designated as a model biomass crop by the U.S. Department of Energy. The self-infertility and high ploidy level make it difficult to study gene function or improve germplasm. To overcome these constraints, we explored the feasibility of using CRISPR/Cas9 for targeted mutagenesis in a tetraploid cultivar 'Alamo' switchgrass. We firs ...[more]

PMID: 28640964

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Project description:BackgroundGlobal warming predictions indicate that temperatures will increase by another 2-6°C by the end of this century. High temperature is a major abiotic stress limiting plant growth and productivity in many areas of the world. Switchgrass (Panicum virgatum L.) is a model herbaceous bioenergy crop, due to its rapid growth rate, reliable biomass yield, minimal requirements of water and nutrients, adaptability to grow on marginal lands and widespread distribution throughout North America. The effect of high temperature on switchgrass physiology, cell wall composition and biomass yields has been reported. However, there is void in the knowledge of the molecular responses to heat stress in switchgrass.ResultsWe conducted long-term heat stress treatment (38°/30°C, day/night, for 50 days) in the switchgrass cultivar Alamo. A significant decrease in the plant height and total biomass was evident in the heat stressed plants compared to controls. Total RNA from control and heat stress samples were used for transcriptome analysis with switchgrass Affymetrix genechips. Following normalization and pre-processing, 5365 probesets were identified as differentially expressed using a 2-fold cutoff. Of these, 2233 probesets (2000 switchgrass unigenes) were up-regulated, and 3132 probesets (2809 unigenes) were down-regulated. Differential expression of 42 randomly selected genes from this list was validated using RT-PCR. Rice orthologs were retrieved for 78.7% of the heat stress responsive switchgrass probesets. Gene ontology (GOs) enrichment analysis using AgriGO program showed that genes related to ATPase regulator, chaperone binding, and protein folding was significantly up-regulated. GOs associated with protein modification, transcription, phosphorus and nitrogen metabolic processes, were significantly down-regulated by heat stress.ConclusionsPlausible connections were identified between the identified GOs, physiological responses and heat response phenotype observed in switchgrass plants. Comparative transcriptome analysis in response to heat stress among four monocots - switchgrass, rice, wheat and maize identified 16 common genes, most of which were associated with protein refolding processes. These core genes will be valuable biomarkers for identifying heat sensitive plant germplasm since they are responsive to both short duration as well as chronic heat stress treatments, and are also expressed in different plant growth stages and tissue types.

Project description:BackgroundSwitchgrass (Panicum virgatum L.) is a warm-season C4 grass that is a target lignocellulosic biofuel species. In many regions, drought stress is one of the major limiting factors for switchgrass growth. The objective of this study was to evaluate the drought tolerance of 49 switchgrass genotypes. The relative drought stress tolerance was determined based on a set of parameters including plant height, leaf length, leaf width, leaf sheath length, leaf relative water content (RWC), electrolyte leakage (EL), photosynthetic rate (Pn), stomatal conductance (g s), transpiration rate (Tr), intercellular CO2 concentration (Ci), and water use efficiency (WUE).ResultsSRAP marker analysis determined that the selected 49 switchgrass genotypes represent a diverse genetic pool of switchgrass germplasm. Principal component analysis (PCA) and drought stress indexes (DSI) of each physiological parameter showed significant differences in the drought stress tolerance among the 49 genotypes. Heatmap and PCA data revealed that physiological parameters are more sensitive than morphological parameters in distinguishing the control and drought treatments. Metabolite profiling data found that under drought stress, the five best drought-tolerant genotypes tended to have higher levels of abscisic acid (ABA), spermine, trehalose, and fructose in comparison to the five most drought-sensitive genotypes.ConclusionBased on PCA ranking value, the genotypes TEM-SEC, TEM-LoDorm, BN-13645-64, Alamo, BN-10860-61, BN-12323-69, TEM-SLC, T-2086, T-2100, T-2101, Caddo, and Blackwell-1 had relatively higher ranking values, indicating that they are more tolerant to drought. In contrast, the genotypes Grif Nebraska 28, Grenville-2, Central Iowa Germplasm, Cave-in-Rock, Dacotah, and Nebraska 28 were found to be relatively sensitive to drought stress. By analyzing physiological response parameters and different metabolic profiles, the methods utilized in this study identified drought-tolerant and drought-sensitive switchgrass genotypes. These results provide a foundation for future research directed at understanding the molecular mechanisms underlying switchgrass tolerance to drought.

Dataset Information

Targeted mutagenesis in tetraploid switchgrass (Panicum virgatum L.) using CRISPR/Cas9.

Publications

Targeted mutagenesis in tetraploid switchgrass (Panicum virgatum L.) using CRISPR/Cas9.

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