Identification of a gene responsible for seedpod spine formation and other phenotypic alterations using whole-genome sequencing analysis in Medicago truncatula.

In nature, some plant species produce seedpods with spines, which is an adaptive biological trait for protecting the seed and helping seed dispersal. However, the molecular mechanism of spine formation is still unclear. While conducting routine tissue culture and transformation in the model legume Medicago truncatula, we identified a smooth seedpod (ssp1) mutant with a suite of other phenotypic changes. Preliminary analysis showed that the mutation was derived from the tissue culture process. Genetic segregation analysis suggested that ssp1 is a recessive mutant. By combining whole-genome sequencing and bioinformatics analysis, we found that the mutant phenotype was caused by a single nucleotide polymorphism and a 30 bp deletion in the gene locus Medtr4g039430, named SSP1. Complementation of the M. truncatula ssp1 and Arabidopsis twd1 mutants showed complete restoration, indicating that SSP1 is an ortholog of Arabidopsis TWD1 which encodes an immunophilin-like FK506-binding protein 42. The formation of spines on seedpods is associated with auxin transport. The method used in this study offers an effective way for detecting genes responsible for somaclonal variations. The results demonstrate, for the first time, that SSP1 plays a crucial role in the determination of spine formation on seedpods.