Abstract
Key message
Recombinations between the parental genomes produced a novel mitochondrial genome in the cytoplasmic male sterile Brassica juncea cybrid Og1. A mitochondrial stoichiometric shift greatly reduced the molecule containing male-sterility-inducing orf138 gene leading to reversion to male fertility.
Abstract
An improved, chlorosis-corrected, cytoplasmic male sterile Brassica juncea cybrid Og1 derived from Ogura cytoplasm shows frequent reversion to male fertility. To determine the nature of mitochondrial recombination in the cybrid and to uncover the molecular mechanism of male fertility reversion, we sequenced the mitochondrial genomes of Og1, its isonuclear parental lines (OgRLM and Brassica juncea RLM198) and the revertant line (Og1-rt). Assembly of Og1 mitochondrial genome gave two circular molecules, Og1a (250.999 kbp) and Og1b (96.185 kbp) sharing two large direct repeat regions capable of recombining to form a single circular molecule. Og1a contains all essential mitochondrial genes, but the male-sterility-causing orf138 was uniquely present in Og1b along with 16 other complete or partial genes already represented in Og1a. Eleven and four recombinations between the parental mitochondrial genomes produced the Og1a and the Og1b molecules, respectively. Five genes were duplicated within Og1a, of which trnfM was inherited from both the parents while the other four genes, atp4, cox1 nad4L and trnM, were inherited from RLM198. RFLP analysis revealed that orf138-containing molecules were less abundant than Og1a in the male-sterile plants while og1b bearing molecules were undetectable in the revertant line. However, orf138 transcripts were amplified in RT-PCR and were also detected in northern blots revealing that Og1b molecules are not completely lost in the revertant plants. This is the first report where the mitochondrial genome of a cybrid is compared with its actual parents. The findings are discussed in the light of previous reports on mitochondrial genome recombination in cybrids.
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Availability of data and materials
The plant materials described here are available from the Director, ICAR-National Institute for Plant Biotechnology, New Delhi. The mitochondrial genome sequences have been deposited in NCBI under the following Accession Numbers. B. juncea cv. RLM198 (MT675103), OgRLM (MT675104) and Og1a, Og1b (MT675105 & MT675106).
Abbreviations
- CMS:
-
Cytoplasmic male sterility
- mt-DNA:
-
Mitochondrial DNA
- RFLP:
-
Restriction fragment length polymorphism
- RT-PCR:
-
Reverse transcription PCR
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VN received financial support from the Indian Council of Agricultural Research, New Delhi, India.
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VN—all lab experiments, mitochondrial genome assembly, annotation, preparation of draft manuscript, VK—mitochondrial DNA isolation, library preparation for sequencing, data analysis, RB—data analysis, financial resources, SRB—planning of experiments, plant material and financial resources, data analysis, manuscript writing and finalization. All authors have read and approved the final manuscript.
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Supplementary Information
Supplementary material 1 (DOCX 50 kb)
Supplementary Table S1 Details of primer sequences used in the study. Supplementary Table S2 Contig size and coverage of de novo assembled mitochondrial genome sequences of the male sterile line Og1 and the revertant, Og1-rt. Supplementary Table S3 Summary of PCR amplicons obtained in Og1 line with inverse primers designed from ends of different contigs of Og1. Supplementary Table S4 Summary of PCR amplicons obtained in og1-rt with inverse primers designed from ends of different contigs of Og1-rt. Supplementary Table S5 Summary of PCR amplicons obtained in Og1 with inverse primers designed from ends of different contigs of Og1 to confirm contig-flanking the repeat regions. Supplementary Table S6 Summary of PCR amplicons obtained in Og1-rt with inverse primers designed from ends of different contigs of Og1-rt to confirm contig-flanking the repeat regions. Supplementary Table S7 Details of repeats found in the mitochondrial genome of Og1 cybrid.
Supplementary material 2 (JPG 331 kb)
Supplementary Fig. S1 Alignment of ORF138 polypeptides of Brassica juncea CMS lines Og1, OgRLM and Ogura. Altered amino acids are highlighted in yellow.
Supplementary material 3 (TIF 519 kb)
Supplementary Fig. S2 Gel pictures showing PCR products obtained in Og1 using inverse primers from different contigs.
Supplementary material 4 (JPG 887 kb)
Supplementary Fig. S3 Pair-wise alignment of mitochondrial genome sequences of Brassica juncea cv. RLM198, OgRLM and Og1 showing co-linearity. Inverted regions are marked with blue colour. Numbers refer to nucleotide sequence.
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Vasupalli, N., Kumar, V., Bhattacharya, R. et al. Analysis of mitochondrial recombination in the male sterile Brassica juncea cybrid Og1 and identification of the molecular basis of fertility reversion. Plant Mol Biol 106, 109–122 (2021). https://doi.org/10.1007/s11103-021-01132-0
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DOI: https://doi.org/10.1007/s11103-021-01132-0