Genetic studies of phenotypic variants in the woodland strawberry, (Fragaria vesca)
The diploid woodland strawberry (Fragaria vesca) is a rapidly developing translational model for members of the family Rosaceae and other plants. This thesis represents some of the first forward genetics studies evaluating putative T-DNA insertional mutants in F. vesca. The observed phenotypes include alterations to floral development, anthocyanin pigmentation and leaf structure.
The floral development mutant named green petal (gp) was not associated with the T-DNA insertions present. Based on similar phenotypes induced by mutation of transcription factors involved in floral development of Arabidopsis thaliana, we used a BLAST search of the F. vesca genome hybrid gene models to identify 30 candidate genes that may have caused the gp phenotype. Expression analysis of these genes revealed that it was due to a 37 bp deletion in a SEPALLATA3-like E-Class MADS box transcription factor. This mutation altered organ structure in the three inner whorls of the flower, affecting fertility and fruit development. The deletion was demonstrated to segregate with the mutant phenotype in a segregating population of 92 individuals, 22 of which had green petals.
The anthocyanin biosynthesis mutant named white runner (wr) lacked red pigmentation in the stems and runners. The T-DNA insertion in this line was located in a highly repetitive LTR retrotransposon region, which complicated analysis. Segregation analysis of the wr lines revealed that the phenotype was unassociated with the T-DNA insertion as well. We used a targeted expression analysis of three critical structural genes in the flavonoid biosynthesis pathway that revealed a 20 bp deletion in the gene encoding flavanone 3-hydroxylase, an enzyme necessary for the production of flavonols, anthocyanins and proanthocyanidins. In an F2 segregating population, this deletion co-segregated with the phenotype.
The third mutant line presented here displayed a curly leaf (cl) phenotype and was found to harbor a T-DNA insertion in a gene encoding a putative erythroblast macrophage attacher protein (EMP). Sequence and protein domain analysis indicated that FvEMP was related to the mammalian EMP protein that functions in cytoskeletal dynamics and red blood cell enucleation. Complementation analysis confirmed that introduction of the wild type FvEMP gene into the cl mutant plants restored wild type leaf phenotype. Further morphological analysis revealed additional pleiotropic effects of the mutation, including abnormalities in seed set and germination, pollen tube growth, adhesion of the abaxial epidermal layer to the mesophyll layer and reduced petiolule length. These phenotypes are consistent with actin binding and microtubule associated protein mutants in other plant species.
Insertional mutagenesis is a critical molecular tool for model crop development. These studies highlight the precautions that must be taken when evaluating insertional mutants. These mutants are excellent tools for studying their respective disrupted gene function. The in depth molecular analysis of the mutants presented in this work was only possible because of the availability of the Fragaria vesca genome which was used extensively to identify T-DNA insertion sites and recover candidate gene sequences for expression analysis.