Aegilops squarrosa typica KU20-2 alloplasmic 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 typica KU20-1 alloplasmic). Raw sequencing reads are deposited here. Assemblies and annotations will be included once available.

核质及细胞质细胞器（如线粒体和质体）之间的恰当交互对于真核细胞功能至关重要。为增进我们对细胞器基因组以及核质相互作用在植物发育与胁迫响应中所起作用的理解，我们的首要目标是调查小麦及其更广泛的 Triticum-Aegilops 复合群体中的细胞器基因组多样性。此研究将继之以对基因组在发育阶段及非生物和生物胁迫响应过程中的动态变化进行评估。此项研究的结果对于改良作物性状具有重要意义。为实现我们的目标，首先建立从有限的起始材料中分离、测序和组装细胞器基因组的方法，且无需全基因组扩增，显得尤为关键。作为概念验证，我们利用具有先前测序数据的小麦品种 Triticum aestivum cv. Chinese Spring 对方法进行了优化。线粒体和叶绿体基因组存在大量重复序列（长度分别为至10kb和20kb）。先前研究已进行全基因组扩增并手动拼接连续序列，以强制形成细胞器基因组的单一主环状构型，但这可能或不一定反映小麦细胞器基因组的真实原生状态。为解决长重复序列并无需全基因组扩增和手动拼接连续序列进行从头组装，我们利用低输入 PacBio 20kb 库制备方法生成长测序读段。总计，我们使用 PacBio 对20个细胞器富集样本进行了测序，包括13个多样化的野生种、T. durum、T. aestivum cv. Chinese Spring 以及三个小麦异源质体系。此外，我们还为许多其他品种、野生种和异源质体系生成了 Illumina 短读段序列。本项目中包含这些样本之一（Aegilops squarrosa typica KU20-1 异源质体）。原始测序读段已存档于此。一旦汇编和注释完成，将包含在内。