Haplotype‐resolved genome assembly of Phanera championii reveals molecular mechanisms of flavonoid synthesis and adaptive evolution

The Plant JournalEarly View Original Article Haplotype-resolved genome assembly of Phanera championii reveals molecular mechanisms of flavonoid synthesis and adaptive evolution Yongbin Lu, Yongbin Lu orcid.org/0000-0002-8635-3354 State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, Nanning, 530004 China Guangxi Key Laboratory of Plant Conservation and Restoration Ecology in Karst Terrain, Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and the Chinese Academy of Sciences, Yanshan, Guilin, 541006 ChinaSearch for more papers by this authorXiao Chen, Xiao Chen Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, 518120 ChinaSearch for more papers by this authorHang Yu, Hang Yu State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, Nanning, 530004 China Key Laboratory of Crop Cultivation and Physiology, Education Department of Guangxi Zhuang Autonomous Region, Guangxi University, Nanning, 530004 ChinaSearch for more papers by this authorChao Zhang, Chao Zhang State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, Nanning, 530004 China Key Laboratory of Crop Cultivation and Physiology, Education Department of Guangxi Zhuang Autonomous Region, Guangxi University, Nanning, 530004 ChinaSearch for more papers by this authorYajie Xue, Yajie Xue State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, Nanning, 530004 China Key Laboratory of Crop Cultivation and Physiology, Education Department of Guangxi Zhuang Autonomous Region, Guangxi University, Nanning, 530004 ChinaSearch for more papers by this authorQiang Zhang, Qiang Zhang Guangxi Key Laboratory of Plant Conservation and Restoration Ecology in Karst Terrain, Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and the Chinese Academy of Sciences, Yanshan, Guilin, 541006 ChinaSearch for more papers by this authorHaifeng Wang, Corresponding Author Haifeng Wang [email protected] orcid.org/0000-0001-7213-927X State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, Nanning, 530004 China Key Laboratory of Crop Cultivation and Physiology, Education Department of Guangxi Zhuang Autonomous Region, Guangxi University, Nanning, 530004 China For correspondence (e-mail [email protected]).Search for more papers by this author Yongbin Lu, Yongbin Lu orcid.org/0000-0002-8635-3354 State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, Nanning, 530004 China Guangxi Key Laboratory of Plant Conservation and Restoration Ecology in Karst Terrain, Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and the Chinese Academy of Sciences, Yanshan, Guilin, 541006 ChinaSearch for more papers by this authorXiao Chen, Xiao Chen Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, 518120 ChinaSearch for more papers by this authorHang Yu, Hang Yu State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, Nanning, 530004 China Key Laboratory of Crop Cultivation and Physiology, Education Department of Guangxi Zhuang Autonomous Region, Guangxi University, Nanning, 530004 ChinaSearch for more papers by this authorChao Zhang, Chao Zhang State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, Nanning, 530004 China Key Laboratory of Crop Cultivation and Physiology, Education Department of Guangxi Zhuang Autonomous Region, Guangxi University, Nanning, 530004 ChinaSearch for more papers by this authorYajie Xue, Yajie Xue State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, Nanning, 530004 China Key Laboratory of Crop Cultivation and Physiology, Education Department of Guangxi Zhuang Autonomous Region, Guangxi University, Nanning, 530004 ChinaSearch for more papers by this authorQiang Zhang, Qiang Zhang Guangxi Key Laboratory of Plant Conservation and