PMAT: an efficient plant mitogenome assembly toolkit using low-coverage HiFi sequencing data

Abstract

Complete mitochondrial genomes (mitogenomes) of plants are valuable resources for nucleocytoplasmic interactions, plant evolution, and plant cytoplasmic male sterile line breeding. However, the complete assembly of plant mitogenomes is challenging due to frequent recombination events and horizontal gene transfers. Previous studies have adopted Illumina, PacBio, and Nanopore sequencing data to assemble plant mitogenomes, but the poor assembly completeness, low sequencing accuracy, and high cost limit the sampling capacity. Here, we present an efficient assembly toolkit (PMAT) for de novo assembly of plant mitogenomes using low-coverage HiFi sequencing data. PMAT has been applied to the de novo assembly of 13 broadly representative plant mitogenomes, outperforming existing organelle genome assemblers in terms of assembly accuracy and completeness. By evaluating the assembly of plant mitogenomes from different sequencing data, it was confirmed that PMAT only requires 1 × HiFi sequencing data to obtain a complete plant mitogenome. The source code for PMAT is available at https://github.com/bichangwei/PMAT. The developed PMAT toolkit will indeed accelerate the understanding of evolutionary variation and breeding application of plant mitogenomes.

Acknowledgements

The work was supported by the National Key Research and Development Plan of China (2021YFD2200202) and the Key Research and Development Project of Jiangsu Province, China (BE2021366). The work was also supported by the Natural Science Foundation of Jiangsu Province (BK20220414), the Natural Science Foundation of the Higher Education Institutions of Jiangsu Province (22KJB220003), the National Natural Science Foundation of China (31901331), and the Innovation Program of Chinese Academy of Agricultural Sciences. We thank Dr Weishu Fan from Kunming Institute of Botany for providing the sample of Lycopodium japonicum.

Author contributions

C.B., F.S., and F.H. planned and designed the research. C.B., F.H., Y.Q., and W.H. wrote the code and processed the data. J.H., K.X., and L.X. provided the materials used in this study. C.B. and F.H. analyzed the data and prepared the figures. C.B. wrote the initial version of the manuscript; F.S., Z.W., and T.Y. revised and provided comments. Z.W. and T.Y. supervised the project.

Data availability

The PacBio HiFi sequencing data of L. japonicum, P. trichocarpa, and S. wilsonii have been submitted to the NCBI Sequence Read Archive (SRA) repository under SRR24785435, SRR3204721, and SRR21570388, respectively. The Illumina sequencing data of the purified mtDNA of P. trichocarpa and M. domestica have been deposited in the SRA repository under SRR24785916 and SRR24789033, respectively. Other datasets used in this study were downloaded from GSA and SRA repositories with the accession numbers listed in Table 1. All de novo assembled and annotated mitogenomes have been submitted to the NCBI Nucleotide Database (https://www.ncbi.nlm.nih.gov/nuccore/) with the accession numbers listed in Table 1. The scripts of PMAT are available at https://github.com/bichangwei/PMAT.

Conflict of interest

The authors declare no conflicts of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Arrieta-Montiel MP, Mackenzie SA. Plant mitochondrial genomes and recombination. In: Kempken F,ed. Plant Mitochondria. New York, NY: Springer, 2011,65-82

[2] Moller IM, Rasmusson AG, Van Aken O. Plant mitochondria - past, present and future. Plant J. 2021; 108 :912-59

[3] Gong J, Chen Y, Xu Y. et al. Tracking organelle activities through efficient and stable root genetic transformation system in woody plants. Hortic Res. 2023;uhad262

[4] Jiang N, Feng M-Q, Cheng L-C. et al. Spatiotemporal profiles of gene activity in stamen delineate nucleo-cytoplasmic interaction in a male-sterile somatic cybrid citrus. Hortic Res. 2023; 10 :uhad105

[5] Logan DC. Plant mitochondrial dynamics. Biochim Biophys Acta. 2006; 1763 :430-41

[6] Wu ZQ, Liao XZ, Zhang XN. et al. Genomic architectural variation of plant mitochondria—a review of multichromosomal structuring. J Syst Evol. 2022; 60 :160-8

