Mutator Transposable Element Causes Double Flowers in Thalictrum

Erin Y. Cot, Kelsey D. Galimba, Vernica S. Di Stilio University of Washington Department of Biology
The ABC Model
The ABC model describes the interaction of three classes of transcription factors that determine floral organ identity. Transcription factors activate or deactivate other genes, which means that they can jump-start a cascade of developmental effects in an organism. The floral organ identity genes are typically turned on early in flower development, resulting in the formation of specific floral organs. The ABC model was based on a hermaphroditic flower with four whorls of organs. A-class genes
alone cause the

Results
We hypothesized that a mutation in the C-class gene AG1 would be a likely cause of the double phenotype. Having not found a coding sequence difference we investigated the genomic sequence of this gene, especially focusing on Intron 1 and 2 which contain many regulatory motifs found in the Arabadopsis thaliana C-class gene AGAMOUS.

Proposed Evolutionary Scenario

Shoafs Double Genomic AG1

formation of sepals. Their Interactions with B class genes lead to the development of petals. B and C class genes together form stamens. The innermost whorl contains the carpels, which result from C class genes alone; lack of C class gene function results in double flowers, where all stamens and carpels are replaced by petals.1

Double Phenotype in Thalictrum

Double flowers were the first floral mutants ever described piquing the curiosity of botanists from Ancient China and Greece over 2,000 years ago. This project focuses on the Thalictrum thalictroides mutants Cameo and Shoafs Double. The double mutant Double White has been previously characterized and has an insertion in the coding sequence of the C-class gene AG1 which causes a non-functional protein.2

The entire AG1 gene in Shoafs Double is 7 kb larger than in wild type. The extra sequence belongs to a DNA transposon in the Mutator family. Mutator elements are characterized by having a 9 bp target site duplication (TSD) and long terminal inverted repeats of 100 to 200 bp which we found in the element above. It also contains sequence which codes for transposase, an enzyme used by this DNA element to jump around the genome, suggesting that this is an active element. In Intron 2 Shoafs Double is missing two regulatory motifs, one CArG box and a LFY binding site which could also cause the double phenotype.

Cameo Genomic AG1

We hypothesize two possible scenarios for the origin of the double flower cultivars. In Shoafs Double either the large insert disrupts the interaction of the AAGAAT boxes it is inserted between or the missing CArG box causes a disruption in cis regulation (CArG boxes are sites of DNA binding for MADS box proteins). We propose that the element jumped into a shared ancestor of Shoafs Double and Cameo. Once this element largely disrupted the function of the AG1 gene, other mutations accumulated, including the one that nulled the CArG box in Intron 2. The two cultivars continued to diverge from one another and eventually the element jumped out of Intron 1 in Cameo, taking some of the gene with it and leaving a footprint deletion. Finally, another mutation disrupted the LFY binding site in Intron 2 of Shoafs Double.

Future Work
We intend to investigate expression of this transposable element using RT-PCR on cDNA. We also intend to develop a transposon based mutagenesis to generate novel flower mutants. Cameo, is missing a 200 bp stretch of DNA in intron 1, in the exact same position as the Shoafs Double insertion. We interpret this as a footprint of Mutator element excision in this mutant. Cameo is also missing the same CArG box as Shoafs Double in Intron 2.

References
1.Causier, B., Schwarz-Sommer, Z. & Davies, B. Floral organ identity: 20 years of ABCs. Semin. Cell Dev. Biol. 21, 7379 (2010). 2.Galimba, K. D. et al. Loss of deeply conserved C-class floral homeotic gene function and C- and E-class protein interaction in a double-flowered ranunculid mutant. Proc. Natl. Acad. Sci. 109, E2267E2275 (2012).

a. Thalictrum thalictroides c. T. thalictroides Shoafs Wild Type Double b. T. thalictroides Double White d. T. thalictroides Cameo