Restoration Ecology in Karst Terrain, Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and the Chinese Academy of Sciences, Yanshan, Guilin, 541006 ChinaSearch for more papers by this authorHaifeng Wang, Corresponding Author Haifeng Wang [email protected] orcid.org/0000-0001-7213-927X State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, Nanning, 530004 China Key Laboratory of Crop Cultivation and Physiology, Education Department of Guangxi Zhuang Autonomous Region, Guangxi University, Nanning, 530004 China For correspondence (e-mail [email protected]).Search for more papers by this author First published: 03 January 2024 https://doi.org/10.1111/tpj.16620Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat SUMMARY Phanera championii is a medicinal liana plant that has successfully adapted to hostile karst habitats. Despite extensive research on its medicinal components and pharmacological effects, the molecular mechanisms underlying the biosynthesis of critical flavonoids and its adaptation to karst habitats remain elusive. In this study, we performed high-coverage PacBio and Hi-C sequencing of P. championii, which revealed its high heterozygosity and phased the genome into two haplotypes: Hap1 (384.60 Mb) and Hap2 (383.70 Mb), encompassing a total of 58 612 annotated genes. Comparative genomes analysis revealed that P. championii experienced two whole-genome duplications (WGDs), with approximately 59.59% of genes originating from WGD events, thereby providing a valuable genetic resource for P. championii. Moreover, we identified a total of 112 genes that were strongly positively selected. Additionally, about 81.60 Mb of structural variations between the two haplotypes. The allele-specific expression patterns suggested that the dominant effect of P. championii was the elimination of deleterious mutations and the promotion of beneficial mutations to enhance fitness. Moreover, our transcriptome and metabolome analysis revealed alleles in different tissues or different haplotypes collectively regulate the synthesis of flavonoid metabolites. In summary, our comprehensive study highlights the significance of genomic and morphological adaptation in the successful adaptation of P. championii to karst habitats. The high-quality phased genomes obtained in this study serve as invaluable genomic resources for various applications, including germplasm conservation, breeding, evolutionary studies, and elucidation of pathways governing key biological traits of P. championii. CONFLICT OF INTEREST The authors declare no conflicts of interest regarding this work. Supporting Information Filename Description tpj16620-sup-0001-FigureS1-S23.pdfPDF document, 51 MB Figure S1. The phenotype and habitat of Phanera championii in karst. Photo provided by Zhaochen Lu. Figure S2. Genome survey of Phanera championii on the distribution of 27-mer. Figure S3. Hi-C interaction heat map showing 14 clusters representing pseudochromosomes in the two haplotypes. Figure S4. Dot-plot of synteny blocks in Phanera championii. The dot-plot is identified and colored based on their Ks values. Figure S5. Dot-plot of synteny blocks between Phanera championii and Bauhinia variegata. The dot-plot is identified and colored based on their Ks values. Figure S6. Violin and box plots showing Ks of the genes originating from five modes of gene duplication. Figure S7. GO functional enrichment of unique gene families in Phanera championii. Figure S8. GO functional enrichment of contracted gene families in Phanera championii. Figure S9. Categories and proportions of different gene duplication modes of the expanded gene families in Phanera championii. Figure S10. The phylogenetic tree of the CHS gene family and its tandem repeats in Phanera championii. (a) The phylogenetic tree of