IOS EDGE: Development of genetic and genomic resources for milkweed, Asclepias syriaca and Asclepias curassavica

乳草植物以其与帝王蝶的标志性互动而闻名。虽然乳草产生的毒素对大多数昆虫都是致命的，但帝王蝶毛虫的抵抗力很强，甚至会储存乳草毒素来防御鸟类等掠食者。正如马利筋属的名字所表明的那样，Asclepias来自希腊的治疗之神Asclepius，美洲原住民使用这些植物来治疗各种医疗疾病。几项研究表明，乳草是新化合物的丰富来源，不仅可以防御昆虫，而且还具有潜在的医学应用。在这个项目中，研究人员将开发新的资源来研究普通乳草和热带乳草中特定基因的功能。将开发以靶向方式敲除或过表达特定马利筋基因的方法。两种乳草属植物的基因组测序，以及乳草属植物基因表达和化学成分的自然变异，将有助于发现与防御食草动物和生产医学相关的植物代谢产物有关的新基因。将通过该项目开发的资源将在产生时公开提供，没有限制其使用的限制。该项目还将对高中生和大学生暑期实习生进行培训，为他们将来在工业、学术界或政府部门从事研究工作做好准备。为了进一步开发马利筋和热带马利筋作为研究模型系统，需要改进研究单个基因功能的工具。在该项目中，将开发乳草特异性方案用于：（i）使用农杆菌渗透的瞬时基因表达，（ii）病毒诱导的内源基因表达沉默，（iii）使用CRISPR/Cas9的靶向诱变，（iv）组织培养中的稳定转化，（v）从愈伤组织再生植物，以及（vi）乳草基因组的遗传修饰。为了便于马利筋基因的功能分析，A。syriaca和A. curassavica基因组将使用太平洋生物科学DNA测序组装。将使用经典遗传图谱和BioNano光学图谱对组装的重叠群进行排序并分配至特定染色体。乳草基因将通过计算机比较和来自不同组织类型的RNA的深度测序来识别和注释。基因型A. syriaca和A.库拉索芦荟遗传作图群体将使得能够对在组成谱系中变化的任何表型或分子性状进行定量性状基因座作图。植物基因表达和代谢物含量在不同组织类型中以及响应环境刺激而显著变化。特别地，昆虫攻击诱导产生多种生物活性植物代谢物。6个A. syriaca和A. curassavica组织类型，有和没有食草动物的处理，将提供这些物种的遗传和代谢潜力的广泛概述。分子协议，基因组序列，转录组数据，以及相关分析连接转录组，代谢组，和其他马利筋表型将显示在项目网站上，www.milkweedbase.net。

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.