Aegilops squarrosa L. organellar-enriched DNA sequencing, assembly, and comparative genomics

Proper interactions between the nucleus and cytoplasmic organelles (mitochondria and plastids) are essential to eukaryotic cellular function. To improve our understanding of the role of organellar genomes and nuclear-cytoplasmic interactions in plant development and stress response, our first aim is to survey organellar genome diversity in wheat and across the broader Triticum-Aegilops complex. This will be followed by work to assess genome dynamics across developmental stages as well as during abiotic and biotic stress response. The results of this work will be important for improving crop traits. To accomplish our goals, it was critical to first establish improved methods for the isolation, sequencing, and assembly of organellar genomes from limited starting material without whole genome amplification. As a proof of concept, we optimized our methods using the Triticum aestivum cv. Chinese Spring, for which there is previous sequencing data available. The mitochondria and chloroplast genomes have large repeats (upto 10kb and 20kb in length, respectively). Previous studies have performed whole genome amplification and have manually stitched contigs to force a single master circle configuration of the organellar genomes, which may or may not reflect the true native state of the wheat organellar genomes. To resolve the long repeats and perform de novo assemblies without whole genome amplification and manual stitching of contigs, we utilized low input PacBio 20kb library preparations to generate long sequencing reads. In total, we sequenced 20 organellar-enriched samples with PacBio, including 13 diverse wild species, T. durum, T. aestivum cv. Chinese Spring, and three wheat alloplasmic lines. In addition we generated Illumina short-read sequences for many additional cultivars, wild species, and alloplasmic lines. This project includes data for one of these samples (Aegilops squarrosa L.). Raw sequencing reads are deposited here. Assemblies and annotations will be included once available.

细胞核与细胞质细胞器（线粒体和质体）之间的正常互作，对真核细胞的功能至关重要。为加深我们对细胞器基因组以及核质互作在植物发育与胁迫响应中作用的理解，本研究的首要目标是调查小麦以及更广范围的小麦-山羊草（Triticum-Aegilops）复合群中的细胞器基因组多样性。后续将开展工作，评估不同发育阶段以及非生物和生物胁迫响应过程中的基因组动态变化。本研究成果对作物性状改良具有重要意义。为达成研究目标，首先需建立优化方法，以在无需全基因组扩增的前提下，从有限起始材料中分离、测序并组装细胞器基因组。作为概念验证，我们以普通小麦（Triticum aestivum cv. Chinese Spring）——已有公开测序数据的材料——优化了实验方法。线粒体与叶绿体基因组均存在大片段重复序列（长度分别可达10kb和20kb）。既往研究曾采用全基因组扩增手段，并通过人工拼接重叠群以强行生成细胞器基因组的单一主环构型，而该构型未必能反映小麦细胞器基因组真实的天然状态。为解析长重复序列并在无需全基因组扩增与人工拼接重叠群的前提下完成从头组装，我们采用低起始量PacBio 20kb文库制备方案以生成长读长测序数据。我们总计利用PacBio测序了20份细胞器富集样本，涵盖13份不同的野生种质、硬粒小麦（T. durum）、普通小麦品种中国春，以及3份小麦异质细胞质系。此外，我们还为大量额外的栽培品种、野生种质与异质细胞质系生成了Illumina短读长测序数据。本项目包含其中一份样本（节节麦Aegilops squarrosa L.）的相关数据。原始测序读段已存档于此。组装结果与注释信息将在完成后公开。