the CHS gene families among 12 selected species. (b) Chromosome locations of tandem CHS genes on Chr02 and Chr05. Figure S11. The phylogenetic tree of the F3H gene family and its tandem repeats in Phanera championii. (a) The phylogenetic tree of the F3H gene families among 12 selected species. (b) Chromosome locations of tandem F3H genes on Chr04. Figure S12. GO functional enrichment of genes subject to positive selection in two haplotype genomes of Phanera championii. Figure S13. Dot-plot of synteny blocks of two haplotype genomes in Phanera championii. The dot-plot is identified and colored based on their Ks values. Figure S14. Visualization of structural variations between haplotypes. Figure S15. PCA analysis among transcriptome samples in Phanera championii. Figure S16. Allele expression in different tissues in the two haplotype genomes of Phanera championii. Figure S17. Comparison of Ka/Ks ratio and Ks value between ASEs and non-ASEs in the leaves. Figure S18. Comparison of Ka/Ks ratio and Ks value between ASEs and non-ASEs in the lianas. Figure S19. Comparison of Ka/Ks ratio and Ks value between ASEs and non-ASEs in the roots. Figure S20. Summarizing the expression of homologous genes related to flavonoid biosynthesis. Figure S21. Flavonoid metabolites between different tissues. Figure S22. Correlation between high-impact alleles and ASEs, P values were tested by the hypergeometric test. Figure S23. Examples of structural variation leading to differential expression of alleles. tpj16620-sup-0002-TableS1-S23.xlsxExcel 2007 spreadsheet , 8.1 MB Table S1. BUSCO assessment of assembly in Phanera championii. Table S2. Annotation of repeats between two haplotypes in Phanera championii. Table S3. Chromosome length and number of genes in haplotype genomes. Table S4. BUSCO assessment of gene annotation. Table S5. Predicted transcription factors in the Phanera championii genome. Table S6. Classification of transcription factors in Phanera championii genome. Table S7. Genomic information for comparative genomic analysis. Table S8. GO functional enrichment of expanded gene families in Phanera championii. Table S9. Classification of duplicated types of structural genes in the flavonoid synthesis pathway in Phanera championii. Table S10. List of genes subject to positive selection in two haplotype genomes. Table S11. GO functional enrichment of genes subject to positive selection in Phanera championii. Table S12. Statistics of structural variations between two haplotypes in Phanera championii. Table S13. Summary of transcriptome sequencing and alignment of different tissues. Table S14. Ka/Ks calculation in ASE pairs across different tissues in Phanera championii. Table S15. Ka/Ks calculation in non-ASE pairs across different tissues in Phanera championii. Table S16. Statistics of flavonoid metabolites between different tissues. Table S17. Information of transcription factors associated with flavonoid biosynthesis. Table S18. Prediction of MYB111 binding sites associated with flavonoid synthesis in Phanera championii. Table S19. Expression correlation analysis between the expression of stomatal opening and closing related genes and transcription factors. Table S20. Expression correlation analysis between intracellular calcium homeostasis-related genes and transcription factors. Table S21. Origins of expanded transcription factors associated with the regulation of stomata. Table S22. Origins of expanded transcription factors associated with intracellular calcium homeostasis. Table S23. Origins of expanded genes regulating intracellular calcium homeostasis. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. REFERENCES Agati, G., Azzarello, E., Pollastri, S. & Tattini, M. (2012) Flavonoids as antioxidants in plants: location and functional significance. Plant Science, 196, 67–76. 