The chromosome-level quality genome provides insights into the evolution of the biosynthesis genes for aroma compounds of Osmanthus fragrans

Abstract

Sweet osmanthus ( Osmanthus fragrans) is a very popular ornamental tree species throughout Southeast Asia and USA particularly for its extremely fragrant aroma. We constructed a chromosome-level reference genome of O. fragrans to assist in studies of the evolution, genetic diversity, and molecular mechanism of aroma development. A total of over 118 Gb of polished reads was produced from HiSeq (45.1 Gb) and PacBio Sequel (73.35 Gb), giving 100× depth coverage for long reads. The combination of Illumina-short reads, PacBio-long reads, and Hi-C data produced the final chromosome quality genome of O. fragrans with a genome size of 727 Mb and a heterozygosity of 1.45%. The genome was annotated using de novo and homology comparison and further refined with transcriptome data. The genome of O. fragrans was predicted to have 45,542 genes, of which 95.68% were functionally annotated. Genome annotation found 49.35% as the repetitive sequences, with long terminal repeats (LTR) being the richest (28.94%). Genome evolution analysis indicated the evidence of whole-genome duplication 15 million years ago, which contributed to the current content of 45,242 genes. Metabolic analysis revealed that linalool, a monoterpene is the main aroma compound. Based on the genome and transcriptome, we further demonstrated the direct connection between terpene synthases (TPSs) and the rich aromatic molecules in O. fragrans. We identified three new flower-specific TPS genes, of which the expression coincided with the production of linalool. Our results suggest that the high number of TPS genes and the flower tissue- and stage-specific TPS genes expressions might drive the strong unique aroma production of O. fragrans.

References

[1] Shang, F. D., Yin, Y. J. & Xiang, Q. B. The culture of sweet osmanthus in China. J. Henan Univ. Nat. Sci. 43, 136-139 (2003).

[2] Hao, R. M., Zang, D. K. & Xiang, Q. B. Investigation on natural resources of Osmanthus fragrans Lour. at Zhou luo cun in Hunan. Acta Hortic. Sin. 32, 926-929 (2005).

[3] Zang, D. K., Xiang, Q. B., Liu, Y. L. & Hao, R. M. The studying history and the application to International Cultivar Registration Authority of sweet osmanthus (Osmanthus fragrans Lour.). J. Plant Resour. Environ. 12, 49-53 (2003).

[4] Deng, C. H., Song, G. X. & Hu, Y. M. Application of HS-SPME and GC-MS to characterization of volatile compounds emitted from Osmanthus flowers. Ann. Chim. 94, 921-927 (2004).

[5] Xin, H. P. et al. Characterization of volatile compounds in flowers from four groups of sweet osmanthus (Osmanthus fragrans) cultivars. Can. J. Plant Sci. 93, 923-931 (2013).

[6] Cai, X. et al. & W, C.Y. Analysis of aroma-active compounds in three sweet osmanthus (Osmanthus fragrans) cultivars by gas-chromatography olfactometry and GC-mass spectrometry. J. Zhejiang. Univ. Sci. B 15, 638-648 (2014).

[7] Hu, C. D. et al. Essential oil composition of Osmanthus fragrans varieties by GC-MS and heuristic evolving latent projections. Chromatographia 70, 1163-1169 (2009).

[8] Wang, L. M. et al. Variations in the components of Osmanthus fragrans Lour. Essential oil at different stages of flowering. Food Chem. 114, 233-236 (2009).

[9] Hu, B. F., Guo, X. L., Xiao, P. & Luo, L. P. Chemical composition comparison of the essential oil from four groups of Osmanthus fragrans Lour. flowers. J. Essent. Oil Plants 15, 832-838 (2012).

[10] Lei, G. M. et al. Water-soluble essential oil components of fresh flowers of Osmanthus fragrans lour.. J. Essent. Oil Res. 28, 177-184 (2016).

[11] Shang, F. D., Yin, Y. J. & Zhang, T. The RAPD analysis of 17 Osmanthus fragrans cultivars in Henan province. Acta Hortic. Sin. 31, 685-687 (2004).

[12] Yuan, W. J., Han, Y. J., Dong, M. F. & Shang, F. D. Assessment of genetic diversity and relationships among Osmanthus fragrans cultivars using AFLP markers. Electron. J. Biotechnol. 14, 2-3 (2011).

