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

乳草植物以其与帝王蝴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，但帝王蝶毛虫具有很强的抵抗力，甚至储存乳草毒素来防御鸟类等捕食者。正如源自希腊治愈之神Asclepius的乳草属名称Asclepias所表明的那样，美洲原住民使用这些植物来治疗各种医疗疾病。多项研究表明，乳草含有丰富的新型化合物，不仅可以防御昆虫，还具有潜在的医疗应用价值。在这个项目中，研究人员将开发新的资源来研究普通马利筋和热带马利筋中特定基因的功能。将开发有针对性地敲除或过度表达特定乳草基因的方法。两种马利筋的基因组测序，加上马利筋基因表达和化学含量的自然变异，将使人们能够发现与防御草食性疾病和产生医学上相关的植物代谢物有关的新基因。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。该项目还将培训高中生和本科生暑期实习生，这将为他们未来在工业、学术界或政府部门的研究职业生涯做好准备。将马尾藻(Asclepias Syriaca)和热带马尾藻(Asclepias Cursavica)进一步发展为研究模型系统，将需要改进工具来研究单个基因的功能。在这个项目中，将开发针对乳草的操作规程：(I)利用农杆菌渗透进行瞬时基因表达，(Ii)病毒诱导的内源基因表达沉默，(Iii)使用CRISPR/Cas9进行靶向突变，(Iv)在组织培养中稳定转化，(V)从愈伤组织中再生植株，以及(Vi)对乳草基因组进行可遗传的修饰。为了方便对马利筋基因的功能分析，将使用太平洋生物科学公司的DNA测序来组装马利筋和马利筋的基因组。组装的重叠群将被排序，并使用经典遗传图谱和生物纳米光学图谱分配到特定的染色体。乳草基因将通过电子比较和对不同组织类型的RNA进行深度测序来识别和注释。黑曲霉和红曲霉的基因定位群体将能够对组成谱系中不同的任何表型或分子性状进行定量的性状基因座定位。植物的基因表达和代谢物含量在不同的组织类型和对环境刺激的反应中有很大的差异。特别是，虫害会诱导产生各种生物活性的植物代谢物。对六种不同组织类型的黑麦草和红曲霉进行转录和代谢组学分析，无论是否进行草食处理，都将提供对这些物种遗传和代谢潜力的广泛概述。分子方案、基因组序列、转录组数据以及将转录组、代谢组和其他马利筋表型联系起来的相关分析将在该项目网站www.milkweedbase.net上展示。

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.