The genome of Hibiscus hamabo reveals its adaptation to saline and waterlogged habitat

Abstract

Hibiscus hamabo is a semi-mangrove species with strong tolerance to salt and waterlogging stress. However, the molecular basis and mechanisms that underlie this strong adaptability to harsh environments remain poorly understood. Here, we assembled a high-quality, chromosome-level genome of this semi-mangrove plant and analyzed its transcriptome under different stress treatments to reveal regulatory responses and mechanisms. Our analyses suggested that H. hamabo has undergone two recent successive polyploidy events, a whole-genome duplication followed by a whole-genome triplication, resulting in an unusually large gene number (107 309 genes). Comparison of the H. hamabo genome with that of its close relative Hibiscus cannabinus, which has not experienced a recent WGT, indicated that genes associated with high stress resistance have been preferentially preserved in the H. hamabo genome, suggesting an underlying association between polyploidy and stronger stress resistance. Transcriptomic data indicated that genes in the roots and leaves responded differently to stress. In roots, genes that regulate ion channels involved in biosynthetic and metabolic processes responded quickly to adjust the ion concentration and provide metabolic products to protect root cells, whereas no such rapid response was observed from genes in leaves. Using co-expression networks, potential stress resistance genes were identified for use in future functional investigations. The genome sequence, along with several transcriptome datasets, provide insights into genome evolution and the mechanism of salt and waterlogging tolerance in H. hamabo, suggesting the importance of polyploidization for environmental adaptation.

References

[1] Sakhanokho HF, Islam-Faridi N, Babiker EM et al. Determination of nuclear DNA content, ploidy, and FISH location of ribosomal DNA in hibiscus hamabo . Sci Hortic. 2020; 264: 109167.

[2] Li J, Liao J, Guan M et al. Salt tolerance of hibiscus hamabo seedlings: a candidate halophyte for reclamation areas. Acta Physiol Plant. 2012; 34: 1747-55.

[3] Fryxell PA, Stelly DM . Documented chromosome numbers 1993:2. Additional chromosome counts in the Malvaceae . SIDA Contributions to Botany. 1993; 15: 639-47.

[4] Takayama K, Ohi-Toma T, Kudoh H et al. Origin and diversification of Hibiscus glaber, species endemic to the oceanic Bonin Islands, revealed by chloroplast DNA polymorphism: origin and diversification of Hibiscus glaber . Mol Ecol. 2005; 14: 1059-71.

[5] Tanmoy AM, Alam MM, Moosa M et al. Corchorus L. and hibiscus L.: molecular phylogeny helps to understand their relative evolution and dispersal routes . Bioresearch Communication. 2015; 1.

[6] Takayama K, Kajita T, Murata J et al. Phylogeography and genetic structure of Hibiscus tiliaceus- speciation of a pantropical plant with sea-drifted seeds: Phylogeography of Hibiscus tiliaceus . Mol Ecol. 2006; 15: 2871-81.

[7] Wu T, Gu S, Zhou H et al. Photosynthetic and physiological responses of native and exotic tidal woody seedlings to simulated tidal immersion. Estuar Coast Shelf Sci. 2013; 135: 280-4.

[8] Wang Z, Xu X, Ni L et al. Efficient virus-induced gene silencing in hibiscus hamabo Sieb. Et Zucc. Using tobacco rattle virus. PeerJ. 2019; 7: e7505.

[9] Lin H, Yang Y, Quan R et al. Phosphorylation of SOS3-LIKE CALCIUM BINDING PROTEIN8 by SOS2 protein kinase stabilizes their protein complex and regulates salt tolerance in Arabidopsis . Plant Cell. 2009; 21: 1607-19.

[10] Yang Y, Guo Y . Elucidating the molecular mechanisms mediating plant salt-stress responses. New Phytol. 2018; 217: 523-39.

[11] Van Zelm E, Zhang Y, Testerink C . Salt tolerance mechanisms of plants. Annu Rev Plant Biol. 2020; 71: 403-33.

[12] Yin D, Sun D, Han Z et al. PhERF2, an ethylene-responsive element binding factor, plays an essential role in waterlogging tolerance of petunia . Hortic Res. 2019; 6: 83.

[13] Zhou W, Chen F, Meng Y et al. Plant waterlogging/flooding stress responses: from seed germination to maturation. Plant Physiol Biochem. 2020; 148: 228-36.