[13] Hu, W., Luo, Y., Yang, Y., Zhang, Z. Y. & Fan, D. M. Genetic diversity and population genetic structure of wild sweet osmanthus revealed by microsatellite markers. Acta Hortic. Sin. 41, 1427-1435 (2014).

[14] Yuan, W. J., Li, Y., Ma, Y. F., Han, Y. J. & Shang, F. D. Isolation and characterization of microsatellite markers for Osmanthus fragrans (Oleaceae) using 454 sequencing technology. Genet. Mol. Res. 14, 17154-17158 (2015).

[15] Han, Y. J. et al. cDNA-AFLP analysis on 2 Osmanthus fragrans cultivars with different flower color and molecular characteristics of MYB1gene. Trees 29, 931-940 (2015).

[16] He, Y. X., Yuan, W. J., Dong, M. F., Han, Y. J. & Shang, F. D. The first genetic map in sweet osmanthus (Osmanthus fragrans Lour.) using specific locus amplified fragment sequencing. Front. Plant Sci. 8, 1621 (2017).

[17] Zhang, X. S., Pei, J. J., Zhao, L. G., Tang, F. & Fang, X. Y. RNA-Seq analysis and comparison of the enzymes involved in ionone synthesis of three cultivars of Osmanthus. J. Asian Nat. Prod. Res. 9, 1-13 (2018).

[18] Yang, X. L. et al. Transcriptomic analysis of the candidate genes related to aroma formation in Osmanthus fragrans. Molecules 23, 1604 (2018).

[19] Xu, C. et al. Cloning and expression analysis of MEP pathway enzyme-encoding genes in Osmanthus fragrans. Genes 7, 78 (2016).

[20] Zeng, X. L. et al. Emission and accumulation of monoterpene and the key terpene synthase (TPS) associated with monoterpene biosynthesis in Osmanthus fragrans Lour.. Front. Plant Sci. 6, 1232 (2015).

[21] Baldermann, S. et al. Functional characterization of a carotenoid cleavage dioxygenase 1 and its relation to the carotenoid accumulation and volatile emission during the floral development of Osmanthus fragrans Lour.. J. Exp. Bot. 61, 2967-2977 (2010).

[22] Baldermann, S., Kato, M., Fleischmann, P. & Watanabe, N. Biosynthesis of α- and β-ionone, prominent scent compounds, in flowers of Osmanthus fragrans. Acta Biochim. Pol. 59, 79-81 (2012).

[23] Han, Y. J., Liu, L. X., Dong, M. F., Yuan, W. J. & Shang, F. D. cDNA cloning of the phytoene synthase (PSY) and expression analysis of PSY and carotenoid cleavage dioxygenase genes in Osmanthus fragrans. Biologia 68, 258-263 (2013).

[24] Han, Y. J. et al. Differential expression of carotenoid-related genes determines diversified carotenoid coloration in flower petal of Osmanthus fragrans. Tree. Genet. Genom. 10, 329-338 (2014).

[25] Zhang, C., Wang, Y. G., Fu, J. X., Bao, Z. Y. & Zhao, H. B. Transcriptomic analysis and carotenogenic gene expression related to petal coloration in Osmanthus fragrans ‘Yanhong Gui’. Trees 30, 1207-1223 (2016).

[26] Mu, H. N. et al. Transcriptome sequencing and analysis of sweet osmanthus (Osmanthus fragrans Lour.). Genes. Genom. 36, 777-788 (2014).

[27] Han, Y. J. et al. Characterization of OfWRKY3, a transcription factor that positively regulates the carotenoid cleavage dioxygenase gene OfCCD4 in Osmanthus fragrans. Plant Mol. Biol. 91, 485-496 (2016).

[28] Wang, L. et al. Analysis of the main active ingredients and bioactivities of essential oil from Osmanthus fragrans Var. thunbergii using a complex network approach. BMC Syst. Biol. 11, 144 (2017).

[29] Guillaume, M. & Carl, K. A fast, lock-free approach for efficient parallel counting of occurrences of K-Mers. Bioinformatics 27, 764-770 (2011).