[7] Arimura SI. Fission and fusion of plant mitochondria, and genome maintenance. Plant Physiol. 2018; 176 :152-61

[8] Bi C, Qu Y, Hou J. et al. Deciphering the multi-chromosomal mitochondrial genome of Populus simonii. Front Plant Sci. 2022; 13 :914635

[9] Christensen AC. Plant mitochondrial genome evolution can be explained by DNA repair mechanisms. Genome Biol Evol. 2013; 5 :1079-86

[10] Nosek J, Tomaska L. Mitochondrial genome diversity: evolution of the molecular architecture and replication strategy. Curr Genet. 2003; 44 :73-84

[11] Sloan DB. One ring to rule them all? Genome sequencing provides new insights into the ‘master circle’ model of plant mitochondrial DNA structure. New Phytol. 2013; 200 :978-85

[12] Kim CK, Kim YK. The multipartite mitochondrial genome of Fallopia multiflora (Caryophyllales: Polygonaceae). Mitochondrial DNA B Resour. 2018; 3 :155-6

[13] Sloan DB, Alverson AJ, Chuckalovcak JP. et al. Rapid evolution of enormous, multichromosomal genomes in flowering plant mitochondria with exceptionally high mutation rates. PLoS Biol. 2012; 10 :e1001241

[14] Varre JS, D’Agostino N, Touzet P. et al. Complete sequence, multichromosomal architecture and transcriptome analysis of the Solanum tuberosum mitochondrial genome. Int J Mol Sci. 2019; 20 :4788

[15] Alverson AJ, Rice DW, Dickinson S. et al. Origins and recombination of the bacterial-sized multichromosomal mitochondrial genome of cucumber. Plant Cell. 2011; 23 :2499-513

[16] Wang S, Li D, Yao X. et al. Evolution and diversification of kiwifruit mitogenomes through extensive whole-genome rearrangement and mosaic loss of intergenic sequences in a highly variable region. Genome Biol Evol. 2019; 11 :1192-206

[17] Guo W, Zhu A, Fan W. et al. Complete mitochondrial genomes from the ferns Ophioglossum californicum and Psilotum nudum are highly repetitive with the largest organellar introns. New Phytol. 2017; 213 :391-403

[18] Li J, Li J, Ma Y. et al. The complete mitochondrial genome of okra ( Abelmoschus esculentus ): using nanopore long reads to investigate gene transfer from chloroplast genomes and rearrangements of mitochondrial DNA molecules. BMC Genomics. 2022; 23 :481

[19] Qu Y, Zhou P, Tong C. et al. Assembly and analysis of the Populus deltoides mitochondrial genome: the first report of a multicircular mitochondrial conformation for the genus Populus. J For Res. 2023; 34 :717-33

[20] Al-Nakeeb K, Petersen TN, Sicheritz-Ponten T. Norgal: extraction and de novo assembly of mitochondrial DNA from whole-genome sequencing data. BMC Bioinformatics. 2017; 18 :510

[21] Yang Z, Ni Y, Lin Z. et al. De novo assembly of the complete mitochondrial genome of sweet potato ( Ipomoea batatas [L.] Lam) revealed the existence of homologous conformations generated by the repeat-mediated recombination. BMC Plant Biol. 2022; 22 :285

[22] Ahmed Z, Fu YB. An improved method with a wider applicability to isolate plant mitochondria for mtDNA extraction. Plant Methods. 2015; 11 :56

[23] Strehle MM, Purfeerst E, Christensen AC. A rapid and efficient method for enriching mitochondrial DNA from plants. Mitochondrial DNA B Resour. 2018; 3 :239-42

[24] Bankevich A, Nurk S, Antipov D. et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012; 19 :455-77

[25] Dierckxsens N, Mardulyn P, Smits G. NOVOPlasty: de novo assembly of organelle genomes from whole genome data. Nucleic Acids Res. 2017; 45 :e18

[26] Jin JJ, Yu WB, Yang JB. et al. GetOrganelle: a fast and versatile toolkit for accurate de novo assembly of organelle genomes. Genome Biol. 2020; 21 :241