[14] Zhao N, Li C, Yan Y et al. Comparative transcriptome analysis of waterlogging-sensitive and waterlogging-tolerant Chrysanthemum morifolium cultivars under waterlogging stress and reoxygenation conditions . Int J Mol Sci. 2018; 19: 1455.

[15] Yuan Y, Brunel C, van Kleunen M et al. Salinity-induced changes in the rhizosphere microbiome improve salt tolerance of hibiscus hamabo . Plant Soil. 2019; 443: 525-37.

[16] Ou S, Chen J, Jiang N . Assessing genome assembly quality using the LTR assembly index (LAI). Nucleic Acids Res. 2018; 46: e126.

[17] Zhang L, Xu Y, Zhang X et al. The genome of kenaf (Hibiscus cannabinus L.) provides insights into bast fibre and leaf shape biogenesis . Plant Biotechnol J. 2018; 18: 1796-809.

[18] Sun P, Jiao B, Yang Y et al. WGDI: a user-friendly toolkit for evolutionary analyses of whole-genome duplications and ancestral karyotypes. Biorxiv. 2021.

[19] Yang Y, Guo Y . Unraveling salt stress signaling in plants: salt stress signaling. J Integr Plant Biol. 2018; 60: 796-804.

[20] Barragán V, Leidi EO, Andrés Z et al. Ion exchangers NHX1 and NHX2 mediate active potassium uptake into vacuoles to regulate cell turgor and stomatal function in Arabidopsis . Plant Cell. 2012; 24: 1127-42.

[21] Kumar V, Khare T, Sharma M et al. ROS-Induced signaling and gene expression in crops under salinity stress. in reactive oxygen species and antioxidant systems in plants: Role and regulation under abiotic stress . Springer Singapore 159-184 (2017).

[22] Teige M, Scheikl E, Eulgem T et al. The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis . Mol Cell. 2004; 15: 141-52.

[23] Xu J, Li Y, Wang Y et al. Activation of MAPK kinase 9 induces ethylene and camalexin biosynthesis and enhances sensitivity to salt stress in Arabidopsis . J Biol Chem. 2008; 283: 26996-7006.

[24] Raghavendra AS, Gonugunta VK, Christmann A et al. ABA perception and signalling. Trends Plant Sci. 2010; 15: 395-401.

[25] Phukan UJ, Mishra S, Shukla RK . Waterlogging and submergence stress: affects and acclimation. Crit Rev Biotechnol. 2016; 36: 956-66.

[26] Van de Peer Y, Maere S, Meyer A . The evolutionary significance of ancient genome duplications. Nat Rev Genet. 2009; 10: 725-32.

[27] Van de Peer Y, Mizrachi E, Marchal K . The evolutionary significance of polyploidy. Nat Rev Genet. 2017; 18: 411-24.

[28] Zhang L, Li X, Ma B et al. The tartary buckwheat genome provides insights into rutin biosynthesis and abiotic stress tolerance. Mol Plant. 2017; 10: 1224-37.

[29] Van de Peer Y, Ashman T-L, Soltis PS et al. Polyploidy: an evolutionary and ecological force in stressful times. Plant Cell. 2021; 33: 11-26.

[30] Zhao C, Zhang H, Song C et al. Mechanisms of plant responses and adaptation to soil salinity. The Innovation. 2020; 1: 100017.

[31] Vidoz ML, Mignolli F, Aispuru HT et al. Rapid formation of adventitious roots and partial ethylene sensitivity result in faster adaptation to flooding in the aerial roots (aer) mutant of tomato. Sci Hortic. 2016; 201: 130-9.

[32] Wang S, Huang J, Wang X et al. PagERF16 of populus promotes lateral root proliferation and sensitizes to salt stress . Front Plant Sci. 2021; 12.

[33] Hinz M, Wilson IW, Yang J et al. Arabidopsis RAP2.2: an ethylene response transcription factor that is important for hypoxia survival . Plant Physiol. 2010; 153: 757-72.

[34] Kim Y-M, Kim S, Koo N et al. Genome analysis of Hibiscus syriacus provides insights of polyploidization and indeterminate flowering in woody plants. DNA Res. 2017; 24: 71-80.

