Developing molecular genetic tools for gene discovery in soybean (glycine max)

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Soybean (Glycine max (L.) Merr.) is one of the most valuable crops grown worldwide for seed protein and oil production and can fix atmospheric nitrogen. The completed genome sequence and the availability of transcriptomics and proteomics data for genomic and physiological studies have made soybean a feasible model legume plant. The soybean genome is large (~978Mb) and highly duplicated, with ~75% of predicted genes present in multiple copies, and 50% of these paralogs are sub-functionalized. In addition, the relatively low genetic diversity in soybean relative to other major crops (e.g., corn) limits the available phenotypic variations that can be exploited for discovering genes, gene function and crop improvement. Thus, developing a feasible, efficient molecular genetic tool for gene discovery in soybean is required. In this dissertation, along with fast neutron mutagenesis, a dual gRNA CRISPR/Cas9 genome editing system was developed as a molecular genetic tool for gene discovery and crop improvement in soybean. In order to demonstrate the efficiency and ability to simultaneous by edit homeologous genes using a CRISPR/Cas9 system in soybean, GmFAD2 genes were chosen as targets for testing. Our CRISPR/Cas9 system showed 100% of T0 transgenic plants, obtained using the cotyledonary node transformation method, harbored induced mutations in either or both GmFAD2 genes. Importantly, 40% of the T0 plants harbored homozygous or biallelic mutations in both genes and these mutations were heritable in T1 and T2 progenies. Seeds derived from double homozygous mutant plants showed a typical high oleic acid (83.3%) phenotype compared to wild-type seeds (20.2%). In addition to generating new alleles for crop improvement, the use of CRISPR/Cas9 in conjunction with fast neutron mutagenesis allowed the identification of causative genes for observed fast neutron-induced phenotypes. Through forward genetics, we identified a deleted DNA fragment in chromosome 17 that co-segregated with the production of big seeds and leaves) in the fast neutron mutant line K83. CRISPR/Cas9 gene editing system was employed to unveil the function of GmKIX1 in controlling seed size in soybean. Loss of function gmkix1 soybean plants obtained by CRISPR/Cas9 mutagenesis phenocopied the seed and leaf phenotypes of mutant K83. In addition to editing genes associated with above-ground traits through stable transformation, this work also demonstrated the efficacy of CRISPR/Cas9 system in mutating genes associated with root traits when coupled with hairy root transformation. Using GmUOX1 and GmXHD1 as targets, we were able to induce homozygous or biallelic mutations in 59% and 69% of transgenic roots, respectively. GmUOX1 and GmXHD1 encode enzymes involved in ureide biosynthesis, the form of fixed nitrogen that is transported from root nodules to other parts of the plant. Characterization of nodule development in roots carrying mutations in GmUOX1, obtained using fast neutron and CRISPR/Cas9 mutagenesis, and GmXDH1, obtained by CRISPR/Cas9 editing, indicated a critical role of the ureide biosynthetic pathway not only in incorporation of fixed nitrogen but in the successful development of functional, nitrogen fixing nodules. Results from these studies showed that the dual-gRNA CRISPR/Cas9 system developed from this dissertation indeed offers a rapid and highly efficient genetic tool to discover unknown genes and molecular mechanisms underlying important agronomic traits such as drought tolerance, yield capacity and seed quality in soybean