Zanthoxylum-specific whole genome duplication and recent activity of transposable elements in the highly repetitive paleotetraploid Z. bungeanum genome

Abstract

Zanthoxylum bungeanum is an important spice and medicinal plant that is unique for its accumulation of abundant secondary metabolites, which create a characteristic aroma and tingling sensation in the mouth. Owing to the high proportion of repetitive sequences, high heterozygosity, and increased chromosome number of Z. bungeanum, the assembly of its chromosomal pseudomolecules is extremely challenging. Here, we present a genome sequence for Z. bungeanum, with a dramatically expanded size of 4.23 Gb, assembled into 68 chromosomes. This genome is approximately tenfold larger than that of its close relative Citrus sinensis. After the divergence of Zanthoxylum and Citrus, the lineage-specific whole-genome duplication event η-WGD approximately 26.8 million years ago (MYA) and the recent transposable element (TE) burst ~6.41 MYA account for the substantial genome expansion in Z. bungeanum. The independent Zanthoxylum-specific WGD event was followed by numerous fusion/fission events that shaped the genomic architecture. Integrative genomic and transcriptomic analyses suggested that prominent species-specific gene family expansions and changes in gene expression have shaped the biosynthesis of sanshools, terpenoids, and anthocyanins, which contribute to the special flavor and appearance of Z. bungeanum. In summary, the reference genome provides a valuable model for studying the impact of WGDs with recent TE activity on gene gain and loss and genome reconstruction and provides resources to accelerate Zanthoxylum improvement.

References

[1] Kubitzki, K., Kallunki, J., Duretto, M. & Wilson, P. Rutaceae. In The families and genera of vascular plants, flowering plants eudicots: sapindales, cucurbitales, myrtaceae (ed. Kubitzki K. ) 276-356 (Springer Verlag, 2011).

[2] Zhang, M. et al. Zanthoxylum bungeanum Maxim. (Rutaceae): a systematic review of its traditional uses, botany, phytochemistry, pharmacology, pharmacokinetics, and toxicology. Int. J. Mol. Sci. 18, 2172 (2017).

[3] Yang, X. Aroma constituents and alkylamides of red and green Huajiao (Zanthoxylum bungeanum and Zanthoxylum schinifolium). J. Agr. Food Chem. 56, 1689-1696 (2008).

[4] Boonen, J. et al. Alkamid database: chemistry, occurrence and functionality of plant N-alkylamides. J. Ethnopharmacol. 142, 563-590 (2012).

[5] Greger, H. Alkamides: structural relationships, distribution and biological activity. Planta Med. 50, 366-375 (1984).

[6] Yasuda, I., Takeya, K. & Itokawa, H. Distribution of unsaturated aliphatic acid amides in Japanese Zanthoxylum species. Phytochemistry 21, 1295-1298 (1982).

[7] Matthias, B., Stark, T. D., Corinna, D., Sofie, L. S. & Thomas, H. All-trans-configuration in Zanthoxylum alkylamides swaps the tingling with a numbing sensation and diminishes salivation. J. Agric. Food Chem. 62, 2479-2488 (2014).

[8] Xiong, Q., Dawen, S., Yamamoto, H. & Mizuno, M. Alkylamides from pericarps of Zanthoxylum bungeanum. Phytochemistry 46, 1123-1126 (1997).

[9] Devkota, K. P. et al. Isobutylhydroxyamides from the pericarp of Nepalese Zanthoxylum armatum inhibit NF1-defective tumor cell line growth. J. Nat. Prod. 76, 59-63 (2013).

[10] Rong, R. et al. Anesthetic constituents of Zanthoxylum bungeanum Maxim. pharmacokinetic study. J. Sep. Sci. 39, 2728-2735 (2016).

[11] Tsunozaki, M. et al. A ‘toothache tree’ alkylamide inhibits Aδ mechanonociceptors to alleviate mechanical pain. J. Physiol. 591, 3325-3340 (2013).

[12] Artaria, C., Maramaldi, G., Bonfigli, A., Rigano, L. & Appendino, G. Lifting properties of the alkamide fraction from the fruit husks of Zanthoxylum bungeanum. Int. J. Cosmet. Sci. 33, 328-333 (2011).

[13] Yamazaki, E., Inagaki, M., Kurita, O. & Inoue, T. Antioxidant activity of Japanese pepper (Zanthoxylum piperitum DC.) fruit. Food Chem. 100, 171-177 (2007).

[14] Li, K. et al. Zanthoxylum bungeanum essential oil induces apoptosis of HaCaT human keratinocytes. J. Ethnopharmacol. 186, 351-361 (2016).

