Establishment of genome-editing system and assembly of a near-complete genome in broomcorn millet

Abstract

The ancient crop broomcorn millet (Panicum miliaceum L.) is an indispensable orphan crop in semi-arid regions due to its short life cycle and excellent abiotic stress tolerance. These advantages make it an important alternative crop to increase food security and achieve the goal of zero hunger, particularly in light of the uncertainty of global climate change. However, functional genomic and biotechnological research in broomcorn millet has been hampered due to a lack of genetic tools such as transformation and genome-editing techniques. Here, we successfully performed genome editing of broomcorn millet. We identified an elite variety, Hongmi, that produces embryogenic callus and has high shoot regeneration ability in in vitro culture. We established an Agrobacterium tumefaciens-mediated genetic transformation protocol and a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome-editing system for Hongmi. Using these techniques, we produced herbicide-resistant transgenic plants and edited phytoene desaturase (PmPDS), which is involved in chlorophyll biosynthesis. To facilitate the rapid adoption of Hongmi as a model line for broomcorn millet research, we assembled a near-complete genome sequence of Hongmi and comprehensively annotated its genome. Together, our results open the door to improving broomcorn millet using biotechnology.

Keywords

broomcorn millet / CRISPR/Cas9 editing / genome assembly / herbicide resistance / transformation system

References

[1] Aganezov, S.,Yan, S.M.,Soto, D.C.,Kirsche, M.,Zarate, S.,Avdeyev, P.,Taylor, D.J.,Shafin, K.,Shumate, A.,Xiao, C., et al. (2022). A complete reference genome improves analysis of human genetic variation. Science 376: eabl3533.

[2] Anjanappa, R.B., and Gruissem, W. (2021). Current progress and challenges in crop genetic transformation. J. Plant Physiol. 261:153411.

[3] Aregawi, K.,Shen, J.,Pierroz, G.,Sharma, M.K.,Dahlberg, J.,Owiti, J., and Lemaux, P.G. (2022). Morphogene-assisted transformation of Sorghum bicolor allows more efficient genome editing. Plant Biotechnol. J. 20:748–760.

[4] Banakar, R.,Schubert, M.,Collingwood, M.,Vakulskas, C.,Eggenberger, A.L., and Wang, K. (2020). Comparison of CRISPR-Cas9/Cas12a ribonucleoprotein complexes for genome editing efficiency in the rice phytoene desaturase (OsPDS) gene. Rice 13:4.

[5] Benson, G. (1999). Tandem repeats finder: A program to analyze DNA sequences. Nucleic Acids Res. 27:573–580.

[6] Bolger, A.M.,Lohse, M., and Usadel, B. (2014). Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120.

[7] Buchholzer, M., and Frommer, W.B. (2023). An increasing number of countries regulate genome editing in crops. New Phytol. 237:12–15.

[8] Cable, J.,Ronald, P.C.,Voytas, D.,Zhang, F.,Levy, A.A.,Takatsuka, A.,Arimura, S.I.,Jacobsen, S.E.,Toki, S.,Toda, E., et al. (2021). Plant genome engineering from lab to field-a Keystone Symposia report. Ann. N. Y. Acad. Sci. 1506:35–54.

[9] Chen, Z.,Debernardi, J.M.,Dubcovsky, J., and Gallavotti, A. (2022). Recent advances in crop transformation technologies. Nat. Plants 8:1343–1351.

[10] Chen, K., and Gao, C. (2014). Targeted genome modification technologies and their applications in crop improvements. Plant Cell Rep. 33:575–583.

[11] Chen, J.,Liu, Y.,Liu, M.,Guo, W.,Wang, Y.,He, Q.,Chen, W.,Liao, Y.,Zhang, W.,Gao, Y., et al. (2023). Pangenome analysis reveals genomic variations associated with domestication traits in broomcorn millet. Nat. Genet. 55:2243–2254.

[12] Cheng, H.,Concepcion, G.T.,Feng, X.,Zhang, H., and Li, H. (2021). Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nat. Methods 18:170–175.

