The Development of Genetic Resources and Methods for Performing Crosses in Setaria viridis - A New Model System for the Grasses

Setaria viridis is an emerging model genetic system for C₄ grasses. However, current challenges include the need for efficient methods of performing crosses and breaking seed dormancy, and to establish a rich diversity of genetic resources. Here we provide the first standard protocol for performing crosses in S. viridis A10.1, by using a heat treatment with warm water for emasculation. After pruning the panicle to 20-30 florets and labeling of flowers, a pre-dawn cold treatment was given to promote uniform flower opening for pollination. After testing a series of heat treatments we have established an optimum temperature and time of 48 °C for 5 min. Using this method, a minimum of 15 crosses can be performed by a single worker per day and an average of 3-5 outcross progeny per panicle can be recovered. We have initiated the construction of six RIL populations by single seed decent and will begin back crosses to obtain NIL populations. We have also developed a method to break seed dormancy, by keeping wrapped seeds in wet Sphagnum moss at 4 ºC for 2-3 weeks, which can achieve a  germination rate at 50-80% in  A10.1 after 2 weeks and 85%-90% after 3 weeks, while the control was 1%-5%. Finally, we have produced 3000 NMU mutant families and are further characterizing a subset of the population. We have also obtained a collection of about 390 natural diverse Setaria accessions in collaboration, some of which have been characterized for phenotypic traits and propagated for seed distribution.