10.1016/j.plantsci.2012.07.014 CASPubMedWeb of Science®Google Scholar Bao, W., Kojima, K.K. & Kohany, O. (2015) Repbase update, a database of repetitive elements in eukaryotic genomes. Mobile DNA, 6, 1–6. 10.1186/s13100-015-0041-9 Web of Science®Google Scholar Bellucci, E., Bitocchi, E., Rau, D., Rodriguez, M., Biagetti, E., Giardini, A. et al. (2014) Genomics of origin, domestication and evolution of Phaseolus vulgaris. In: R. Tuberosa, A. Graner & E. Frison (Eds.) Genomics of plant genetic resources. Dordrecht: Springer, pp. 483–507. 10.1007/978-94-007-7572-5_20 Google Scholar Bertioli, D.J., Cannon, S.B., Froenicke, L., Huang, G., Farmer, A.D., Cannon, E.K. et al. (2016) The genome sequences of Arachis duranensis and Arachis ipaensis, the diploid ancestors of cultivated peanut. Nature Genetics, 48, 438–446. 10.1038/ng.3517 CASPubMedWeb of Science®Google Scholar Birney, E., Clamp, M. & Durbin, R. (2004) GeneWise and genomewise. Genome Research, 14, 988–995. 10.1101/gr.1865504 CASPubMedWeb of Science®Google Scholar Boratyn, G.M., Camacho, C., Cooper, P.S., Coulouris, G., Fong, A., Ma, N. et al. (2013) BLAST: a more efficient report with usability improvements. Nucleic Acids Research, 41, W29–W33. 10.1093/nar/gkt282 PubMedWeb of Science®Google Scholar Bruneau, A., Mercure, M., Lewis, G.P. & Herendeen, P.S. (2008) Phylogenetic patterns and diversification in the caesalpinioid legumes. Botany, 86, 697–718. 10.1139/B08-058 CASWeb of Science®Google Scholar Calvillo-Canadell, L. & Cevallos-Ferriz, S. (2005) Diverse assemblage of Eocene and Oligocene leguminosae from Mexico. International Journal of Plant Sciences, 166, 671–692. 10.1086/430096 Web of Science®Google Scholar Cantalapiedra, C.P., Hernández-Plaza, A., Letunic, I., Bork, P. & Huerta-Cepas, J. (2021) eggNOG-mapper v2: functional annotation, orthology assignments, and domain prediction at the metagenomic scale. Molecular Biology and Evolution, 38, 5825–5829. 10.1093/molbev/msab293 CASPubMedWeb of Science®Google Scholar Castro-Mondragon, J.A., Riudavets-Puig, R., Rauluseviciute, I., Berhanu Lemma, R., Turchi, L., Blanc-Mathieu, R. et al. (2022) JASPAR 2022: the 9th release of the open-access database of transcription factor binding profiles. Nucleic Acids Research, 50, D165–D173. 10.1093/nar/gkab1113 CASPubMedWeb of Science®Google Scholar Chalhoub, B., Denoeud, F., Liu, S., Parkin, I.A., Tang, H., Wang, X. et al. (2014) Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome. Science, 345, 950–953. 10.1126/science.1253435 CASPubMedWeb of Science®Google Scholar Chen, C., Chen, H., Zhang, Y., Thomas, H.R., Frank, M.H., He, Y. et al. (2020) TBtools: an integrative toolkit developed for interactive analyses of big biological data. Molecular Plant, 13, 1194–1202. 10.1016/j.molp.2020.06.009 CASPubMedWeb of Science®Google Scholar Chen, M., Ma, Y., Wu, S., Zheng, X., Kang, H., Sang, J. et al. (2021) Genome warehouse: a public repository housing genome-scale data. Genomics, Proteomics & Bioinformatics, 19, 584–589. 10.1016/j.gpb.2021.04.001 PubMedWeb of Science®Google Scholar Chen, N. (2004) Using repeat masker to identify repetitive elements in genomic sequences. Current Protocols in Bioinformatics, 5, 4.10.1–14.10.14. 10.1002/0471250953.bi0410s05 Google Scholar Chen, S., Zhou, Y., Chen, Y. & Gu, J. (2018) fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics, 34, i884–i890. 10.1093/bioinformatics/bty560 PubMedWeb of Science®Google Scholar Chen, T., Chen, X., Zhang, S., Zhu, J., Tang, B., Wang, A. et al. (2021) The genome sequence archive family: toward explosive data growth and diverse data types. Genomics, Proteomics & Bioinformatics, 19, 578–583. 