Improving Lignocellulose Saccharification by High Throughput Mutant Screening and Large Scale Mutant Re-Sequencing

To achieve cost efficient conversion of lignocellulosic biomass into biofuels, basic knowledge on the genes that control cell wall recalcitrance and enhanced saccharification is needed. We are using rice, a tractable model grass species, to identify genes involved cell wall biosynthesis and modification and degradation. As part of this project, we generated two fast-neutron mutant populations of 100,000 lines and screened screen 5,000 M2 lines from 1,592 M1 plants using a high throughput rice lignocellulose saccharification assay. For the screening, AIR (alcohol insoluble residue) samples were prepared from the mature rice stem and the saccharification assays were done with a high throughput digestibility platform. From this screen, we identified 154 mutant candidates with altered saccharification efficiency. Of these, 53 mutant candidates showed increased saccharification efficiency and 101 mutant candidates showed reduced saccharification efficiency. One of the mutants, rcs60, was further characterized. The rcs60 mutant segregated for reduced saccharification efficiency in the M2 generation. The rcs60 progeny were dwarfed and impaired in fertility.

We have also initiated a whole genome sequencing project of 2000 of the fast neutron mutants in collaboration with the Joint Genome Institue (JGI). The purpose of this project is to create a publicly available resource that will facilitate identification of genes governing key biological processes in grasses. To date we have re-sequenced 47 lines and data are being analyzed. Our results on rcs60 mutant characterization and our whole genome sequencing strategy to identify other mutants altered in cell wall biosynthesis and modification will be presented.