Milkweed plants are well-known for their iconic interactions with monarch butterflies. Although milkweeds produce toxins that are lethal to most insects, monarch butterfly caterpillars are quite resistant and even store milkweed toxins for their own defense against predators like birds. As is indicated by the milkweed genus name, Asclepias, which is derived from Asclepius, the Greek god of healing, Native Americans used these plants to treat a variety of medical ailments. Several studies have shown that milkweeds are rich sources of novel chemical compounds that not only provide defense against insects, but also have potential medical applications. In this project the investigators will develop new resources to investigate the function of specific genes in both common milkweed and tropical milkweed. Methods will be developed to knock out or overexpress specific milkweed genes in a targeted manner. Genome sequencing of two milkweed species, together with natural variation in milkweed gene expression and chemical content, will enable the discovery of new genes involved in defense against herbivory and the production of medically relevant plant metabolites. The resources that will be developed through this project will be made publicly available as they are being generated, with no restrictions limiting their use. The project also will involve training high school students and undergraduate summer interns, which will prepare them for future research careers in industry, academia, or government service.
The further development of Asclepias syriaca (common milkweed) and Asclepias curassavica (tropical milkweed) as research model systems will require improved tools for studying the functions of individual genes. In this project, milkweed-specific protocols will be developed for: (i) transient gene expression using Agrobacterium infiltration, (ii) virus-induced silencing of endogenous gene expression, (iii) targeted mutagenesis using CRISPR/Cas9, (iv) stable transformation in tissue culture, (v) regeneration of plants from callus, and (vi) heritable modification of the milkweed genome. To facilitate functional analysis of milkweed genes, the A. syriaca and A. curassavica genomes will be assembled using Pacific Biosciences DNA sequencing. Assembled contigs will be ordered and assigned to specific chromosomes using classical genetic maps and BioNano optical maps. Milkweed genes will be identified and annotated through in silico comparisons and deep sequencing of RNA from different tissue types. Genotyped A. syriaca and A. curassavica genetic mapping populations will enable quantitate trait locus mapping of any phenotypic or molecular trait that varies in the constituent lineages. Plant gene expression and metabolite content varies dramatically in different tissue types and in response to environmental stimuli. In particular, insect attack induces the production of a wide variety of biologically active plant metabolites. Transcriptomic and metabolomic analysis of six A. syriaca and A. curassavica tissue types, with and without herbivory treatment, will provide a broad overview of the genetic and metabolic potential of these species. Molecular protocols, genome sequences, transcriptome data, and correlative analyses linking the transcriptomes, metabolomes, and other milkweed phenotypes will be displayed on the project website, www.milkweedbase.net.
