Date of Award
Doctor of Philosophy
Switchgrass (Panicum virgatum) is an important bioenergy crop in United States. During past decades, numerous research studies on switchgrass breeding, genetics and biology have been conducted to improve the biomass yield of switchgrass. It has been shown that tiller number is positively correlated with biomass yield in switchgrass. To facilitate breeding improvement for tiller production, it is urgent to understand the tillering mechanism at the molecular level in switchgrass. The teosinte branched 1 (tb1) gene is a well-studied branching related gene in grasses. Yet, the function of tb1-like genes in switchgrass is still not clear. Characterizing the roles of Panicum virgatum tb1 (Pvtb1) genes in switchgrass will provide valuable information for us to understand tillering mechanism in switchgrass. Reverse genetics research using gene knockout mutants is a powerful tool to characterize gene function. CRISPR/Cas9-based gene editing tools have been wildly in various species. Homozygous targeted mutants could be obtained in one generation using this effective gene-editing tool. In this research, we established a CRISPR/Cas9-based gene editing system for switchgrass. A homozygous Phosphoglycerate Mutase (PGM) mutant was obtained in T0 generation in switchgrass. Using a single construct with two target sequences targeting conserved regions of Pvtb1a and Pvtb1b genes, primary mutants with targeted mutations were obtained at frequencies of 95.5% (Pvtb1a) and 11% (Pvtb1b). The mutant nature of Pvtb1 genes primary mutants was characterized applying Next Generation Sequencing. Two chimeric mutants (35-2 and 52-1) and one heterozygous mutant (97-2) were determined among the primary mutants. Solid mutants with various allelic compositions of Pvtb1 genes were successfully isolated from the chimeric primary mutants using micropropagation. Further, we obtained T1 progeny containing Cas9/gRNA-induced mutations from the primary mutants, which demonstrated that Cas9/gRNA-induced mutations could transmit to next generation in switchgrass. Additionally, through investigating the tiller numbers of these mutants and wild type plants, we found that Pvtb1 genes negatively control tiller production in switchgrass, where Pvtb1b plays a major role. Analysis of the transcriptomic profiling of mutant 52-1 and the wild type plant WT-1 revealed that Pvtb1 genes integrate multiple pathways to control tillering in switchgrass.
Liu, Yang, "Increase tiller production in switchgrass by CRISPR/Cas9" (2018). Graduate Theses and Dissertations. 17247.