[13] Danecek, P.,Bonfield, J.K.,Liddle, J.,Marshall, J.,Ohan, V.,Pollard, M.O.,Whitwham, A.,Keane, T.,McCarthy, S.A.,Davies, R.M., et al. (2021). Twelve years of SAMtools and BCFtools. Gigascience 10: giab008.

[14] Deng, Y.,Liu, S.,Zhang, Y.,Tan, J.,Li, X.,Chu, X.,Xu, B.,Tian, Y.,Sun, Y.,Li, B., et al. (2022). A telomere-to-telomere gap-free reference genome of watermelon and its mutation library provide important resources for gene discovery and breeding. Mol. Plant 15:1268–1284.

[15] Flynn, J.M.,Hubley, R.,Goubert, C.,Rosen, J.,Clark, A.G.,Feschotte, C., and Smit, A.F. (2020). RepeatModeler2 for automated genomic discovery of transposable element families. Proc. Natl. Acad. Sci. U. S. A. 117:9451–9457.

[16] Gao, C. (2021). Genome engineering for crop improvement and future agriculture. Cell 184:1621–1635.

[17] Ghannoum, O.,Caemmerer, S.V., and Conroy, J.P. (2002). The effect of drought on plant water use efficiency of nine NAD-ME and nine NADP-ME Australian C4 grasses. Funct. Plant Biol. 29:1337–1348.

[18] Goron, T.L., and Raizada, M.N. (2015). Genetic diversity and genomic resources available for the small millet crops to accelerate a New Green Revolution. Front. Plant Sci. 6:157.

[19] Hamoud, M.A.,Haroun, S.A.,Macleod, R.D., and Richards, A.J. (1994). Cytological relationships of selected species of Panicum L. Biol. Plant. 36:37–45.

[20] Holt, C., and Yandell, M. (2011). MAKER2: An annotation pipeline and genome-database management tool for second-generation genome projects. BMC Bioinform. 12:491.

[21] Hunt, H.V.,Rudzinski, A.,Jiang, H.,Wang, R.,Thomas, M.G., and Jones, M.K. (2018). Genetic evidence for a western Chinese origin of broomcorn millet (Panicum miliaceum). Holocene 28:1968–1978.

[22] Jain, C.,Rhie, A.,Hansen, N.F.,Koren, S., and Phillippy, A.M. (2022). Long-read mapping to repetitive reference sequences using Winnowmap2. Nat. Methods 19:705–710.

[23] Jones, P.,Binns, D.,Chang, H.Y.,Fraser, M.,Li, W.,McAnulla, C.,McWilliam, H.,Maslen, J.,Mitchell, A.,Nuka, G., et al. (2014). InterProScan 5: Genome-scale protein function classification. Bioinformatics 30:1236–1240.

[24] Kamburova, V.S.,Nikitina, E.V.,Shermatov, S.E.,Buriev, Z.T.,Kumpatla, S.P.,Emani, C., and Abdurakhmonov, I.Y. (2017). Genome editing in plants: An overview of tools and applications. Int. J. Agron. 2017:7315351.

[25] Kim, D.,Paggi, J.M.,Park, C.,Bennett, C., and Salzberg, S.L. (2019). Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat. Biotechnol. 37:907–915.

[26] Kolmogorov, M.,Yuan, J.,Lin, Y., and Pevzner, P.A. (2019). Assembly of long, error-prone reads using repeat graphs. Nat. Biotechnol. 37:540–546.

[27] Korf, I. (2004). Gene finding in novel genomes. BMC Bioinform. 5:59.

[28] Kovaka, S.,Zimin, A.V.,Pertea, G.M.,Razaghi, R.,Salzberg, S.L., and Pertea, M. (2019). Transcriptome assembly from long-read RNA-seq alignments with StringTie2. Genome Biol. 20:278.

[29] Kumar, K.,Gambhir, G.,Dass, A.,Tripathi, A.K.,Singh, A.,Jha, A.K.,Yadava, P.,Choudhary, M., and Rakshit, S. (2020). Genetically modified crops: Current status and future prospects. Planta 251:91.

[30] Lee, K., and Wang, K. (2023). Strategies for genotype-flexible plant transformation. Curr. Opin. Biotechnol. 79:102848.

