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Identification of Genes Required for Male and Female Gametophyte Function in Maize

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Identification of Genes Required for Male and Female Gametophyte Function in Maize

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    1. Sierra Wolfenbarger John Fowler Rex Cole Identification of Genes Required for Male and Female Gametophyte Function in Maize

    2. Like most plants it completes an “alternation of generation” Diploid to Haploid to Diploid Haploid stage is called the gametophyte stage (e.g.: pollen and embryo sac) Maize Life Cycle

    3. Corn is a model organism and discoveries could relate to other plants Maize is one of the top three food crops in the world With a better understanding of the fertilization process, higher yields from this crop could be pursued, resulting in more corn for everyone Why Study Maize Gametophytes

    4. Haploid gametophytes require active gene functions Pollination and Fertilization

    5. Not much is known about gametophyte genes Due to haploidy, mutations in active genes are often lethal Impossible to study if mutation is not heritable Genes needed for Gametophyte

    6. Rescuing the Gene, part 1

    7. Rescuing the Gene, part 2

    8. The trisomes monitored by kernel marker Example translucent kernel Rescuing the Gene, part 3

    9. An Activator transposon (Ac) is used to disrupt the coding sequence of genes Ac is monitored by a separate marker Example purple spotting Creating the Mutations

    10. The Ac can duplicate, creating a new transposon insertion monitored by spotting phenotype of the kernels New mutation potentially interesting Creating the Mutation

    11. Isolate and identify newly found gametophyte genes Adapt TAIL-PCR protocol to Maize Gather more data to support gametophyte mutant phenotypes Verify plant genotypes using PCR Perform crosses with mutants to confirm effect on gametophytes Focus for Summer

    12. The lab has already recovered a number of mutations (duplicated Ac’s), with trisomes present These plants were crossed with a *tester line, and then 2Ac + trisome seeds were selected by phenotype First Step (completed)

    13. These trisome +2Ac seeds are a set of putative gametophyte mutants The progeny of these seeds are evaluated to see if a transmission phenotype is present Based on ratios of kernel phenotype Second Step (completed)

    14. Categories of the Screening Process

    15. Categories of the Screening Process

    16. Categories of the Screening Process

    17. Putative gametophyte mutants were planted 3 with trisome 9s and 7 with trisome 1L Experiment: Cross PCR-verified plants to confirm mutant effects on gametophytes Third Step (in progress)

    18. Plants are checked to make sure they contain trisome Polymerase Chain Reaction (PCR) Verification of Genotype

    19. Trisome PCR

    20. Loss of Trisome

    21. Only 2 of 10 mutant lines contain the trisome Loss of Trisome

    22. Recombination

    23. Identify the mutant gene using Thermal Asymmetric Interlaced PCR (TAIL-PCR) Adapt procedure to maize Ac TAIL-PCR uses both nested Ac-specific primers and degenerate arbitrary primers alternating high and low annealing temperature cycles Fourth Step (early stages)

    24. Second step of TAIL-PCR (TAIL-2) Uses diluted DNA from 1st Tail reaction (TAIL-1) with nested Ac primer TAIL-PCR

    25. Gel from TAIL2-PCR

    26. TAIL-2 Product Sequence Analysis

    27. Mapping of Sequence

    28. Mapping of Sequence

    29. Trisome confirmed in 2 of 10 mutant lines Tester crosses made with these 2 lines Future: harvest and sort seeds to confirm mutant transmission ratios Summary

    30. Trisome confirmed in 2 of 10 mutant lines Tester crosses made with these 2 lines Future: harvest and sort seeds to confirm mutant transmission ratios TAIL-PCR protocol adapted to Maize Mapped 2 new transposon sites using TAIL-PCR Future: additional TAIL-PCR to find Ac’s of interest (from 2 gametophyte mutant lines) Summary

    31. Trisome confirmed in 2 of 10 mutant lines Tester crosses made with these 2 lines Future: harvest and sort seeds to confirm mutant transmission ratios TAIL-PCR protocol adapted to Maize (Ac-TAIL) Mapped 2 new transposon sites using Ac-TAIL Future: additional TAIL PCR to find Ac’s of interest (from 2 gametophyte mutant lines) OVERALL: Ac-TAIL will be very useful as more gametophyte mutants are found Summary

    32. Howard Hughes Medical Institute National Science Foundation John Fowler Kevin Ahern Fowler Lab: Rex Cole Zuzana Vejlupkova Maria Ivanchenko Chintan Joshi Nathan Synder Luisa Snyder Acknowledgements

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