STTR Phase II: Novel Analysis Tools for Production of Higher Indican Yielding Plants for Bio-based Indigo

这项小型企业技术转移（STTR）第二阶段项目将开发一种新的工具，用于分析靛蓝植物的原位籼稻前体，当与选择性培育的靛蓝作物的基因组分析和遗传连锁图谱相结合时，将产生高籼稻产量的育种亲本系，并最终获得具有价格竞争力的天然靛蓝染料。此外，通过了解和利用对籼稻合成和其他对靛蓝总产量有重要意义的方面至关重要的基因，鉴定遗传标记将加速进一步的作物改良。这些进步将使客户和牛仔布厂受益，因为它们将带来更可靠、更低成本的靛蓝原料供应。这个多阶段STTR项目的成功将使牛仔布染色过程具有成本竞争力，更清洁，更可持续，同时大大扩大国内生产的天然靛蓝的市场。商业化生产一种更稳定、产量更高的美国种植靛蓝植物，生产高纯度的靛蓝粉，可以取代目前标准的合成、进口靛蓝粉。虽然目前市场对植物衍生靛蓝的需求很大，但由于每英亩每株植物的产量低，每磅成本高，因此目前仅用于优质牛仔布产品。这项研究的直接结果将是为纺织行业开辟新的细分市场，扩大美国种植和制造的生物基靛蓝的现有市场渗透率，这是一个18.6亿美元的潜在市场。通过该项目开发的方法和技术可以直接进入商业市场，该行业已准备好支持生物基纺织染料，如植物衍生的靛蓝。在本项目中，将构建靛蓝原料作物的参考基因组资源，构建青花苜蓿、青花苜蓿和suffruticosa品种的F1图谱群体，并通过实验室分析验证手持式快速检测装置的设计。采用Pacific Biosciences公司的SMRT测序和组装技术构建参比基因组。除了这个参考基因组外，还将对现有的鹅毛苣苔和鹅毛苣苔品种进行全基因组重测序。对不同靛蓝品种间的核苷酸变异和变异效应进行预测。这一变异将用于开发评价品种间杂交的标记。结合已开发的资源，对3个品种进行温室控制杂交，形成F1定位群体，用于构建遗传图谱和连锁图谱。将选择具有明显的商业表型性状或显着的染料产量差异的种内品种进行杂交以产生F1群体。基于实验室的荧光测量设备的结果将评估功效，然后与从叶子生物量中提取的靛蓝染料分析进行比较。将根据这些发现构建最终原型，并通过在实验室和实地使用进行验证。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This Small Business Technology Transfer (STTR) Phase II project will develop a new tool for the analysis of in situ indican precursor in indigo plants, which when combined with genomic analysis and genetic linkage mapping in selectively bred indigo crops, will lead to high indican yielding breeding parental lines and ultimately competitively price natural indigo dye. Additionally, characterization of genetic markers will accelerate further crop improvement by understanding and harnessing the genes crucial to indican synthesis and other aspects of significance to overall indigo yield. These advancements will benefit customers, denim mills, by leading to a more reliable, lower cost plant-derived indigo supply. The success of this multiphase STTR project will enable a cost-competitive, cleaner, and more sustainable denim dyeing process, while greatly expanding the market for domestically produced natural indigo. Commercialization of a more consistent and higher yielding US-grown indigo plant that produces high purity indigo powder can replace the current standard of synthetic, imported indigo powder. While in demand by the marketplace today, plant-derived indigo is currently only used in premium denim products due to the high cost per pound resulting from low yields per plant per acre. The direct result of this research will be to open new market segments and expand existing market penetration for US-grown and manufactured biobased indigo for the textile industry, an addressable market of $1.86B. The methods and technology developed through this project have a direct path to the commercial marketplace and the industry is ready to support biobased textile dyes such as plant-derived indigo.During this project, reference genome resources will be constructed for indigo feedstock crops, F1 mapping populations will be constructed for P. tinctoria, I. tinctoria, and I. suffruticosa varieties, and design of the handheld rapid assay device will be validated through laboratory analysis. The reference genome for I. suffruticosa will be built using Pacific Biosciences SMRT sequencing and assembly. In addition to this reference genome, whole-genome resequencing will be performed on available I. suffruticosa and I. tinctoria varieties. Nucleotide variation and variant effect prediction will be made between the Indigofera-based indigo varieties. This variation will be used to develop markers to evaluate intervarietal crosses. Controlled greenhouse crosses of three species will be made to create F1 mapping populations for use in constructing a genetic map and linkage mapping, in combination with the resources developed. Intraspecies varieties exhibiting distinct phenotypic traits of commercial interest or notable dye yield differences will be selected for crossing to generate the F1 populations. Results from laboratory-based fluorometry equipment will be evaluated for efficacy and then compared against extractive indigo dye analysis from the leaf biomass. A final prototype will be constructed based on these findings and validated through use in the laboratory and in the field.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.