10.1016/j.gpb.2021.08.001 PubMedWeb of Science®Google Scholar Cheng, H., Concepcion, G.T., Feng, X., Zhang, H. & Li, H. (2021) Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nature Methods, 18, 170–175. 10.1038/s41592-020-01056-5 CASPubMedWeb of Science®Google Scholar Cingolani, P., Platts, A., Wang, L.L., Coon, M., Nguyen, T., Wang, L. et al. (2012) A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly, 6, 80–92. 10.4161/fly.19695 CASPubMedWeb of Science®Google Scholar Clements, R., Sodhi, N.S., Schilthuizen, M. & Ng, P.K. (2006) Limestone karsts of Southeast Asia: imperiled arks of biodiversity. Bioscience, 56, 733–742. 10.1641/0006-3568(2006)56[733:LKOSAI]2.0.CO;2 Web of Science®Google Scholar CNCB-NGDC Members and Partners. (2022) Database resources of the National Genomics Data Center, China National Center for Bioinformation in 2022. Nucleic Acids Research, 50, D27–D38. 10.1093/nar/gkab951 PubMedWeb of Science®Google Scholar CNCB-NGDC Members and Partners. (2023) Database resources of the National Genomics Data Center, China National Center for Bioinformation in 2023. Nucleic Acids Research, 51, D18–D28. 10.1093/nar/gkac1073 PubMedWeb of Science®Google Scholar Danecek, P., Bonfield, J.K., Liddle, J., Marshall, J., Ohan, V., Pollard, M.O. et al. (2021) Twelve years of SAMtools and BCFtools. GigaScience, 10, giab008. 10.1093/gigascience/giab008 PubMedWeb of Science®Google Scholar De Bie, T., Cristianini, N., Demuth, J.P. & Hahn, M.W. (2006) CAFE: a computational tool for the study of gene family evolution. Bioinformatics, 22, 1269–1271. 10.1093/bioinformatics/btl097 CASPubMedWeb of Science®Google Scholar Doyle, J. (1987) Genomic plant DNA preparation from fresh tissue-CTAB method. Phytochemical Bulletin, 19, 11. 10.1007/s10592-005-9073-x CASWeb of Science®Google Scholar Edgar, R.C. (2021) MUSCLE v5 enables improved estimates of phylogenetic tree confidence by ensemble bootstrapping. bioRxiv. https://doi.org/10.1101/2021.06.20.449169 10.1101/2021.06.20.449169 Google Scholar Emms, D.M. & Kelly, S. (2019) OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biology, 20, 1–14. 10.1186/s13059-019-1832-y PubMedWeb of Science®Google Scholar Feng, C., Wang, J., Wu, L., Kong, H., Yang, L., Feng, C. et al. (2020) The genome of a cave plant, Primulina huaijiensis, provides insights into adaptation to limestone karst habitats. New Phytologist, 227, 1249–1263. 10.1111/nph.16588 CASPubMedWeb of Science®Google Scholar Flynn, J.M., Hubley, R., Goubert, C., Rosen, J., Clark, A.G., Feschotte, C. et al. (2020) RepeatModeler2 for automated genomic discovery of transposable element families. Proceedings of the National Academy of Sciences of the United States of America, 117, 9451–9457. 10.1073/pnas.1921046117 CASPubMedWeb of Science®Google Scholar Fraga, C.G., Clowers, B.H., Moore, R.J. & Zink, E.M. (2010) Signature-discovery approach for sample matching of a nerve-agent precursor using liquid chromatography-mass spectrometry, XCMS, and chemometrics. Analytical Chemistry, 82, 4165–4173. 10.1021/ac1003568 CASPubMedWeb of Science®Google Scholar Goel, M., Sun, H., Jiao, W.B. & Schneeberger, K. (2019) SyRI: finding genomic rearrangements and local sequence differences from whole-genome assemblies. Genome Biology, 20, 1–13. 10.1186/s13059-019-1911-0 PubMedWeb of Science®Google Scholar Guk, J.Y., Jang, M.J., Choi, J.W., Lee, Y.M. & Kim, S. (2022) De novo phasing resolves haplotype sequences in complex plant genomes. Plant Biotechnology Journal, 20, 1031–1041. 10.1111/pbi.13815 CASPubMedWeb of Science®Google Scholar Guo, Z., Song, G., Liu, Z., Qu, X., Chen, R., Jiang, D. et al. (2015) Global epigenomic analysis indicates that epialleles contribute to allele-specific expression via allele-specific histone modifications in hybrid rice. BMC Genomics, 16, 1–9. 