Project description:About one third of all angiosperm species produce flowers with petals fused into a corolla tube. As an important element of the tremendous diversity of flower morphology, the corolla tube plays a critical role in many specialized interactions between plants and animal pollinators (e.g., beeflies, hawkmoths, hummingbirds, nectar bats), which in turn drives rapid plant speciation. Despite its clear significance in plant reproduction and evolution, the corolla tube remains one of the least understood plant structures from a developmental genetics perspective. Through mutant analyses and transgenic experiments, here we show that the tasiRNA-ARF pathway is required for corolla tube formation in the monkeyflower species Mimulus lewisii. Loss-of-function mutations in the M. lewisii orthologs of ARGONAUTE7 and SUPRESSOR OF GENE SILENCING 3 cause a dramatic decrease in tasiARF abundance and a moderate up-regulation of Auxin Response Factor 3 (ARF3) and ARF4, which lead to inhibition of lateral expansion of the bases of petal primordia and complete arrest of the upward growth of the inter-primordial regions, resulting in unfused corollas. Integrating our molecular and phenotypic analyses of the tasiRNA-ARF pathway in Mimulus with historical insights from morphological and anatomical studies in various sympetalous species, we propose a new conceptual model for the developmental genetic control of corolla tube formation.
Project description:Phenotype-driven forward genetic experiments are among the most powerful approaches for linking biology and disease to genomic elements. Although widely used in a range of model organisms, positional cloning of causal variants is still a very laborious process. Here, we describe a novel universal approach, named fast forward genetics that combines traditional bulk segregant techniques with next-generation sequencing technology and targeted genomic enrichment, to dramatically improve the process of mapping and cloning multiple mutants in a single experiment. In a two-step procedure the mutation is first roughly mapped by ‘light’ sequencing of the bulk segregant pool, followed by genomic enrichment and deep-sequencing of the mutant pool for the linked genomic region. The latter step allows for simultaneous fine-mapping and mutation discovery. We successfully applied this approach to three Arabidopsis mutants, but the method can in principle be applied to any model organism of interest and is largely independent of the genome size. Moreover, we show that both steps can be performed in multiplex using barcoded samples, thereby increasing efficiency enormously. Inducible overexpression of the RETINOBLASTOMA-RELATED (RBR-OE) gene in Arabidopsis roots causes the complete differentiation of stem cells and premature differentiation of daughter cells, leading to a full exhaustion of the primary root meristem. In order to identify regulators of RBR function in cell differentiation, RBR-OE plants in the Columbia background (Col0) were treated with EMS mutagenesis and a set of genetic suppressors of RBR-OE, which restores root growth capacity, were isolated. In this study, we used one the identified suppressor lines, which segregated as a recessive mutation. Mapping populations were generated by outcrossing to Ler ecotype. Seedlings from the F2 population were grown for 15 days post germination (dpg). A pool of 60 seedlings each with a clear suppressor phenotype (homozygous for suppressor mutation) and of 60 seedlings showing RBOE phenotype (Heterozygous for the suppressor mutation) were prepared and genomic DNA was isolated with the RNeasy Plant Mini Kit from QIAGEN according to manufacturer's protocol. The other two, mutants 136 and 193 were obtained in fluorescence based mutant screen and a QCmarker based mutagenesis, respectively. Mutants were generated by chemical mutagenesis (EMS) in Colombia (Col) genetic background. Mutants were subsequently crossed to the Landsberg (Ler) ecotype to create the mapping populations. Bulk-segregant pools of about 200 mutant as well as wild-type plants were generated for every mutant line.