[35] Vanneste K, Baele G, Maere S et al. Analysis of 41 plant genomes supports a wave of successful genome duplications in association with the cretaceous-Paleogene boundary. Genome Res. 2014; 24: 1334-47.

[36] Birchler JA, Veitia RA . Gene balance hypothesis: connecting issues of dosage sensitivity across biological disciplines. Proc Natl Acad Sci. 2012; 109: 14746-53.

[37] Conant GC, Birchler JA, Pires JC . Dosage, duplication, and diploidization: clarifying the interplay of multiple models for duplicate gene evolution over time. Curr Opin Plant Biol. 2014; 19: 91-8.

[38] Shang H. Studies on Mechanism of Salt Ions Transportation and Distribution in Hibiscus Hamabo Sieb.Et Zucc . Shandong Normal University; 2010.

[39] Bo P. Effects of NaCl Stress on Seeds Germination and Seedlings Physiological Characteristics of Hibiscus Hamabo Sieb.Et Zucc . Shandong Normal University; 2008.

[40] Bo P, Sun X, Sun T et al. Antioxidative system and osmotic regulation in seedlings of annual hibiscus hamabo sieb.Et Zucc under salt stress. Acta Botanica Boreali. 2008; 28: 0113-8.

[41] Bielach A, Hrtyan M, Tognetti V . Plants under stress: involvement of auxin and cytokinin. Int J Mol Sci. 2017; 18: 1427.

[42] Sahu SK, Thangaraj M, Kathiresan K . DNA extraction protocol for plants with high levels of secondary metabolites and polysaccharides without using liquid nitrogen and phenol. ISRN Molecular Biology. 2012; 2012: 205049.

[43] Walker BJ, Abeel T, Shea T et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One. 2014; 9: e112963.

[44] Li H . Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018; 34: 3094-100.

[45] Simão FA, Waterhouse RM, Ioannidis P et al. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015; 31: 3210-2.

[46] Li H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv:13033997 [q-bio]. 2013.

[47] McKenna A, Hanna M, Banks E et al. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010; 20: 1297-303.

[48] Xu Z, Wang H . LTR_FINDER: an efficient tool for the prediction of full-length LTR retrotransposons. Nucleic Acids Res. 2007; 35: W265-8.

[49] Ou S, Jiang N . LTR_retriever: a highly accurate and sensitive program for identification of long terminal repeat retrotransposons. Plant Physiol. 2018; 176: 1410-22.

[50] Tarailo-Graovac M, Chen N . Using repeatMasker to identify repetitive elements in genomic sequences. Curr Protoc Bioinformatics. 2009; 25.

[51] Jurka J . Repbase update: a database and an electronic journal of repetitive elements. Trends Genet. 2000; 16: 418-20.

[52] Bao Z . Automated De novo identification of repeat sequence families in sequenced genomes. Genome Res. 2002; 12: 1269-76.

[53] Price AL, Jones NC, Pevzner PA . De novo identification of repeat families in large genomes. Bioinformatics. 2005; 21: i351-8.

[54] Benson G. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res. 1999; 27: 573-80.

[55] Stanke M, Steinkamp R, Waack S et al. AUGUSTUS: a web server for gene finding in eukaryotes. Nucleic Acids Res. 2004; 32: W309-12.

[56] Stanke M, Morgenstern B . AUGUSTUS: a web server for gene prediction in eukaryotes that allows user-defined constraints. Nucleic Acids Res. 2005; 33: W465-7.

[57] Stanke M, Keller O, Gunduz I et al. AUGUSTUS: ab initio prediction of alternative transcripts. Nucleic Acids Res. 2006; 34: W435-9.

[58] Burge C, Karlin S . Prediction of complete gene structures in human genomic DNA. J Mol Biol. 1997; 268: 78-94.

[59] Trapnell C, Pachter L, Salzberg SL . TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 2009; 25: 1105-11.

[60] Trapnell C, Williams BA, Pertea G et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010; 28: 511-5.

[61] Cantarel BL, Korf I, Robb SMC et al. MAKER: an easy-to-use annotation pipeline designed for emerging model organism genomes. Genome Res. 2007; 18: 188-96.

[62] Boeckmann B. The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003. Nucleic Acids Res. 2003; 31: 365-70.