10.1186/s12864-015-1454-z PubMedWeb of Science®Google Scholar Gutiérrez, F., Parise, M., De Waele, J. & Jourde, H. (2014) A review on natural and human-induced geohazards and impacts in karst. Earth-Science Reviews, 138, 61–88. 10.1016/j.earscirev.2014.08.002 Web of Science®Google Scholar Hamilton, J.P. & Robin Buell, C. (2012) Advances in plant genome sequencing. The Plant Journal, 70, 177–190. 10.1111/j.1365-313X.2012.04894.x CASPubMedWeb of Science®Google Scholar Hane, J.K., Ming, Y., Kamphuis, L.G., Nelson, M.N., Garg, G., Atkins, C.A. et al. (2017) A comprehensive draft genome sequence for lupin (Lupinus angustifolius), an emerging health food: insights into plant–microbe interactions and legume evolution. Plant Biotechnology Journal, 15, 318–330. 10.1111/pbi.12615 CASPubMedWeb of Science®Google Scholar Hasing, T., Tang, H., Brym, M., Khazi, F., Huang, T. & Chambers, A.H. (2020) A phased Vanilla planifolia genome enables genetic improvement of flavour and production. Nature Food, 1, 811–819. 10.1038/s43016-020-00197-2 CASPubMedGoogle Scholar Herendeen, P.S. & Manchester, S.R. (2004) Fruits and foliage of Cercis from the Late Eocene of Oregon (abstract). Botany 2004 meeting, Salt Lake City, Utah. Available at: http://www.2004.botonyconterene.org. Google Scholar Herendeen, P.S. (1992) The fossil history of the Leguminosae: phylogenetic and biogeographic implications. In: Advances in legume systematics, part 4. The fossil record. Kew: Royal Botanic Gardens, pp. 303–316. Google Scholar Hui, Y., Jianhong, L., Jiarui, C. & Jianhua, C. (2015) Soil calcium speciation at different geomorphological positions in the Yaji karst experimental site in Guilin, China. Journal of Resources and Ecology, 6, 224–229. 10.5814/j.issn.1674-764X.2015.04.005 Google Scholar Jiang, Z., Lian, Y. & Qin, X. (2014) Rocky desertification in Southwest China: impacts, causes, and restoration. Earth-Science Reviews, 132, 1–12. 10.1016/j.earscirev.2014.01.005 Web of Science®Google Scholar Jiao, Y. (2018) Double the genome, double the fun: genome duplications in angiosperms. Molecular Plant, 11, 357–358. 10.1016/j.molp.2018.02.009 CASPubMedWeb of Science®Google Scholar Jin, J., Tian, F., Yang, D., Meng, Y., Kong, L., Luo, J. et al. (2016) PlantTFDB 4.0: toward a central hub for transcription factors and regulatory interactions in plants. Nucleic Acids Research, 45, gkw982. Web of Science®Google Scholar Kang, M., Tao, J., Wang, J., Ren, C., Qi, Q., Xiang, Q.Y. et al. (2014) Adaptive and nonadaptive genome size evolution in karst endemic flora of China. New Phytologist, 202, 1371–1381. 10.1111/nph.12726 CASPubMedWeb of Science®Google Scholar Kang, S.-H., Pandey, R.P., Lee, C.-M., Sim, J.-S., Jeong, J.-T., Choi, B.-S. et al. (2020) Genome-enabled discovery of anthraquinone biosynthesis in Senna tora. Nature Communications, 11, 5875. 10.1038/s41467-020-19681-1 CASPubMedWeb of Science®Google Scholar Katoh, K. & Standley, D.M. (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution, 30, 772–780. 10.1093/molbev/mst010 CASPubMedWeb of Science®Google Scholar Kim, D., Paggi, J.M., Park, C., Bennett, C. & Salzberg, S.L. (2019) Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nature Biotechnology, 37, 907–915. 10.1038/s41587-019-0201-4 CASPubMedWeb of Science®Google Scholar Klopfenstein, D., Zhang, L., Pedersen, B.S., Ramírez, F., Warwick Vesztrocy, A., Naldi, A. et al. (2018) GOATOOLS: a python library for gene ontology analyses. Scientific Reports, 8, 1–17. 10.1038/s41598-018-28948-z CASPubMedWeb of Science®Google Scholar Kozlov, A.M., Darriba, D., Flouri, T., Morel, B. & Stamatakis, A. (2019) RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics, 35, 4453–4455. 10.1093/bioinformatics/btz305 CASPubMedWeb of Science®Google Scholar Lan, Y., Sun, J., Tian, R., Bartlett, D.H., Li, R., Wong, Y.H. et al. (2017) Molecular adaptation in the world's deepest-living animal: insights from transcriptome sequencing of the hadal amphipod Hirondellea gigas. Molecular Ecology, 26, 3732–3743. 