Rapid identification of mutations caused by fast neutron bombardment in Medicago truncatula

Fast neutron bombardment (FNB) is a very effective approach for mutagenesis and has been widely used in generating mutant libraries in many plant species. The main type of mutations of FNB mutants are deletions of DNA fragments ranging from few base pairs to several hundred kilobases, thus usually leading to the null mutation of genes. Despite its efficiency in mutagenesis, identification of the mutation sites is still challenging in many species. The traditional strategy of positional cloning is very effective in identifying the mutation but time-consuming. With the availability of genome sequences, the array-based comparative genomic hybridization (CGH) method has been developed to detect the mutation sites by comparing the signal intensities of probes between wild-type and mutant plants. Though CGH method is effective in detecting copy number variations (CNVs), the resolution and coverage of CGH probes are not adequate to identify mutations other than CNVs.

We report a new strategy and pipeline to sensitively identify the mutation sites of FNB mutants by combining deep-coverage whole-genome sequencing (WGS), polymorphism calling, and customized filtering in Medicago truncatula. Initially, we performed a bulked sequencing for a FNB white nodule (wn) mutant and its wild-type like plants derived from a backcross population. Following polymorphism calling and filtering, validation by manual check and Sanger sequencing, we identified that SymCRK is the causative gene of white nodule mutant. We also sequenced an individual FNB mutant yellow leaves 1 (yl1) and wild-type plant. We identified that ETHYLENE-DEPENDENT GRAVITROPISM-DEFICIENT AND YELLOW-GREEN 1 (EGY1) is the candidate gene for M. truncatula yl1 mutant.

Our results demonstrated that the method reported here is rather robust in identifying the mutation sites for FNB mutants.