Project Methods Methods: (1) The identity of collected species will be accomplished using recently developed taxonomic keys and by microsatellite PCR. Examine candidate gene function (apomeiosis, parthenogenesis and autonomous or pseudogamous endosperm formation) in Boechera, Arabidopsis and sorghum (milestone, Jun 2018 deliverables, transgenic plants expressing apomixis or apomixis-like developmental processes) Verify gene expression in ovules and endosperm of 16-20 species and select candidate genes and gene networks (milestone, Dec 2017 deliverables, evidence-based rationale for proceeding to candidate gene analyses) 7. Compare ovule and endosperm mRNA and sRNA of sexual and apomictic Boechera (milestone, Dec 2017 deliverables, evidence for the genetic and epigenetic causes of the differences identified in 4 above) 6. Obtain deep sequence data for small and large RNAs during early ovule development and during early endosperm formation for sexual and apomictic Boechera (milestone, Dec, 2016 deliverables, large database identifying differences in gene expression during seed formation between sexual and apomictic Boechera) 5. Compare DNA and histone methylation levels in eggs and central cells of the 16-20 sexual and apomictic Boechera species (milestone, Dec 2016 deliverables, knowledge base concerning possible differences between apomicts and sexuals in DNA methylation of eggs and central cells) 4. Screen seeds of the apomictic Boechera for tolerance to exceptional maternal to paternal (MP) genome ratios in endosperm and for autonomous endosperm formation (milestone, Jun 2016 deliverables, verified modes of endosperm formation including possible departures from normal that could be of agronomic value) 3. Collect 8-10 putatively sexual diploid and 8-10 putatively apomictic diploid Boechera species and verify their reproductive mode (milestone, Dec 2015 deliverables, seed and living plant collections of verified sexual and apomictic Boechera) 2. Goals / Objectives Goals and objectives: 1. Functional analyses of identified candidate genes will then be conducted by up and down regulating them transgenically in model species and in crop plants, the results of which may enable the engineering of crops that possess the agronomic impacts of apomixis as outlined above (milestone, Jun 2018). These studies will provide information concerning the putative roles of genetics and epigenetics in the evolution and regulation of apomixis (milestone, Dec, 2017). Genetic and epigenetic mechanisms of apomictic reproduction will be evaluated by sequencing small and large RNA pools from appropriately staged ovules of sexual and apomictic species and by identifying consistent differences among modes of reproduction in these RNA. Mode of reproduction will be evaluated cytologically by analysis of at least 50 appropriately staged ovules per species, to determine mode of embryo formation (milestone, Dec 2015), and by flow cytometry of at least 200 seeds per species, to determine mode of endosperm formation (milestone, Jun 2016). Methods and evaluation plan: Eight to 10 species of putatively sexual and putatively apomictic Boechera will be collected. This could make hybrid seed production feasible for essentially all crops. A second goal is to engineer crops with genes that may induce apomixis so that hybrids of these crops pass their hybrid vigor on through succeeding seed generations. One of our goals is to elucidate the molecular mechanisms of apomixis in wild plants. Apomixis does not occur in major world crops. These plants are essentially high-yielding natural hybrids that pass hybrid vigor on to succeeding generations by asexual seed formation (apomixis). Certain wild plants possess this capacity, including blackberries, dandelions, St John's wart, crab apples and Kentucky bluegrass. Such seed could be produced commercially if these plants were engineered to produce their seed clonally. Additionally, the floral anatomy of wheat and rice prevents most seed companies from commercially producing hybrid seed of these crops. Hence, seed companies must produce hybrid seed every year, a process costing approximately $1 billion annually in the U.S. In crops that are grown as hybrids, such as corn, high yields occur in the first season only. Production of hybrid seed, which could close this gap, is not economically feasible for most major world crops including rice and wheat. Yet yields for wheat and rice remain 15-30% below their biological potentials even when grown in ideal soils and climates using ideal irrigation and cultivation practices. Non Technical Summary Need for the project: Grains, principally rice and wheat, provide the majority of calories in the diets of people worldwide, ranging from 23% in the U.S.
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