[31] Li, C.,Liu, C.,Qi, X.,Wu, Y.,Fei, X.,Mao, L.,Cheng, B.,Li, X., and Xie, C. (2017). RNA-guided Cas9 as an in vivo desired-target mutator in maize. Plant Biotechnol. J. 15:1566–1576.

[32] Li, H. (2013). Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv>:1303.3997v2.

[33] Li, H. (2018). Minimap2: Pairwise alignment for nucleotide sequences. Bioinformatics 34:3094–3100.

[34] Li, X., and Siddique, K.H.M. (2020). Future Smart Food: Harnessing the potential of neglected and underutilized species for Zero Hunger. Matern. Child Nutr. 16: e13008.

[35] Liu, M.X.,Qiao, Z.J.,Zhang, S.,Wang, Y.Y., and Lu, P. (2015). Response of broomcorn millet (Panicum miliaceum L.) genotypes from semiarid regions of China to salt stress. Crop J. 3:57–66.

[36] Lu, H.,Zhang, J.,Liu, K.B.,Wu, N.,Li, Y.,Zhou, K.,Ye, M.,Zhang, T.,Zhang, H.,Yang, X., et al. (2009). Earliest domestication of common millet (Panicum miliaceum) in East Asia extended to 10, 000 years ago. Proc. Natl. Acad. Sci. U. S. A. 106:7367–7372.

[37] Manni, M.,Berkeley, M.R.,Seppey, M.,Simao, F.A., and Zdobnov, E.M. (2021). BUSCO update: Novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol. Biol. Evol. 38:4647–4654.

[38] Marone, D.,Mastrangelo, A.M., and Borrelli, G.M. (2023). From transgenesis to genome editing in crop improvement: Applications, marketing, and legal issues. Int. J. Mol. Sci. 24:7122.

[39] McKenna, A.,Hanna, M.,Banks, E.,Sivachenko, A.,Cibulskis, K.,Kernytsky, A.,Garimella, K.,Altshuler, D.,Gabriel, S.,Daly, M., et al. (2010). The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20:1297–1303.

[40] Ou, S.,Chen, J., and Jiang, N. (2018). Assessing genome assembly quality using the LTR Assembly Index (LAI). Nucleic Acids Res. 46: e126.

[41] Ou, S., and Jiang, N. (2018). LTR_retriever: A highly accurate and sensitive program for identification of long terminal repeat retrotransposons. Plant Physiol. 176:1410–1422.

[42] Pedersen, B.S., and Quinlan, A.R. (2018). Mosdepth: Quick coverage calculation for genomes and exomes. Bioinformatics 34:867–868.

[43] Puchta, H. (2017). Applying CRISPR/Cas for genome engineering in plants: The best is yet to come. Curr. Opin. Plant Biol. 36:1–8.

[44] Rhie, A.,Walenz, B.P.,Koren, S., and Phillippy, A.M. (2020). Merqury: Reference-free quality, completeness, and phasing assessment for genome assemblies. Genome Biol. 21:245.

[45] Salt, D.E. (2023). GMO or non-GMO? That is the question. New Phytol. 237:7–8.

[46] Seghatoleslami, M.J.,Kafi, M., and Majidi, E. (2008). Effect of drought stress at different growth stages on yield and water use efficiency of five proso millet (Panicum miliaceum L.) genotypes. Pak. J. Bot. 40:1427–1432.

[47] Servant, N.,Varoquaux, N.,Lajoie, B.R.,Viara, E.,Chen, C.J.,Vert, J.P.,Dekker, J.,Heard, E., and Barillot, E. (2015). HiC-Pro: An optimized and flexible pipeline for Hi-C processing. Genome Biol. 16:259.

[48] Shang, L.,He, W.,Wang, T.,Yang, Y.,Xu, Q.,Zhao, X.,Yang, L.,Zhang, H.,Li, X.,Lv, Y., et al. (2023). A complete assembly of the rice Nipponbare reference genome. Mol. Plant 16:1232–1236.

[49] Shi, J.,Ma, X.,Zhang, J.,Zhou, Y.,Liu, M.,Huang, L.,Sun, S.,Zhang, X.,Gao, X.,Zhan, W., et al. (2019). Chromosome conformation capture resolved near complete genome assembly of broomcorn millet. Nat. Commun. 10:464.

