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<p>Organelle genomes govern bioenergetics and stress responses, yet bulk sequencing masks heterogeneity. We establish single-organelle DNA-sequencing by adapting a droplet-based workflow to mitochondria and chloroplasts, using a sorbitol-based isolation that preserves organelles for encapsulation, amplification, and sorting. In <i>Arabidopsis thaliana</i> leaves, 313 of 384 single-organelle amplified genome libraries passed quality control (261 mitochondrial, 52 chloroplast). Genome coverage breadth at 1000 × sequencing depth showed medians of 59.5% and 84.5%, with maxima of 97.2% and 100%. Read-depth profiles were more fragmented for mitochondria and more uniform for chloroplasts, consistent with mitochondrial heterogeneity. This workflow enables analyses of recombination, rearrangements, and heteroplasmy.</p>

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Single-organelle DNA-sequencing of chloroplasts and mitochondria in Arabidopsis thaliana

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月26日

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  • Zikai Xiang,
  • Kazuki Takahashi,
  • Chang Zhou,
  • Hideki Takanashi,
  • Shin-ichi Arimura,
  • Masahito Hosokawa

摘要

Organelle genomes govern bioenergetics and stress responses, yet bulk sequencing masks heterogeneity. We establish single-organelle DNA-sequencing by adapting a droplet-based workflow to mitochondria and chloroplasts, using a sorbitol-based isolation that preserves organelles for encapsulation, amplification, and sorting. In Arabidopsis thaliana leaves, 313 of 384 single-organelle amplified genome libraries passed quality control (261 mitochondrial, 52 chloroplast). Genome coverage breadth at 1000 × sequencing depth showed medians of 59.5% and 84.5%, with maxima of 97.2% and 100%. Read-depth profiles were more fragmented for mitochondria and more uniform for chloroplasts, consistent with mitochondrial heterogeneity. This workflow enables analyses of recombination, rearrangements, and heteroplasmy.