[15] Patiño, L., Prieto, R. & Cuca, S. Zanthoxylum genus as potential source of bioactive compounds. In Bioactive Compounds in Phytomedicine (ed. Rasooli I.) 185-218 (InTech, 2012).

[16] Tang, M. et al. A novel drug candidate for alzheimer’s disease treatment: gx-50 derived from Zanthoxylum bungeanum. J. Alzheimers Dis. 34, 203-213 (2013).

[17] Zhu, H., Huang, Y., Ji, X., Su, T. & Zhou, Z. Continuous existence of Zanthoxylum (Rutaceae) in Southwest China since the Miocene. Quatern. Int. 392, 224-232 (2016).

[18] Chinese Pharmacopoeia Commission . Chinese Pharmacopoeia (in Chinese) Shanghai: Science and Technology Press of Shanghai. 275 (1977).

[19] Chinese Pharmacopoeia Commission . Chinese Pharmacopoeia (in Chinese) Shanghai: Science and Technology Press of Shanghai. 149 (2010).

[20] Chinese Pharmacopoeia Commission . Chinese Pharmacopoeia (in Chinese) Shanghai: Science and Technology Press of Shanghai. 159-160 (2015).

[21] Xu, Q. et al. The draft genome of sweet orange (Citrus sinensis). Nat. Genet. 45, 59-66 (2013).

[22] Wang, X. et al. Genomic analyses of primitive, wild and cultivated citrus provide insights into asexual reproduction. Nat. Genet. 49, 765-772 (2017).

[23] Wu, G. A. et al. Genomics of the origin and evolution of Citrus. Nature 554, 1-20 (2018).

[24] Hu, J., Fan, J., Sun, Z. & Liu, S. NextPolish: a fast and efficient genome polishing tool for long read assembly. Bioinformatics 36, 2253-2255 (2019).

[25] Zeng, L. et al. Whole genomes and transcriptomes reveal adaptation and domestication of pistachio. Genome Biol. 20, 79 (2019).

[26] Yan, L. et al. The genome of Dendrobium officinale illuminates the biology of the important traditional Chinese orchid herb. Mol. Plant 8, 922-934 (2015).

[27] Zhang, J. et al. Allele-defined genome of the autopolyploid sugarcane Saccharum spontaneum L. Nat. Genet. 50, 1565-1573 (2018).

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

[29] Jaillon, O. et al. The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 449, 463-467 (2007).

[30] Jiao, Y. et al. Ancestral polyploidy in seed plants and angiosperms. Nature 473, 97-100 (2011).

[31] Sierro, N. et al. The tobacco genome sequence and its comparison with those of tomato and potato. Nat. Commun. 5, 3833- 3833 (2014).

[32] Bai, R. et al. Resequencing of 429 chickpea accessions from 45 countries provides insights into genome diversity, domestication and agronomic traits. Nat. Genet. 51, 857-864 (2019).

[33] Rizhsky, L. et al. Integrating metabolomics and transcriptomics data to discover a biocatalyst that can generate the amine precursors for alkamide biosynthesis. Plant J. 88, 775-793 (2016).

[34] Wang, Y. et al. Isobutylhydroxyamides from Zanthoxylum bungeanum and their suppression of NO production. Molecules 21, 1416 (2016).

[35] Wang, Y. et al. Isolation, structural characterization and neurotrophic activity of alkylamides from Zanthoxylum bungeanum. Nat. Prod. Commun. 12, 1121-1124 (2017).

[36] Kim, S. et al. Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species. Nat. Genet. 46, 270-278 (2014).

[37] Sugai, E., Morimitsu, Y. & Kubota, K. Quantitative analysis of sanshool compounds in Japanese pepper (Zanthoxylum piperitum DC.) and their pungent characteristics. Biosci. Biotech. Bioch. 69, 1958-1962 (2005).

[38] De Pascualteresa, S. & Sanchezballesta, M. T. Anthocyanins: from plant to health. Phytochem. Rev. 7, 281-299 (2008).

[39] Huang, D. et al. Subfunctionalization of the Ruby2-Ruby1 gene cluster during the domestication of citrus. Nat. Plants 4, 930-941 (2018).

[40] Gong, Y. et al. Chemical composition and antifungal activity of the fruit oil of Zanthoxylum bungeanum Maxim. (Rutaceae) from China. J. Essent. Oil Res. 21, 174-178 (2009).