[50] Springer, N.M. (2010). Isolation of plant DNA for PCR and genotyping using organic extraction and CTAB. Cold Spring Harb. Protoc. 2010: db.prot5515.

[51] Stanke, M.,Diekhans, M.,Baertsch, R., and Haussler, D. (2008). Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics 24:637–644.

[52] Sun, Y.,Liu, Y.,Shi, J.,Wang, L.,Liang, C.,Yang, J.,Chen, J., and Chen, M. (2023). Biased mutations and gene losses underlying diploidization of the tetraploid broomcorn millet genome. Plant J. 113:787–801.

[53] Varshney, R.K.,Bohra, A.,Roorkiwal, M.,Barmukh, R.,Cowling, W.A.,Chitikineni, A.,Lam, H.M.,Hickey, L.T.,Croser, J.S.,Bayer, P.E., et al. (2021). Fast-forward breeding for a food-secure world. Trends Genet. 37:1124–1136.

[54] Vaser, R.,Sović I.,Nagarajan, N., and Šikić M. (2017). Fast and accurate de novo genome assembly from long uncorrected reads. Genome Res. 27:737–746.

[55] Walker, B.J.,Abeel, T.,Shea, T.,Priest, M.,Abouelliel, A.,Sakthikumar, S.,Cuomo, C.A.,Zeng, Q.,Wortman, J.,Young, S.K., et al. (2014). Pilon: An integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 9: e112963.

[56] Wang, Z.,Huang, S.,Yang, Z.,Lai, J.,Gao, X., and Shi, J. (2023). A high-quality, phased genome assembly of broomcorn millet reveals the features of its subgenome evolution and 3D chromatin organization. Plant Commun. 4:100557.

[57] Wang, S.,Gao, S.,Nie, J.,Tan, X.,Xie, J.,Bi, X.,Sun, Y.,Luo, S.,Zhu, Q.,Geng, J., et al. (2022). Improved 93-11 genome and time-course transcriptome expand resources for rice genomics. Front. Plant Sci. 12:769700.

[58] Washburn, J.D.,Schnable, J.C.,Davidse, G., and Pires, J.C. (2015). Phylogeny and photosynthesis of the grass tribe Paniceae. Am. J. Bot. 102:1493–1505.

[59] Wu, C., and Sui, Y. (2019). Efficient and fast production of transgenic rice plants by Agrobacterium-mediated transformation. Methods Mol. Biol. 1864:95–103.

[60] Yang, Z.,Zhang, H.,Li, X.,Shen, H.,Gao, J.,Hou, S.,Zhang, B.,Mayes, S.,Bennett, M.,Ma, J., et al. (2020). A mini foxtail millet with an Arabidopsis-like life cycle as a C4 model system. Nat. Plants 6:1167–1178.

[61] Yaqoob, H.,Tariq, A.,Bhat, B.A.,Bhat, K.A.,Nehvi, I.B.,Raza, A.,Djalovic, I.,Prasad, P.V., and Mir, R.A. (2023). Integrating genomics and genome editing for orphan crop improvement: A bridge between orphan crops and modern agriculture system. GM Crops Food 14:1–20.

[62] Ye, C.Y., and Fan, L. (2021). Orphan crops and their wild relatives in the genomic era. Mol. Plant 14:27–39.

[63] Zhang, X.,Zhang, S.,Zhao, Q.,Ming, R., and Tang, H. (2019). Assembly of allele-aware, chromosomal-scale autopolyploid genomes based on Hi-C data. Nat. Plants 5:833–845.

[64] Zhang, D.,Zhang, Z.,Unver, T., and Zhang, B. (2020). CRISPR/Cas: A powerful tool for gene function study and crop improvement. J. Adv. Res. 29:207–221.

[65] Zou, C.,Li, L.,Miki, D.,Li, D.,Tang, Q.,Xiao, L.,Rajput, S.,Deng, P.,Peng, L.,Jia, W., et al. (2019). The genome of broomcorn millet. Nat. Commun. 10:436.

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