Cytoplasmic male sterility (CMS) and seedlessness are highly desirable traits for citrus. However, a high-quality reference mitochondrial genome of citrus is unavailable, and the mechanisms of CMS remain to be elucidated in citrus. We previously generated a male sterile somatic cybrid of pummelo (G1+HBP) by fusing protoplasts from CMS ‘Guoqing No. 1’ Satsuma mandarin (G1) and fertile ‘Hirado Buntan’ pummelo (HBP). Here, we sequenced and assembled the mitochondrial genome of G1, G1+HBP, and HBP into circular molecules of 521.6 kb, 538.4 kb, and 518.3 kb, respectively. Mitochondrial genome of citrus encodes 35–36 proteins, three ribosomal RNAs, and 21 tRNAs. Comparative analysis indicated that nuclear genome and chloroplast genome of the cybrid are from HBP, whereas the mitochondrial genome sequences of the cybrid and G1 have 96.9% synteny, and that the cybrid has an extra recombinant mitochondrial DNA fragment of only 16.8 kb. Compared with the fertile HBP mitochondrial genome, 21 and 20 CMS-specific mitotype-specific sequences (MSSs), and 89 and 70 CMS-specific ORFs were identified in the cybrid and G1 mitochondrial genomes, respectively. Among them, 57 ORFs that co-transcribed with the same mitochondrial genes in the cybrid and G1 were identified as the CMS-specific ORFs. The assembly of mitochondrial genomes and the identification of the CMS-specific ORFs provide basis for determination of the CMS-associated gene and elucidation of CMS mechanisms in citrus.

细胞质的雄性不育（CMS）和无核是柑橘的理想特性。然而，柑橘的高质量参考线粒体基因组是不可用的，CMS的机制尚待阐明。我们以前通过融合CMS'国庆1号'萨摩柑（G1）和可育的'Hirado Buntan'柚（HBP）的原生质体，生成了一个雄性不育的柚子混合体（G1 + HBP）。在这里，我们对G1，G1 + HBP和HBP的线粒体基因组进行了测序和组装，分别形成了521.6 kb，538.4 kb和518.3 kb的环状分子。柑橘的线粒体基因组编码35-36个蛋白质，三个核糖体RNA和21个tRNA。比较分析表明，杂种的核基因组和叶绿体基因组均来自HBP，而杂种和G1的线粒体基因组序列具有96.9％的同义性，并且该杂种具有仅16.8 kb的额外重组线粒体DNA片段。与可育的HBP线粒体基因组相比，在杂种和G1线粒体基因组中分别鉴定出21和20个CMS特异性线型特异性序列（MSS），以及89和70个CMS特异性线粒体特异性ORF。其中，与cybrid和G1中的相同线粒体基因共转录的57个ORF被确定为CMS特异性ORF。线粒体基因组的组装和CMS特定ORF的鉴定为确定CMS相关基因和阐明柑橘中CMS机制提供了基础。在混合和线粒体G1基因组中分别鉴定出89和70种CMS特异的ORF。其中，与cybrid和G1中的相同线粒体基因共转录的57个ORF被确定为CMS特异性ORF。线粒体基因组的组装和CMS特定ORF的鉴定为确定CMS相关基因和阐明柑橘中CMS机制提供了基础。在混合和线粒体G1基因组中分别鉴定出89和70种CMS特异的ORF。其中，与cybrid和G1中的相同线粒体基因共转录的57个ORF被确定为CMS特异性ORF。线粒体基因组的组装和CMS特定ORF的鉴定为确定CMS相关基因和阐明柑橘中CMS机制提供了基础。