Organelle genome assembly uncovers the dynamic genome reorganization and cytoplasmic male sterility associated genes in tomato

Abstract

To identify cytoplasmic male sterility (CMS)-associated genes in tomato, we determined the genome sequences of mitochondria and chloroplasts in three CMS tomato lines derived from independent asymmetric cell fusions, their nuclear and cytoplasmic donors, and male fertile weedy cultivated tomato and wild relatives. The structures of the CMS mitochondrial genomes were highly divergent from those of the nuclear and cytoplasmic donors, and genes of the donors were mixed up in these genomes. On the other hand, the structures of CMS chloroplast genomes were moderately conserved across the donors, but CMS chloroplast genes were unexpectedly likely derived from the nuclear donors. Comparative analysis of the structures and contents of organelle genes and transcriptome analysis identified three genes that were uniquely present in the CMS lines, but not in the donor or fertile lines. RNA-sequencing analysis indicated that these three genes transcriptionally expressed in anther, and identified different RNA editing levels in one gene, orf265, that was partially similar to ATP synthase subunit 8, between fertile and sterile lines. The orf265 was a highly potential candidate for CMS-associated gene. This study suggests that organelle reorganization mechanisms after cell fusion events differ between mitochondria and chloroplasts, and provides insight into the development of new F1 hybrid breeding programs employing the CMS system in tomato.

References

[1] Bohra, A., Jha, U. C., Adhimoolam, P., Bisht, D. & Singh, N. P. Cytoplasmic male sterility (CMS) in hybrid breeding in field crops. Plant Cell Rep. 35, 967-93 (2016).

[2] Petrova, M. et al. Characterisation of a cytoplasmic male-sterile hybrid line between Lycopersicon peruvianum Mill. ×Lycopersicon pennellii Corr. and its crosses with cultivated tomato. Theor. Appl. Genet. 98, 825-830 (1999).

[3] Sandhu, A. P., Abdelnoor, R. V. & Mackenzie, S. A. Transgenic induction of mitochondrial rearrangements for cytoplasmic male sterility in crop plants. Proc. Natl Acad. Sci. USA 104, 1766-70 (2007).

[4] Melchers, G., Mohri, Y., Watanabe, K., Wakabayashi, S. & Harada, K. One-step generation of cytoplasmic male sterility by fusion of mitochondrial-inactivated tomato protoplasts with nuclear-inactivated Solanum protoplasts. Proc. Natl Acad. Sci. USA 89, 6832-6 (1992).

[5] Shikanai, T., Kaneko, H., Nakata, S., Harada, K. & Watanabe, K. Mitochondrial genome structure of a cytoplasmic hybrid between tomato and wild potato. Plant Cell Rep. 17, 832-836 (1998).

[6] Shikanai, T., Nakata, S., Harada, K. & Watanabe, K. Analysis of the heterogeneous transcripts of the highly edited orf206 in tomato mitochondria. Plant Cell Physiol. 37, 692-6 (1996).

[7] Chen, L. & Liu, Y. G. Male sterility and fertility restoration in crops. Annu Rev. Plant Biol. 65, 579-606 (2014).

[8] Igarashi, K., Kazama, T., Motomura, K. & Toriyama, K. Whole genomic sequencing of RT98 mitochondria derived from Oryza rufipogon and north- ern blot analysis to uncover a cytoplasmic male sterility-associated gene. Plant Cell Physiol. 54, 237-43 (2013).

[9] Okazaki, M., Kazama, T., Murata, H., Motomura, K. & Toriyama, K. Whole mitochondrial genome sequencing and transcriptional analysis to uncover an RT102-type cytoplasmic male sterility-associated candidate Gene Derived from Oryza rufipogon. Plant Cell Physiol. 54, 1560-8 (2013).

[10] Makarenko, M. S. et al. Characterization of the mitochondrial genome of the MAX1 type of cytoplasmic male-sterile sunflower. BMC Plant Biol. 19, 51 (2019).

[11] Li, S. et al. The comparison of four mitochondrial genomes reveals cytoplasmic male sterility candidate genes in cotton. BMC Genomics 19, 775 (2018).

[12] Wu, Z. et al. Mitochondrial genome and transcriptome analysis of five allo- plasmic male-sterile lines in Brassica juncea. BMC Genomics 20, 348 (2019).

[13] Wang, Z. et al. Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing. Plant Cell 18, 676-87 (2006).