[27] Liu H, Wu S, Li A. et al. SMARTdenovo: a de novo assembler using long noisy reads. Gigabyte. 2021; 2021 :gigabyte15

[28] Hu J, Wang Z, Sun Z. et al. An efficient error correction and accurate assembly tool for noisy long reads. bioRxiv. 2023; 2023.03.09.531669

[29] Koren S, Walenz BP, Berlin K. et al. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017; 27 :722-36

[30] Cheng H, Concepcion GT, Feng X. et al. Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nat Methods. 2021; 18 :170-5

[31] Han F, Qu Y, Chen Y. et al. Assembly and comparative analysis of the complete mitochondrial genome of Salix wilsonii using PacBio HiFi sequencing. Front Plant Sci. 2022; 13 :1031769

[32] Zuo Y, Verheecke-Vaessen C, Molitor C. et al. De novo genome assembly and functional annotation for Fusarium langsethiae. BMC Genomics. 2022; 23 :158

[33] Yang J, Ling C, Zhang H. et al. A comparative genomics approach for analysis of complete mitogenomes of five Actinidiaceae plants. Genes (Basel). 2022; 13 :1827

[34] Miao Y, Chen H, Xu W. et al. Cistanche species mitogenomes suggest diversity and complexity in Lamiales-order mitogenomes. Genes (Basel). 2022; 13 :1791

[35] Li J, Xu Y, Shan Y. et al. Assembly of the complete mitochondrial genome of an endemic plant, Scutellaria tsinyunensis, revealed the existence of two conformations generated by a repeat-mediated recombination. Planta. 2021; 254 :36

[36] Cheng Y, He X, Priyadarshani S. et al. Assembly and comparative analysis of the complete mitochondrial genome of Suaeda glauca. BMC Genomics. 2021; 22 :1-15

[37] Wang X, Zhang R, Yun Q. et al. Comprehensive analysis of complete mitochondrial genome of Sapindus mukorossi Gaertn.: an important industrial oil tree species in China. Ind Crop Prod. 2021; 174 :114210

[38] He W, Xiang K, Chen C. et al. Master graph: an essential integrated assembly model for the plant mitogenome based on a graph-based framework. Bioinformatics. 2023; 24 :bbac522

[39] Kurtzer GM, Sochat V, Bauer MW. Singularity: scientific containers for mobility of compute. PLoS One. 2017; 12 :e0177459

[40] Unseld M, Marienfeld JR, Brandt P. et al. The mitochondrial genome of Arabidopsis thaliana contains 57 genes in 366,924 nucleotides. Nat Genet. 1997; 15 :57-61

[41] Goremykin VV, Lockhart PJ, Viola R. et al. The mitochondrial genome of Malus domestica and the import-driven hypothesis of mitochondrial genome expansion in seed plants. Plant J. 2012; 71 :615-26

[42] Grassa CJ, Ebert DP, Kane NC. et al. Complete mitochondrial genome sequence of sunflower ( Helianthus annuus L.). Genome Announce. 2016; 4 :e00981-16

[43] Makarenko MS, Usatov AV, Tatarinova TV. et al. Organization features of the mitochondrial genome of sunflower ( Helianthus annuus L.) with ANN2-type male-sterile cytoplasm. Plants (Basel). 2019; 8 :439

[44] Sloan DB, Wu Z, Sharbrough J. Correction of persistent errors in Arabidopsis reference mitochondrial genomes. Plant Cell. 2018; 30 :525-7

[45] Xiong X, Gou J, Liao Q. et al. The Taxus genome provides insights into paclitaxel biosynthesis. Nature Plants. 2021; 7 :1026-36

[46] Kan SL, Shen TT, Gong P. et al. The complete mitochondrial genome of Taxus cuspidata (Taxaceae): eight protein-coding genes have transferred to the nuclear genome. BMC Ecol Evol. 2020; 20 :10

[47] Groth-Malonek M, Wahrmund U, Polsakiewicz M. et al. Evolution of a pseudogene: exclusive survival of a functional mitochondrial nad7 gene supports Haplomitrium as the earliest liverwort lineage and proposes a secondary loss of RNA editing in Marchantiidae. Mol Biol Evol. 2007; 24 :1068-74

