Setaria viridis CO2 compensation-point mutant genome sequencing

Wild-type Setaria seeds were treated with N-nitroso-N-methylurea (NMU) and grown to obtain M1 plants. Seeds were harvested from each individual plant to generate M2 lines; each line was assigned a unique four-digit mutant line number (NM000 to NM08880). Seedlings from the M3 generation were screened for altered CO2 compensation points. Plants were exposed to low carbon dioxide (CO2 ) concentrations in custom-made growth chambers designed to create a closed system for rapid screening of CO2 compensation points. Further, maximum quantum efficiency of PSII photochemistry (Fv /Fm) was also measured, and any mutant plant with a reduced Fv/Fm compared with its siblings and the WT Setaria were classified as a reduced Fv /Fm candidate. Candidate mutant plants that produced viable seed were advanced to the M4 generation. A mutant plant was classified as a candidate if the decline in the F v /F m at low CO 2 was greater than the candidate threshold. This approach enabled mutant plants with an increased sensitivity to low CO 2 to be identified. In all generations, progeny where ~50% or more of the sibling were classified as candidates were prioritised and advanced to the next generation. For each mutant line the single best performing progeny (most responsive to low CO 2 and able to produce viable seed) was selected. For further details about mutant identification, see Coe et al. Funct Plant Biol . 2018 Oct;45(10):1017-1025.Finally three best mutants were selected: NM04534, NM03677 and NM03966. In order to discover the underlying gene (function) which was mutated, the mutants were backcrossed and selfed to obtain segregating populations (BC1F2). The genomic DNA of segregants with similar phenotype were bulked, and were subjected to Whole genome shotgun sequencing.