Department of Genetics, Washington University School of Medicine, Saint Louis, MO 63110, USA.
Department of Genetics, Washington University School of Medicine, Saint Louis, MO 63110, USA.
Plant Commun. 2023 Mar 13;4(2):100493. doi: 10.1016/j.xplc.2022.100493. Epub 2022 Nov 17.
Genomic assemblies of the unicellular green alga Chlamydomonas reinhardtii have provided important resources for researchers. However, assembly errors, large gaps, and unplaced scaffolds as well as strain-specific variants currently impede many types of analysis. By combining PacBio HiFi and Oxford Nanopore long-read technologies, we generated a de novo genome assembly for strain CC-5816, derived from crosses of strains CC-125 and CC-124. Multiple methods of evaluating genome completeness and base-pair error rate suggest that the final telomere-to-telomere assembly is highly accurate. The CC-5816 assembly enabled previously difficult analyses that include characterization of the 17 centromeres, rDNA arrays on three chromosomes, and 56 insertions of organellar DNA into the nuclear genome. Using Nanopore sequencing, we identified sites of cytosine (CpG) methylation, which are enriched at centromeres. We analyzed CRISPR-Cas9 insertional mutants in the PF23 gene. Two of the three alleles produced progeny that displayed patterns of meiotic inviability that suggested the presence of a chromosomal aberration. Mapping Nanopore reads from pf23-2 and pf23-3 onto the CC-5816 genome showed that these two strains each carry a translocation that was initiated at the PF23 gene locus on chromosome 11 and joined with chromosomes 5 or 3, respectively. The translocations were verified by demonstrating linkage between loci on the two translocated chromosomes in meiotic progeny. The three pf23 alleles display the expected short-cilia phenotype, and immunoblotting showed that pf23-2 lacks the PF23 protein. Our CC-5816 genome assembly will undoubtedly provide an important tool for the Chlamydomonas research community.
单细胞绿藻莱茵衣藻的基因组组装为研究人员提供了重要资源。然而，组装错误、大缺口、未定位的支架以及菌株特异性变体目前阻碍了许多类型的分析。通过结合 PacBio HiFi 和 Oxford Nanopore 长读技术，我们为源自 CC-125 和 CC-124 杂交的 CC-5816 菌株生成了从头基因组组装。多种评估基因组完整性和碱基对错误率的方法表明，最终的端粒到端粒组装非常准确。CC-5816 组装使以前难以进行的分析成为可能，包括 17 个着丝粒、三个染色体上的 rDNA 阵列以及 56 个细胞器 DNA 插入核基因组的分析。使用 Nanopore 测序，我们确定了胞嘧啶 (CpG) 甲基化的位点，这些位点在着丝粒处富集。我们分析了 PF23 基因的 CRISPR-Cas9 插入突变体。三个等位基因中的两个产生的后代显示出减数分裂不育的模式，这表明存在染色体异常。将 pf23-2 和 pf23-3 的 Nanopore 读数映射到 CC-5816 基因组上表明，这两个菌株各自携带一个易位，该易位始于 11 号染色体上的 PF23 基因座，并分别与 5 号或 3 号染色体连接。通过证明在减数分裂后代中两个易位染色体上的位点之间的连锁，验证了易位的存在。这三个 pf23 等位基因表现出预期的短纤毛表型，免疫印迹显示 pf23-2 缺乏 PF23 蛋白。我们的 CC-5816 基因组组装无疑将为衣藻研究社区提供一个重要的工具。