[41] Njoroge, S. M., Koaze, H., Karanja, P. N. & Sawamura, M. Volatile constituents of redblush grapefruit (Citrus paradisi) and pummelo (Citrus grandis) peel essential oils from Kenya. J. Agr. Food Chem. 53, 9790-9794 (2005).

[42] Fujita, Y. et al. Biosynthesis of volatile terpenes that accumulate in the secretory cavities of young leaves of Japanese pepper (Zanthoxylum piperitum): isolation and functional characterization of monoterpene and sesquiterpene synthase genes. Plant Biotechnol. 34, 17-28 (2017).

[43] Rodríguez, A. et al. Terpene down-regulation triggers defense responses in transgenic orange leading to resistance against fungal pathogens. Plant Physiol. 164, 321-339 (2014).

[44] Rodríguez, A. et al. Engineering d-limonene synthase down-regulation in orange fruit induces resistance against the fungus Phyllosticta citricarpa through enhanced accumulation of monoterpene alcohols and activation of defence. Mol. Plant Pathol. 19, 2077-2093 (2018).

[45] Feng, S. et al. De novo transcriptome assembly of Zanthoxylum bungeanum using Illumina sequencing for evolutionary analysis and simple sequence repeat marker development. Sci. Rep. 7, 16754 (2017).

[46] Bennetzen, J. L. Mechanisms and rates of genome expansion and contraction in flowering plants. Genetica 115, 29-36 (2002).

[47] De Peer, Y. V., Maere, S. & Meyer, A. The evolutionary significance of ancient genome duplications. Nat. Rev. Genet. 10, 725-732 (2009).

[48] Schnable, P. S. et al. The B73 maize genome: complexity, diversity, and dynamics. Science 326, 1112-1115 (2009).

[49] Sun, X. et al. A chromosome-level genome assembly of garlic (Allium sativum) provides insights into genome evolution and allicin biosynthesis. Mol. Plant 13, 1328-1339 (2020).

[50] Qin, C. et al. Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization. Proc. Natl Acad. Sci. USA 111, 5135-5140 (2014).

[51] Tang, C. et al. The rubber tree genome reveals new insights into rubber production and species adaptation. Nat. Plants 2, 16073 (2016).

[52] The, B. T. et al. The draft genome of tropical fruit durian (Durio zibethinus). Nat. Genet. 49, 1633-1641 (2017).

[53] Zhang, J., Yu, C., Krishnaswamy, L., Peterson, T. Transposable elements as catalysts for chromosome rearrangements. In Plant Chromosome Engineering. Methods in Molecular Biology (Methods and Protocols) (ed. Birchler J) 701 (Humana Press, Totowa, NJ, 2011).

[54] Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler Transform. Bioinformatics 26, 589-595 (2009).

[55] Xu, H. et al. FastUniq: a fast de novo duplicates removal tool for paired short reads. PLoS ONE 7, e52249 (2012).

[56] Simao, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V. & Zdobnov, E. M. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 31, 3210-3212 (2015).

[57] Parra, G., Bradnam, K. & Korf, I. CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics 23, 1061-1067 (2007).

[58] Qu, S., Chen, J. & Jiang, N. Assessing genome assembly quality using the LTR Assembly Index (LAI). Nucleic Acids Res. 46, e126 (2018).

[59] Xu, Z. & Wang, H. LTR_FINDER: an efficient tool for the prediction of full-length LTR retrotransposons. Nucleic Acids Res. 35, 265-268 (2007).

[60] Han, Y. & Wessler, S. R. MITE-Hunter: a program for discovering miniature inverted-repeat transposable elements from genomic sequences. Nucleic Acids Res. 38, e199 (2010).

[61] Price, A. L., Jones, N. C. & Pevzner, P. A. De novo identification of repeat families in large genomes. Bioinformatics 21, i351-i358 (2005).

[62] Edgar, R. C. & Myers, E. W. PILER: identification and classification of genomic repeats. Bioinformatics 21, 152-158 (2005).

[63] Wicker, T. et al. A unified classification system for eukaryotic transposable elements. Nat. Revi. Genet. 8, 973-982 (2007).

[64] Bao, W., Kojima, K. & Kohany, O. Repbase Update, a database of repetitive elements in eukaryotic genomes. Mobile. DNA 6, 11- 11 (2015).

[65] Chen, N. Using repeatMasker to identify repetitive elements in genomic sequences. Curr. Protoc. Bioinformatics 25, 10.11-14.10.14 (2004).

[66] Burge, C. & Karlin, S. Prediction of complete gene structures in human genomic DNA. J. Mol. Biol. 268, 78-94 (1997).