[63] Mitchell A, Chang H-Y, Daugherty L et al. The InterPro protein families database: the classification resource after 15 years. Nucleic Acids Res. 2015; 43: D213-21.

[64] Kanehisa M, Goto S, Sato Y et al. KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res. 2012; 40: D109-14.

[65] Altschul S. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997; 25: 3389-402.

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

[67] Mistry J, Bateman A, Finn RD . Predicting active site residue annotations in the Pfam database. BMC Bioinformatics. 2007; 8: 298.

[68] Jones P, Binns D, Chang H-Y et al. InterProScan 5: genome-scale protein function classification. Bioinformatics. 2014; 30: 1236-40.

[69] Punta M, Coggill PC, Eberhardt RY et al. The Pfam protein families database. Nucleic Acids Res. 2012; 40: D290-301.

[70] Conesa A, Götz S . Blast2GO: a comprehensive suite for functional analysis in plant genomics. Int J Plant Genom. 2008; 2008: 1-12.

[71] Ashburner M, Ball CA, Blake JA et al. Gene ontology: tool for the unification of biology. Nat Genet. 2000; 25: 25-9.

[72] Michael TP, Jupe F, Bemm F et al. High contiguity Arabidopsis thaliana genome assembly with a single nanopore flow cell. Nat Commun. 2018; 9: 541.

[73] Sarkar D, Mahato AK, Satya P et al. The draft genome of Corchorus olitorius cv. JRO-524 (Navin) . Genomics Data. 2017; 12: 151-4.

[74] Udall JA, Long E, Hanson C et al. De novo genome sequence assemblies of Gossypium raimondii and Gossypium turneri . G3 Genes|Genomes|Genetics. 2019; 9: 3079-85.

[75] Argout X, Salse J, Aury J-M et al. The genome of Theobroma cacao . Nat Genet. 2011; 43: 101-8.

[76] Gao Y, Wang H, Liu C et al. De novo genome assembly of the red silk cotton tree (Bombax ceiba) . GigaScience. 2018; 7.

[77] Teh BT, Lim K, Yong CH et al. The draft genome of tropical fruit durian (Durio zibethinus) . Nat Genet. 2017; 49: 1633-41.

[78] Hofmeister BT, Denkena J, Colomé-Tatché M et al. A genome assembly and the somatic genetic and epigenetic mutation rate in a wild long-lived perennial Populus trichocarpa . Genome Biol. 2020; 21: 259.

[79] Badouin H, Velt A, Gindraud F et al. The wild grape genome sequence provides insights into the transition from dioecy to hermaphroditism during grape domestication. Genome Biol. 2020; 21: 223.

[80] Islam MS, Saito JA, Emdad EM et al. Comparative genomics of two jute species and insight into fibre biogenesis. Nat Plants. 2017; 3: 1-7.

[81] Shen Y, Liu J, Geng H et al. De novo assembly of a Chinese soybean genome. Sci China Life Sci. 2018; 61: 871-84.

[82] Takei H, Shirasawa K, Kuwabara K et al. De novo genome assembly of two tomato ancestors, Solanum pimpinellifolium and Solanum lycopersicum var. cerasiforme, by long-read sequencing . DNA Res. 2021; 28: dsaa029.

[83] Emms DM, Kelly S . OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol. 2019; 20: 238.

[84] Edgar RC . MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004; 32: 1792-7.

[85] Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30:1312-3.

[86] Han MV, Thomas GWC, Lugo-Martinez J et al. Estimating gene gain and loss rates in the presence of error in genome assembly and annotation using CAFE 3. Mol Biol Evol. 2013; 30: 1987-97.

[87] Zwaenepoel A, Van de Peer Y . Wgd-simple command line tools for the analysis of ancient whole-genome duplications. Bioinformatics. 2019; 35: 2153-5.

[88] Rozewicki J, Li S, Amada KM et al. MAFFT-DASH: integrated protein sequence and structural alignment. Nucleic Acids Res. 2019; 47: W5-W10.

[89] Price MN, Dehal PS, Arkin AP . FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol Biol Evol. 2009; 26: 1641-50.

[90] Yang Z . PAML: a program package for phylogenetic analysis by maximum likelihood. Bioinformatics. 1997; 13: 555-6.

[91] Tang H, Bowers JE, Wang X et al. Synteny and collinearity in plant genomes. Science. 2008; 320: 486-8.