[30] Luo, R. et al. SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. Gigascience 27, 18 (2012).

[31] Hu, X. et al. pIRS: Profile-based Illumina pair-end reads simulator. Bioinformatics 28, 1533-1535 (2012).

[32] Chin, C. S. et al. Phased diploid genome assembly with single-molecule real-time sequencing. Nat. Methods 13, 1050-1054 (2016).

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

[34] Pryszcz, L. P. & Gabaldón, T. Redundans: an assembly pipeline for highly heterozygous genomes. Nucleic Acids Res. 44, e113 (2016).

[35] Simão, 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).

[36] Stanke, M., Steinkamp, R., Waack, S. & Morgenstern, B. AUGUSTUS: a web server for gene finding in eukaryotes. Nucleic Acids Res. 32, W309-W312 (2004).

[37] Chen, N. Using RepeatMasker to identify repetitive elements in genomic sequences. Curr. Protoc. Bioinforma. 4, 10 (2004).

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

[39] Nussbaumer, T. et al. MIPS PlantsDB: a database framework for comparative plant genome research. Nucleic Acids Res. 41, D1144-D1151 (2013).

[40] Kalvari, I. et al. Rfam 13.0: shifting to a genome-centric resource for non-coding RNA families. Nucleic Acids Res. 46, D335-D342 (2017).

[41] Camacho, C. et al. BLAST+: architecture and applications. BMC Bioinforma. 10, 421 (2009).

[42] 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).

[43] Lagesen, K., Hallin, P., Rødland, E. A., Staerfeldt, H. H. & Rognes, T. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 35, 3100-3108 (2007).

[44] Birney, E. & Durbin, R. Using GeneWise in the Drosophila annotation experiment. Genome Res. 10, 547-548 (2000).

[45] Blanco, E., Parra, G. & Guigó, R. Using geneid to identify genes. Curr. Protoc. Bioinforma. 4, 1-28 (2007).

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

[47] Bromberg, Y. & Rost, B. SNAP: predict effect of non-synonymous polymorphisms on function. Nucleic Acids Res. 35, 3823-3835 (2007).

[48] Haas, B. J. et al. Automated eukaryotic gene structure annotation using EVidenceModeler and the program to assemble spliced alignments. Genome Biol. 9, R7 (2008).

[49] Moriya, Y., Itoh, M., Okuda, S., Yoshizawa, A. C. & Kanehisa, M. KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res. 35, W182-W185 (2007).

[50] Quevillon, E. et al. InterProScan: protein domains identifier. Nucleic Acids Res. 33, 116-120 (2005).

[51] De Bodt, S., Maere, S. & Van de Peer, Y. Genome duplication and the origin of angiosperms. Trends Ecol. Evol. 20, 591-597 (2005).

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

[53] Ciu, L. Y. et al. Widespread genome duplications throughout the history of flowering plants. Genome Res. 16, 738-749 (2006).

[54] Casneuf, T., De Bodt, S., Raes, J., Maere, S. & Van de Peer, Y. Nonrandom divergence of gene expression following gene and genome duplications in the flowering plants Arabidopsis thaliana. Genome Biol. 7, R13 (2006).

[55] Xu, Y. C. et al. The differentiation and development of pistils of hermaphrodites and pistillodes of males in androdioecious Osmanthus fragrans L. and implications for the evolution to androdioecy. Plant Syst. Evol. 300, 843-849 (2014).

[56] Huang, X. et al. High-throughput genotyping by whole-genome resequencing. Genome Res. 19, 1068-1076 (2009).

[57] Tholl, D. Terpene synthases and the regulation, diversity and biological roles of terpene metabolism. Curr. Opin. Plant. Biol. 9, 297-304 (2006).

[58] Kaplan, N. & Dekker, J. High-throughput genome scaffolding from in vivo DNA interaction frequency. Nat. Biotechnol. 31, 1143-1147 (2013).

[59] Marie-Nelly, H. et al. High-quality genome (re) assembly using chromosomal contact data. Nat. Commun. 5, 5695 (2014).

[60] Jibran, R. et al. Chromosome-scale scaffolding of the black raspberry (Rubus occidentalis L.) genome based on chromatin interaction data. Hortic. Res. 5, 18008 (2018).