[67] Stanke, M. & Waack, S. Gene prediction with a hidden Markov model and a new intron submodel. Bioinformatics 19, ii215-ii225 (2003).

[68] Majoros, W. H., Pertea, M. & Salzberg, S. L. TigrScan and GlimmerHMM: two open source ab initio eukaryotic gene-finders. Bioinformatics 20, 2878-2879 (2004).

[69] Blanco, E., Parra, G. & Guigó, R. Using geneid to identify genes. Curr. Protoc. Bioinformatics 4, 4-3 (2007).

[70] Keilwagen, J. et al. Using intron position conservation for homology-based gene prediction. Nucleic Acids Res. 44, e89 (2016).

[71] Kent, W. J. BLAT-the BLAST-like alignment tool. Genome Res. 12, 656-664 (2002).

[72] Haas, B. J. et al. Improving the Arabidopsis genome annotation using maximal transcript alignment assemblies. Nucleic Acids Res. 31, 5654-5666 (2003).

[73] Tang, S., Lomsadze, A. & Borodovsky, M. Identification of protein coding regions in RNA transcripts. Nucleic Acids Res. 43, e78 (2015).

[74] Haas, B. J. et al. Automated eukaryotic gene structure annotation using EVidenceModeler and the Program to Assemble Spliced Alignments. Genome Biol. 9, 1-22 (2008).

[75] Tatusov, R. L. et al. The COG database: an updated version includes eukaryotes. BMC Bioinformatics 4, 41- 41 (2003).

[76] Kanehisa, M. & Goto, S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27-30 (1999).

[77] Boeckmann, B. et al. The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003. Nucleic Acids Res. 31, 365-370 (2003).

[78] Conesa, A. et al. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21, 3674-3676 (2005).

[79] Finn, R. D. et al. Pfam: the protein families database. Nucleic Acids Res. 42, 222-230 (2014).

[80] Lowe, T. M. & Eddy, S. R. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25, 955-964 (1997).

[81] Nawrocki, E. P. & Eddy, S. R. Infernal 1.1: 100-fold faster RNA homology searches. Bioinformatics 29, 2933-2935 (2013).

[82] Griffiths-Jones, S., Bateman, A., Marshall, M., Khanna, A. & Eddy, S. R. Rfam: an RNA family database. Nucleic Acids Res. 31, 439-441 (2003).

[83] Li, L., Stoeckert, C. J. & Roos, D. S. OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res. 13, 2178-2189 (2003).

[84] Guindon, S. et al. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic. Biol 59, 307-321 (2010).

[85] Yang, Z. PAML 4: phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 24, 1586-1591 (2007).

[86] Wang, Y. et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 40, e49 (2012).

[87] Hasegawa, M., Kishino, H. & Yano, T. Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol. 22, 160-174 (1985).

[88] Liang, Q. et al. The genome assembly and annotation of yellowhorn (Xanthoceras sorbifolium Bunge). GigaScience 8, 1-15 (2019).

[89] Arabidopsis Genome Initiative . Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796-815 (2000).

[90] Tang, H. et al. Synteny and collinearity in plant genomes. Science 320, 486-488 (2008).

[91] Bolot, S. et al. The ‘inner circle’ of the cereal genomes. Curr. Opin. Plant biol. 12, 119-125 (2009).

[92] Murat, F., Armero, A., Pont, C., Klopp, C. & Salse, J. Reconstructing the genome of the most recent common ancestor of flowering plants. Nat. Genet. 49, 490-496 (2017).

[93] Gaut, B. S., Morton, B. R., McCaig, B. C. & Clegg, M. T. Substitution rate comparisons between grasses and palms: synonymous rate differences at the nuclear gene Adh parallel rate differences at the plastid gene rbcL. Proc. Natl Acad. Sci. USA 93, 10274-10279 (1996).

[94] Kong, Y. Btrim: a fast, lightweight adapter and quality trimming program for next-generation sequencing technologies. Genomics 98, 152-153 (2011).

[95] Pollier, J., Rombauts, S. & Goossens, A. Analysis of RNA-Seq data with TopHat and Cufflinks for genome-wide expression analysis of Jasmonate-Treated plants and plant cultures. Methods Mol. Biol. 1011, 305-315 (2013).

[96] Huang, H. et al. Global increase in DNA methylation during orange fruit development and ripening. Proc. Natl Acad. Sci. USA 116, 1430-1436 (2019).

[97] Punta, M. et al. The Pfam protein families database. Nucleic Acids Res. 30, 276-280 (2000).