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

马利筋植物因其与帝王蝶的标志性互动而闻名。虽然马利筋产生的毒素对大多数昆虫都是致命的，但帝王蝶毛虫却很有抵抗力，甚至会储存马利筋毒素来防御鸟类等捕食者。正如乳草属名称Asclepias所表明的那样，它来源于希腊的治疗之神Asclepius，美洲原住民用这些植物来治疗各种各样的疾病。一些研究表明，乳草是新化合物的丰富来源，不仅提供防御昆虫，而且有潜在的医疗应用。在本项目中，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表达特定乳草基因的方法。对两种乳草物种的基因组测序，以及乳草基因表达和化学含量的自然变异，将有助于发现参与抵御草食和产生医学相关植物代谢物的新基因。将通过该项目开发的资源将在产生时向公众提供，对其使用没有任何限制。该项目还将培训高中生和本科生暑期实习生，为他们未来在工业界、学术界或政府部门的研究工作做好准备。将叙利亚乳草（Asclepias syriaca）和热带乳草（Asclepias curassavica）作为研究模型系统的进一步发展将需要改进研究单个基因功能的工具。在这个项目中，将为乳草制定特定的方案：(i)使用农杆菌浸润的瞬时基因表达，（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)愈伤组织再生植株，以及（vi）乳草基因组的可遗传修饰。为了便于马利筋基因的功能分析，将使用太平洋生物科学公司的DNA测序技术组装A. syriaca和A. curassavica的基因组。使用经典遗传图谱和BioNano光学图谱，组装的contigs将被排序并分配到特定的染色体上。马利筋基因将通过硅比较和来自不同组织类型的RNA深度测序来鉴定和注释。基因分型的叙利亚和curassavica遗传作图群体将使任何表型或分子性状在组成谱系中变化的定量性状位点作图成为可能。植物基因表达和代谢物含量在不同的组织类型和环境刺激下变化很大。特别是，昆虫的攻击诱导了多种生物活性植物代谢物的产生。对六种叙利亚和curassavica组织类型进行转录组学和代谢组学分析，在有无草食处理的情况下，将为这些物种的遗传和代谢潜力提供一个广泛的概述。分子协议、基因组序列、转录组数据以及连接转录组、代谢组和其他乳草表型的相关分析将显示在项目网站www.milkweedbase.net上。

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.