[14] Yang, J., Liu, X., Yang, X. & Zhang, M. Mitochondrially-targeted expression of a cytoplasmic male sterility-associated orf220 gene causes male sterility in Brassica juncea. BMC Plant Biol. 10, 231 (2010).

[15] Kazama, T. et al. Curing cytoplasmic male sterility via TALEN-mediated mito- chondrial genome editing. Nat. Plants 5, 722-730 (2019).

[16] Arimura, S. I. et al. Targeted gene disruption of ATP synthases 6-1 and 6-2 in the mitochondrial genome of Arabidopsis thaliana by mitoTALENs. Plant J. 104, 1459-1471 (2020).

[17] Harada, K., Watabe, K. & Kondo, K. A method for producing a tomato plant with restored fertility. Japanese Patent No. 3386292.(2003).

[18] Bonnema, A. B., Melzer, J. M. & O’Connell, M. A. Tomato cybrids with mito- chondrial DNA from Lycopersicon pennelli. Theor. Appl Genet 81, 339-48 (1991).

[19] Sidorov, V. A., Menczel, L., Nagy, F. & Maliga, P. Chloroplast transfer in Nicotiana based on metabolic complementation between irradiated and iodoacetate treated protoplasts. Planta 152, 341-5, https://doi.org/10.1007/BF00388259 (1981).

[20] Morgan, A. & Maliga, P. Rapid chloroplast segregation and recombination of mitochondrial DNA in Brassica cybrids. Mol. Gen. Genet 209, 240-6 (1987).

[21] Touzet, P. & Meyer, E. H. Cytoplasmic male sterility and mitochondrial meta- bolism in plants. Mitochondrion 19 Pt B, 166-71 (2014).

[22] Fujii, S., Kazama, T., Yamada, M. & Toriyama, K. Discovery of global genomic re- organization based on comparison of two newly sequenced rice mitochon- drial genomes with cytoplasmic male sterility-related genes. BMC Genomics 11, 209 (2010).

[23] Shinada, T., Kikuchi, Y., Fujimoto, R. & Kishitani, S. An alloplasmic male-sterile line of Brassica oleracea harboring the mitochondria from Diplotaxis muralis expresses a novel chimeric open reading frame, orf72. Plant Cell Physiol. 47, 549-53 (2006).

[24] Fujii, S. & Small, I. The evolution of RNA editing and pentatricopeptide repeat genes. N. Phytol. 191, 37-47 (2011).

[25] Takei, H. et al. De novo genome assembly of two tomato ancestors, solanum pimpinellifolium and S. lycopersicum var. cerasiforme, by long-read sequen- cing. DNA Res. https://doi.org/10.1093/dnares/dsaa029 (2021).

[26] Scott, J.W. & Harbaugh, B.K. Micro-tom: A Miniature Dwarf Tomato (Agricultural Experiment Station, Institute of Food and Agricultural Sciences, University of Florida, 1989).

[27] Soorni, A., Haak, D., Zaitlin, D. & Bombarely, A. Organelle_PBA, a pipeline for assembling chloroplast and mitochondrial genomes from PacBio DNA sequencing data. BMC Genomics 18, 49 (2017).

[28] Koren, S. et al. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 27, 722-736 (2017).

[29] Kurtz, S. et al. Versatile and open software for comparing large genomes. Genome Biol. 5, R12 (2004).

[30] Fu, L., Niu, B., Zhu, Z., Wu, S. & Li, W. CD-HIT: accelerated for clustering the next- generation sequencing data. Bioinformatics 28, 3150-2 (2012).

[31] Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. L. Predicting trans- membrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305, 567-80 (2001).

[32] Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389-402 (1997).

[33] Chen, S., Zhou, Y., Chen, Y. & Gu, J. fastp: an ultra-fast all-in-one FASTQ pre- processor. Bioinformatics 34, i884-i890 (2018).

[34] Pertea, M., Kim, D., Pertea, G. M., Leek, J. T. & Salzberg, S. L. Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ball- gown. Nat. Protoc. 11, 1650-67 (2016).

[35] Bairoch, A. & Apweiler, R. The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000. Nucleic Acids Res. 28, 45-8 (2000).

[36] Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357- 9 (2012).

[37] Danecek, P. et al. Twelve years of SAMtools and BCFtools. Gigascience 10, https://doi.org/10.1093/gigascience/giab008 (2021).