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

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 speltoides). Raw sequencing reads are deposited here. Assemblies and annotations will be included once available.

细胞核与细胞质细胞器（线粒体与质体）的正常互作，对真核细胞的功能发挥至关重要。为加深我们对细胞器基因组（organellar genome）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研小麦以及更广范围的小麦-山羊草复合群（Triticum-Aegilops complex）中的细胞器基因组多样性。后续将开展研究，评估不同发育阶段以及非生物、生物胁迫响应过程中的基因组动态变化。本研究成果对于作物性状改良具有重要意义。为实现上述研究目标，首先需建立一套适用于有限起始材料、无需全基因组扩增（whole genome amplification）的细胞器基因组分离、测序与组装优化方法，该步骤是完成研究的关键前提。作为概念验证，我们以已有公开测序数据的普通小麦中国春（Triticum aestivum cv. Chinese Spring）为材料优化了实验方法。线粒体与叶绿体基因组均携带大型重复序列，其长度分别可达10kb与20kb。既往相关研究通过全基因组扩增与手动拼接重叠群（contig），强行将细胞器基因组组装为单一主环构型，但该构型未必能反映小麦细胞器基因组的真实天然状态。为解析长重复序列，并在无需全基因组扩增与手动拼接重叠群的前提下完成从头组装（de novo assembly），我们采用低起始量PacBio 20kb文库制备策略以生成长读长测序数据。本研究共对20份细胞器富集样本开展PacBio测序，样本涵盖13份不同的野生种质、硬粒小麦（Triticum durum）、普通小麦中国春以及3份小麦异质细胞质系（alloplasmic line）。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 speltoides）的测序数据。原始测序reads已在此处存档上传。组装结果与注释信息将在完成后同步上传公开。