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Do Maize Transposable Elements Induce Gene Amplification?

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Do Maize Transposable Elements Induce Gene Amplification? ASA, CSSA, and CSA International Annual Meeting November 5, 2018 Department of Genetics, Development, and Cell Biology, Department of Agronomy Marjorie Hanneman, Sharu Paul Sharma, and Dr. Thomas Peterson Do Maize Transposable Elements Induce Gene Amplification? Abstract: Materials and Methods: Results: As the agricultural industry introduces and increases use of herbicides, weeds • Kernels with small spots were planted and seedling DNA was extracted. Figure 5 shows a Southern blot using Kpn1 digestion and hybridization with a probe develop resistance to the chemicals. An incredibly adaptive weed, Palmer • PCR was performed using primers designed to test junctions of Ac and from the p1 sequence near the Ac element. The B54 allele has two bands, while 353 amaranth, has attained strong resistance to glyphosate through the flanking DNA has 3 bands of different sizes, one of which appears to be doubled in intensity amplification of the EPSPS gene (Gaines et al. 2010; Koo et al. 2018). EPSPS amplification could possibly be induced by Transposable Elements, however • PCR products were analyzed by gel electrophoresis and DNA (arrows). This suggests that sequences flanking Ac are also amplified. the exact mechanism has not been found. Transposons were first discovered by sequencing. Sequences were analyzed by BLAST and aligned to the Barbara McClintock by studying variances in maize kernel phenotypes. The Ac progenitor p1-wwB54 allele. element has a negative dosage effect, meaning that increasing copies of Ac produce fewer transposition events. In this research, the Ac negative dosage 353 B54 effect is used as a visual indicator for amplification of Ac copy number, Southern Blots: possibly by a rolling circle replication as proposed by Zhang et al. 2014. From • Genomic DNA was digested with restriction enzymes, run on agarose the p1-wwB54 allele that contains a single copy of Ac, 97 ears were screened B54 for kernels with tiny spots indicating high Ac dosage. These kernels were gels, transferred to membranes and hybridized with radioactive probes planted, DNA was extracted from the seedlings and then utilized in PCR and a 15 b Southern Blot Hybridizations to map the gene structure and determine Ac copy number. The results will be presented and are expected to provide new insight into the mechanism of transposon-induced gene amplification. Results: 11,495 bp Figure 5 Background: A B Ears of p1-wwB54 were screened and kernels with small C D purple spots were planted and analyzed. Selection of B54 Structure Conclusions: kernels with small spots should enrich for events with Results to date indicate that Ac and flanking sequences are repeated at least twice in 11,00 bp multiple copies of Ac. Figure 1: B54 allele the 353 allele. A working model structure is shown below and will be further tested. Figure 2: Small spotted kernels on 353 Determining the complete structure of the amplified region should help to explain its Colorless 353 Partial Structure A C D B origin. The results may reveal novel mechanisms of gene amplification. A C D B Figure 3 Plants Large Figure 3: Top portion shows structure of progenitor p1-wwB54 allele, with Agronomic Application: Spot Single transposable elements Ac and fAc (arrows), and insertion site AB. Lower Spots Gene dosage of 5-enolpyruvoylshikimate-3-phosphate portion shows structure of small-spot 353 allele with insertion at AB, and synthase, or EPSPS, in Palmer Amaranth has led to glyphosate inversion of sequences between C to A, and B to D. resistance. This may possibly be induced by Transposable C A B D Elements. The mechanism for multiple copies of Ac in a Figure 1 Figure 2 this project could be applied to herbicide resistance. B54 Structure Hypothesis: Acknowledgements: This research is supported by the USDA National Institute of Food and Because of the Ac negative dosage effect, we expect that 353 Partial Structure Agriculture Hatch project number IOW05282, and by State of Iowa funds. We thank Terry Olson Figure 4 for technical assistance kernels with small spots will have multiple copies of Ac as Figure 4: In the 353 allele, Ac is located adjacent to a new sequence Gaines TA, et al. (2010) Gene amplification confers glyphosate resistance in Amaranthus palmeri. Proc Natl Acad Sci USA 107:1029– shown below. Possible mechanisms for this are 1034. (yellow box) located 11.5 kb downstream of the original Ac location. Ac Koo et al. 2018 Extrachromosomal circular DNA-based amplification and transmission of herbicide resistance in crop weed Amaranthus hypothesized to include inversions and rolling circle palmeri. Proc Natl Acad Sci USA 115: 3332-3337 Ac Ac Ac was also found adjacent to sequence B, indicating that there are at least two Natural Resources Conservation Service. (n.d.). Retrieved from tandem repeats. copies of Ac present in the 353 allele. https://www.nrcs.usda.gov/wps/portal/nrcs/ia/technical/ecoscience/invasive/palmer amaranth/ Acknowledgements Zhang, J., Zuo, T., wang, D., and Peterson, T. 2014. Transposition-mediated DNA re-replication in maize. eLife 2014;3:e03724.
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