Browsing by Author "Wisser, Randall J."
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Item Characterization of integration sites and transfer DNA structures in Agrobacterium-mediated transgenic events of maize inbred B104(G3: Genes | Genomes | Genetics, 2023-07-31) Neelakandan, Anjanasree K.; Kabahuma, Mercy; Yang, Qin; Lopez, Miriam; Wisser, Randall J.; Balint-Kurti, Peter; Lauter, NickIn maize, the community-standard transformant line B104 is a useful model for dissecting features of transfer DNA (T-DNA) integration due to its compatibility with Agrobacterium-mediated transformation and the availability of its genome sequence. Knowledge of transgene integration sites permits the analysis of the genomic environment that governs the strength of gene expression and phenotypic effects due to the disruption of an endogenous gene or regulatory element. In this study, we optimized a fusion primer and nested integrated PCR (FPNI-PCR) technique for T-DNA detection in maize to characterize the integration sites of 89 T-DNA insertions in 81 transformant lines. T-DNA insertions preferentially occurred in gene-rich regions and regions distant from centromeres. Integration junctions with and without microhomologous sequences as well as junctions with de novo sequences were detected. Sequence analysis of integration junctions indicated that T-DNA was incorporated via the error-prone repair pathways of nonhomologous (predominantly) and microhomology-mediated (minor) end-joining. This report provides a quantitative assessment of Agrobacterium-mediated T-DNA integration in maize with respect to insertion site features, the genomic distribution of T-DNA incorporation, and the mechanisms of integration. It also demonstrates the utility of the FPNI-PCR technique, which can be adapted to any species of interest.Item Multiple physiological mechanisms involved in reproduction could be targeted for breeding heat tolerance in lima bean(Crop Science, 2024-01-24) Ernest, Emmalea; Wisser, Randall J.With global warming, the impact of high-temperature stress on crop production is one of the major issues facing agriculture. Combining a series of field and controlled environment experiments, the current study aimed at understanding causes of yield loss due to heat stress in lima bean (Phaseolus lunatus L.) in order to guide breeding for heat tolerance. A growth-stage analysis of multi-year field trials suggested that yields were reduced by sensitivity to heat during floral transition. High nighttime temperatures during this period were also associated with delayed pod set and subsequent harvest. These effects were validated in controlled environments, with additional tests revealing how nighttime heat stress generally reduces seeds per pod but can also reduce the number of pods set. Using an intra-plant dual temperature treatment design for vegetative and reproductive organs, it was determined that effects on pollen viability and release are primary factors underlying heat stress-associated yield losses in lima bean. Elevated nighttime temperatures did not reduce aboveground biomass, indicating the physiological basis for temperature sensitivity was not driven by a photosynthate deficit, but instead could be attributed to changes in source–sink dynamics. Still, the basis for sensitivity to heat differed among genotypes, opening new targets for a multi-mechanistic breeding approach for heat tolerance. Abbreviations BFB ‘Bush Florida Butter’ CelSel ‘C-elite Select’ FAO Food and Agriculture Organization FH242 ‘Fordhook 242’ GDD growing degree days HSI heat susceptibility index PGM pollen germination medium UAN urea ammonium nitrateItem ThermoAlign: a genome-aware primer design tool for tiled amplicon resequencing(Nature Publishing Group, 2017-03-16) Francis, Felix; Dumas, Michael D.; Wisser, Randall J.; Felix Francis, Michael D. Dumas & Randall J. Wisser; Francis, Felix; Dumas, Michael D.; Wisser, Randall J.Isolating and sequencing specific regions in a genome is a cornerstone of molecular biology. This has been facilitated by computationally encoding the thermodynamics of DNA hybridization for automated design of hybridization and priming oligonucleotides. However, the repetitive composition of genomes challenges the identification of target-specific oligonucleotides, which limits genetics and genomics research on many species. Here, a tool called ThermoAlign was developed that ensures the design of target-specific primer pairs for DNA amplification. This is achieved by evaluating the thermodynamics of hybridization for full-length oligonucleotide-template alignments — thermoalignments — across the genome to identify primers predicted to bind specifically to the target site. For amplificationbased resequencing of regions that cannot be amplified by a single primer pair, a directed graph analysis method is used to identify minimum amplicon tiling paths. Laboratory validation by standard and long-range polymerase chain reaction and amplicon resequencing with maize, one of the most repetitive genomes sequenced to date (≈85% repeat content), demonstrated the specificity-by-design functionality of ThermoAlign. ThermoAlign is released under an open source license and bundled in a dependency-free container for wide distribution. It is anticipated that this tool will facilitate multiple applications in genetics and genomics and be useful in the workflow of high-throughput targeted resequencing studies.