[48] Zumkeller S, Polsakiewicz M, Knoop V. Rickettsial DNA and a trans-splicing rRNA group I intron in the unorthodox mitogenome of the fern Haplopteris ensiformis. Commun Biol. 2023; 6 :296

[49] Cao J, Yang X, Wang Y. et al. Characterization of the complete mitochondrial genome of the Pohlia nutans M211 from Antarctica. Mitochondrial DNA B Resour. 2020; 5 :2516-7

[50] Zhao N, Wang Y, Hua J. The roles of mitochondrion in intergenomic gene transfer in plants: a source and a pool. Int J Mol Sci. 2018; 19 :547.

[51] Adams KL, Qiu YL, Stoutemyer M. et al. Punctuated evolution of mitochondrial gene content: high and variable rates of mitochondrial gene loss and transfer to the nucleus during angiosperm evolution. Proc Natl Acad Sci USA. 2002; 99 :9905-12

[52] Bergthorsson U, Adams KL, Thomason B. et al. Widespread horizontal transfer of mitochondrial genes in flowering plants. Nature. 2003; 424 :197-201

[53] Rice DW, Alverson AJ, Richardson AO. et al. Horizontal transfer of entire genomes via mitochondrial fusion in the angiosperm Amborella. Science. 2013; 342 :1468-73

[54] Borthakur D, Busov V, Cao XH. et al. Current status and trends in forest genomics. For Res. 2022; 2 :11

[55] Kozik A, Rowan BA, Lavelle D. et al. The alternative reality of plant mitochondrial DNA: one ring does not rule them all. PLoS Genet. 2019; 15 :e1008373

[56] Wu ZQ, Cuthbert JM, Taylor DR. et al. The massive mitochondrial genome of the angiosperm Silene noctiflora is evolving by gain or loss of entire chromosomes. PNAS Nexus. 2015; 112 :10185-91

[57] Uliano-Silva M, Ferreira J, Krasheninnikova K. et al. MitoHiFi: a python pipeline for mitochondrial genome assembly from PacBio high fidelity reads. BMC Bioinformatics. 2023; 24 :288

[58] Wang DY, Zhang Q, Liu Y. et al. The levels of male gametic mitochondrial DNA are highly regulated in angiosperms with regard to mitochondrial inheritance. Plant Cell. 2010; 22 :2402-16

[59] Ye N, Wang X, Li J. et al. Assembly and comparative analysis of complete mitochondrial genome sequence of an economic plant Salix suchowensis. PeerJ. 2017; 5 :e3148

[60] Ma Q, Wang Y, Li S. et al. Assembly and comparative analysis of the first complete mitochondrial genome of Acer truncatum Bunge: a woody oil-tree species producing nervonic acid. BMC Plant Biol. 2022; 22 :1-17

[61] Nederbragt AJ. On the middle ground between open source and commercial software - the case of the Newbler program. Genome Biol. 2014; 15 :113

[62] Margulies M, Egholm M, Altman WE. et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature. 2005; 437 :376-80

[63] Iorizzo M, Senalik D, Szklarczyk M. et al. De novo assembly of the carrot mitochondrial genome using next generation sequencing of whole genomic DNA provides first evidence of DNA transfer into an angiosperm plastid genome. BMC Plant Biol. 2012; 12 :61

[64] Zhang T, Zhang X, Hu S. et al. An efficient procedure for plant organellar genome assembly, based on whole genome data from the 454 GS FLX sequencing platform. Plant Methods. 2011; 7 :38

[65] Camacho C, Coulouris G, Avagyan V. et al. BLAST + : architecture and applications. BMC Bioinformatics. 2009; 10 :421

[66] Wick RR, Schultz MB, Zobel J. et al. Bandage: interactive visualization of de novo genome assemblies. Bioinformatics. 2015; 31 :3350-2

[67] Tillich M, Lehwark P, Pellizzer T. et al. GeSeq - versatile and accurate annotation of organelle genomes. Nucleic Acids Res. 2017; 45 :W6-11

[68] Schattner P, Brooks AN, Lowe TM. The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res. 2005; 33 :W686-9