10.1111/mec.14149 CASPubMedWeb of Science®Google Scholar Lavin, M., Herendeen, P.S. & Wojciechowski, M.F. (2005) Evolutionary rates analysis of Leguminosae implicates a rapid diversification of lineages during the tertiary. Systematic Biology, 54, 575–594. 10.1046/j.1095-8312.2002.00091.x PubMedWeb of Science®Google Scholar Li, B., Fan, R., Guo, S., Wang, P., Zhu, X., Fan, Y. et al. (2019) The Arabidopsis MYB transcription factor, MYB111 modulates salt responses by regulating flavonoid biosynthesis. Environmental and Experimental Botany, 166, 103807. 10.1016/j.envexpbot.2019.103807 CASWeb of Science®Google Scholar Li, H. (2018) Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics, 34, 3094–3100. 10.1093/bioinformatics/bty191 CASPubMedWeb of Science®Google Scholar Li, H. & Durbin, R. (2009) Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics, 25, 1754–1760. 10.1093/bioinformatics/btp324 CASPubMedWeb of Science®Google Scholar Li, H., Wu, L., Dong, Z., Jiang, Y., Jiang, S., Xing, H. et al. (2021) Haplotype-resolved genome of diploid ginger (Zingiber officinale) and its unique gingerol biosynthetic pathway. Horticulture Research, 8, 189. 10.1038/s41438-021-00627-7 CASPubMedWeb of Science®Google Scholar Li, J., Shen, J., Wang, R., Chen, Y., Zhang, T., Wang, H. et al. (2023) The nearly complete assembly of the Cercis chinensis genome and Fabaceae phylogenomic studies provide insights into new gene evolution. Plant Communications, 4, 100422. 10.1016/j.xplc.2022.100422 CASPubMedGoogle Scholar Li, Q., Yu, L., Deng, Y., Li, W., Li, M. & Cao, J. (2007) Leaf epidermal characters of Lonicera japonica and Lonicera confuse and their ecology adaptation. Journal of Forestry Research, 18, 103–108. 10.1007/s11676-007-0020-1 CASGoogle Scholar Lian, Y., You, G.J.Y., Lin, K., Jiang, Z., Zhang, C. & Qin, X. (2015) Characteristics of climate change in southwest China karst region and their potential environmental impacts. Environmental Earth Sciences, 74, 937–944. 10.1007/s12665-014-3847-8 CASWeb of Science®Google Scholar Liu, J., Li, M., Zhang, Q., Wei, X. & Huang, X. (2020) Exploring the molecular basis of heterosis for plant breeding. Journal of Integrative Plant Biology, 62, 287–298. 10.1111/jipb.12804 PubMedWeb of Science®Google Scholar Liu, M., Cao, B., Zhou, S. & Liu, Y. (2012) Responses of the flavonoid pathway to UV-B radiation stress and the correlation with the lipid antioxidant characteristics in the desert plant Caryopteris mongolica. Acta Ecologica Sinica, 32, 150–155. 10.1016/j.chnaes.2012.04.004 Google Scholar Love, M.I., Huber, W. & Anders, S. (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 15, 1–21. 10.1186/s13059-014-0550-8 PubMedWeb of Science®Google Scholar MacGinitie, H.D. (1953) Fossil plants of the Florissant beds. Colorado: Carnegie institution of Washington. Google Scholar Manni, M., Berkeley, M.R., Seppey, M., Simão, F.A. & 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. Molecular Biology and Evolution, 38, 4647–4654. 10.1093/molbev/msab199 CASPubMedWeb of Science®Google Scholar Marçais, G., Delcher, A.L., Phillippy, A.M., Coston, R., Salzberg, S.L. & Zimin, A. (2018) MUMmer4: a fast and versatile genome alignment system. PLoS Computational Biology, 14, e1005944. 10.1371/journal.pcbi.1005944 PubMedWeb of Science®Google Scholar Marçais, G. & Kingsford, C. (2011) A fast, lock-free approach for efficient parallel counting of occurrences of k-mers. Bioinformatics, 27, 764–770. 10.1093/bioinformatics/btr011 CASPubMedWeb of Science®Google Scholar Mo, L., Huang, Y., Qin, J., Wang, x., Lu, S. & Yuan, W. (2008) A preliminary study on the water physiology of four plant species in karst area of Southwest Chi