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Cross bred species such as switchgrass may benefit from advantageous breeding strategies requiring inbred lines. Doubled haploid production methods offer several ways that these lines can be produced that often involve uniparental genome elimination as the rate limiting step. We have used a centromere-mediated genome elimination strategy in which modified CENH3 is expressed to induce the process. Transgenic tetraploid switchgrass lines coexpressed Cas9, a poly-cistronic tRNA-gRNA tandem array containing eight guide RNAs that target two CENH3 genes, and different chimeric versions of CENH3 with alterations to the N-terminal tail region. Genotyping of CENH3 genes in transgenics identified edits including frameshift mutations and deletions in one or both copies of the two CENH3 genes. Flow cytometry of T

Switchgrass (Panicum virgatum L.) has been subject to breeding to improve its yield and composition for bioenergy. Increased tolerance of environmental stress is another factor that can improve yields over the life span of a field plot. Natural ploidy series act as somewhat distinct gene pools in switchgrass that maintain differences with respect to tolerance of environmental stress and which can only be bridged through whole genome reduction or duplication events. In order to document potential wide-ranging effects of whole genome duplication, we examined the effects of water stress on growth, physiology, and gene expression in individual tetraploid clones of the switchgrass cultivar “Liberty” as well as neo-octoploid lines derived from it. The neo-octoploids behaved similarly to Liberty under water stress and recovery conditions: Water-stressed plants had approximately 50% or greater reduction of biomass, plant height, and node numbers. Tiller number and photosynthesis rates of stressed plants were reduced by up to 50%, while proline levels were significantly higher in stressed plants than well-watered plants. A total of 6134 differentially expressed genes (8% of the annotated genes with detectable expression in crown tissue) were detected under water-deficit stress, while 3310 differentially expressed genes were detected in crown tissue after 1 week of recovery from water-deficit stress relative to well-watered treatments. Only 187 genes were identified as being differentially expressed between 4x Liberty and its 8x derivatives. This study indicates neo-octoploids could be viable 8x-equivalent Liberty stand-ins for future breeding assays.

Quantitation of leaf surface wax on a population of switchgrass identified three significant QTL present across six environments that contribute to leaf glaucousness and wax composition and that show complex genetic × environmental (G × E) interactions. The C4 perennial grass Panicum virgatum (switchgrass) is a native species of the North American tallgrass prairie. This adaptable plant can be grown on marginal lands and is useful for soil and water conservation, biomass production, and as a forage. Two major switchgrass ecotypes, lowland and upland, differ in a range of desirable traits, and the responsible underlying loci can be localized efficiently in a pseudotestcross design. An outbred four-way cross (4WCR) mapping population of 750 F2 lines was used to examine the genetic basis of differences in leaf surface wax load between two lowland (AP13 and WBC) and two upland (DAC and VS16) tetraploid cultivars. The objective of our experiments was to identify wax compositional variation among the population founders and to map underlying loci responsible for surface wax variation across environments. GCMS analyses of surface wax extracted from 4WCR F0 founders and F1 hybrids reveal higher levels of wax in lowland genotypes and show quantitative differences of β-diketones, primary alcohols, and other wax constituents. The full mapping population was sampled over two seasons from four field sites with latitudes ranging from 30 to 42 °N, and leaf surface wax was measured. We identified three high-confidence QTL, of which two displayed significant G × E effects. Over 50 candidate genes underlying the QTL regions showed similarity to genes in either Arabidopsis or barley known to function in wax synthesis, modification, regulation, and transport.

Purpose: The polyamine analog bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) depletes cellular polyamines and inhibits malignant cell growth. We have previously shown that BE-4-4-4-4 inhibits nucleosome condensation on supercoiled DNA in a cell-free system. Here we sought to determine whether BE-4-4-4-4 inhibits nucleosome condensation in cells, and whether that effect alters the expression of specific genes. Methods: We used the simian virus 40 (SV-40) minichromosome as a model system and studied the expression of the viral late genes. It is known that the SV-40 late genes are regulated by the steroid receptor elements that, in turn, control gene expression by altering nucleosomal organization. Results: We observed a more than six fold increase in SV-40 late gene expression in cells pretreated with BE-4-4-4-4 for 18 h. The polyamine analog bisethyl norspermine (BE-3-3-3), that does not affect nucleosomal condensation in cell free systems and has little effect on chromatin structure in cultured human tumor cells, had a negligible effect on SV-40 late gene expression under treatment conditions identical to those used with BE-4-4-4-4. Conclusion: Similar to the findings in the cell-free system, the polyamine analog BE-4-4-4-4 inhibited nucleosome formation and, thereby, altered the expression of specific genes in a cellular system.