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ARS Home Pacific West Area Albany, California Plant Gene Expression Center Research





- Fletcher, Jennifer
- Harmon, Frank
- 2023
- Publications
- Plant Genetic Resources, Genomics and Genetic Improvement (301)

Research Project: **Location:
                            Plant Gene Expression Center** Project Number: 2030-21210-001-000-D
                        
                        Project Type: In-House Appropriated Start Date: Mar 19, 2023
                        
                        End Date: Mar 18, 2028 **Objective:**
                        
                        Objective 1: Perform genome editing of CLAVATA (CLV) pathway genes to improve yield traits in pennycress. Characterize and stack mutations in CLV pathway and other yield-regulating genes to further enhance pennycress productivity. Characterize functional role of UNUSUAL FLORAL ORGANS (UFO) in Arabidopsis stem cell maintenance to increase understanding of yield regulation in brassicas and grasses.

Sub-objective 1.A: Perform genetic engineering of yield traits in pennycress.

Sub-objective 1.B: Characterize and stack mutations in the pennycress CLV pathway. 

Sub-objective 1.C: Characterize the functional role of UFO in stem cell maintenance. 

Objective 2: Identify sorghum floral activator genes and their function. Elucidate genetic and biochemical pathways controlling flowering-related growth in sorghum. Identify how reduced day:night temperature differentials change sorghum growth and development. 

Sub-objective 2.A: Identify sorghum floral activator genes and their function. 

Sub-objective 2.B: Elucidate genetic and biochemical pathways controlling flowering-related growth in sorghum. 

Sub-objective 2.C: Identify how reduced day:night temperature differentials change sorghum growth and development. 

Objective 3: Develop genetic and molecular strategies to accelerate crop breeding including the control of juvenility and rejuvenation; characterize the role of miR156 in the regulation of the juvenile phase and rejuvenation in woody crops.

Sub-objective 3.A: Create miR156 target mimicry lines to determine the contribution to reproductive competence in woody crops. 

Sub-objective 3.B: Characterize the function of individual MIR156 genes in woody plants. 

Sub-objective 3.C: Develop graft-based methods for manipulating juvenility and maturation. **Approach:**
                        
                        Objective 1
Hypothesis: Genetic manipulation of CLV gene function will lead to pennycress yield increases; identifying stem cell maintenance factors and combining stem cell mutations can further increase pennycress yield; UFO regulates microRNA gene expression to control shoot stem cell activity.
  
Experimental Approaches/Procedures: Target pennycress CLV3 and CLV1 for mutagenesis using genome editing. Transform constructs, identify mutants by genotyping, perform RT-qPCR to quantify expression, measure yield traits. Conduct molecular mapping-by-sequencing of pennycress 158 and 246 mutants. Generate Taclv2 158 and Taclv2 246 mutants, measure harvest index. Analyze ufo and ufo clv3 mutants by microscopy, identify genetic pathway, quantify miRNA expression by RT-qPCR.
  
Contingencies: If we are unable to identify conserved CLV3 promoter sequences, gRNAs spanning the promoter will be generated. If molecular mapping fails to identify a mutation in the 158/246 lines, a larger gene interval will be sequenced. If RT-qPCR does not yield results, RNA-seq will be conducted.
 
Objective 2
Hypothesis: Novel late flowering sorghum mutants alter the function of floral activator genes; Sorghum GIGANTEA regulates catabolism of gibberellin phytohormone as part of flowering-related growth; identify how reduced day:night temperature differentials change sorghum growth and development.
  
Experimental Approaches/Procedures: Mapping and mutant analysis to identify causal mutants for late flowering sorghum mutants. Determine gene expression changes to floral regulatory networks in these mutant lines. Gene expression analysis and hormone treatments to identify factors promoting stem growth in sorghum and other grasses. Measure progression of growth and development of sorghum plants exposed to high and low day:night temperature differentials.
  
Contingencies: Late flowering will be screened in the greenhouse if longer growing seasons are needed. Testing growth promotion by phytohormone auxin will be the alternative to gibberellin. More narrow day/night temperature differentials will be tested if no changes in sorghum phenology are initially observed.
 
Objective 3
Hypothesis/Goals: Reduction of miR156 levels will accelerate maturity; identify the most important MIR156 genes for reproductive competence and juvenile traits to aid in improvement strategies utilizing miR156; grafting mature scions onto rootstocks overexpressing miR156 and TFL/ATC will rejuvenate new growth and enhance propagation traits.

Experimental Approaches/Procedures: Measure time to first-flowering and other molecular phenotypes in transgenic plants with elevated and reduced levels of miR156. Use deep sequencing by RNA-seq to generate transcript models for MIR156 genes across a broad phylogenetic sampling of woody crops. Heterograft mature scions with transgenic rootstocks overexpressing miR156/TFL/ATC.

Contingencies: Use RNA-seq and qRT-PCR if transgenic plants fail to produce visible maturation related phenotypes. Explore different types and depths of RNA-seq libraries for ranking MIR156 importance. If miR156/TFL/ATC transcripts show mobility, fuse tRNA-like sequences to their 3’ ends.


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