Construction of the super pan-genome for the genus Actinidia reveals structural variations linked to phenotypic diversity

Abstract

Kiwifruits, belonging to the genus Actinidia, are acknowledged as one of the most successfully domesticated fruits in the twentieth century. Despite the rich wild resources and diverse phenotypes within this genus, insights into the genomic changes are still limited. Here, we conducted whole-genome sequencing on seven representative materials from highly diversified sections of Actinidia, leading to the assembly and annotation of 14 haplotype genomes with sizes spanning from 602.0 to 699.6 Mb. By compiling these haplotype genomes, we constructed a super pan-genome for the genus. We identified numerous structural variations (SVs, including variations in gene copy number) and highly diverged regions in these genomes. Notably, significant SV variability was observed within the intronic regions of the MED25 and TTG1 genes across different materials, suggesting their potential roles in influencing fruit size and trichome formation. Intriguingly, our findings indicated a high genetic divergence between two haplotype genomes, with one individual, tentatively named Actinidia × leiocacarpae, from sect. Leiocacarpae. This likely hybrid with a heterozygous genome exhibited notable genetic adaptations related to resistance against bacterial canker, particularly through the upregulation of the RPM1 gene, which contains a specific SV, after infection by Pseudomonas syringae pv. actinidiae. In addition, we also discussed the interlineage hybridizations and taxonomic treatments of the genus Actinidia. Overall, the comprehensive pan-genome constructed here, along with our findings, lays a foundation for examining genetic compositions and markers, particularly those related to SVs, to facilitate hybrid breeding aimed at developing desired phenotypes in kiwifruits.

Acknowledgments

This study was supported by the Second Tibetan Plateau Scientific Expedition and Research program (No. 2019QZKK0502), Natural Science Innovative Foundation Research Group of Sichuan Province, Research and Demonstration of Key Technology Innovations for Cangxi’s Characteristic Agricultural Industry (No. 25NSFTD0107).

Author Contributions

Q.H., D.L., and J.L. designed the research, H.W., W.Y., and G.D. collected the materials and performed the genome sequencing, H.W., W.Y., Q.H., and G.D. performed the analyses and experiments, and H.W., W.Y., Q.H., D.L., and J.L. wrote the manuscript. All authors contributed to the manuscript and approved the final version.

Data availability

All raw sequence data and genome assembly data have been deposited in National Genomics Data Center, Beijing Institute of Genomics, Chinese Academy of Sciences and China National Center for Bioinformation, under accession number PRJCA031175.

Conflict of interest statement

No conflict of interest declared.

Supplementary Data

Supplementary data is available at Horticulture Research online.

References

[1] Xu X, Chen Y, Li B. et al. Molecular mechanisms underlying multi-level defense responses of horticultural crops to fungal pathogens. Hortic Res. 2022; 9:9

[2] Surjadinata BB, Jacobo-Velázquez DA, Cisneros-Zevallos L. UVA, UVB and UVC light enhances the biosynthesis of phenolic antioxidants in fresh-cut carrot through a synergistic effect with wounding. Molecules. 2017; 22:668

[3] Cho MH, Lee SW. Phenolic phytoalexins in rice: biological func-tions and biosynthesis. Int J Mol Sci. 2015; 16:29120-33

[4] Sharma A, Shahzad B, Rehman A. et al. Response of phenyl-propanoid pathway and the role of polyphenols in plants under abiotic stress. Molecules. 2019; 24:2452

[5] Kumari S, Nazir F, Maheshwari C. et al. Plant hormones and secondary metabolites under environmental stresses: enlightening defense molecules. Plant Physiol Biochem. 2024; 206: 108238

[6] Brunetti C, Fini A, Sebastiani F. et al. Modulation of phyto-hormone signaling: a primary function of flavonoids in plant-environment interactions. Front Plant Sci. 2018; 9:9

[7] Mierziak J, Kostyn K, Kulma A. Flavonoids as important molecules of plant interactions with the environment. Molecules. 2014; 19:16240-65

[8] Tang Y, Lu L, Sheng Z. et al. An R2R3-MYB network modulates stem strength by regulating lignin biosynthesis and secondary cell wall thickening in herbaceous peony. Plant J. 2023; 113: 1237-58

[9] Lv S, Lin Z, Shen J. et al. OsTCP19 coordinates inhibition of lignin biosynthesis and promotion of cellulose biosynthesis to modify lodging resistance in rice. JExp Bot. 2024; 75:123-36

[10] Lv M, Zhang L, Wang Y. et al. Floral volatile benzenoids/phenylpropanoids: biosynthetic pathway, regulation and ecolog-ical value. Hortic Res. 2024;11:uhae220

[11] Ehlting J, Hamberger B, Million-Rousseau R. et al. Cytochromes P450 in phenolic metabolism. Phytochem Rev. 2006; 5:239-70

[12] Matsuno M, Compagnon V, Schoch GA. et al. Evolution of a novel phenolic pathway for pollen development. Science. 2009; 325: 1688-92

[13] Alber A, Ehlting J.Cytochrome P450s in lignin biosynthesis. In: Alber A, Ehlting J ( MA,eds.), Advances in Botanical Research, vol. 61. Cambridge, USA: Academic Press, 2012, 113-43

[14] Mizutani M. Impacts of diversification of cytochrome P450 on plant metabolism. Biol Pharm Bull. 2012; 35:824-32

[15] Nelson DR, Koymans L, Kamataki T. et al. The P450 superfamily: update on new sequences, gene mapping, and recommended nomenclature. DNA Cell Biol. 1991; 10:1-14

[16] Denisov IG, Makris TM, Sligar SG. et al. Structure and chemistry of cytochrome P450. Chem Rev. 2005; 105:2253-78

[17] Hasemann CA, Ravichandran KG, Peterson JA. et al. Crystal struc-ture and refinement of cytochrome P450terp at 2.3 a resolution. J Mol Biol. 1994; 236:1169-85

[18] Nelson D, Werck-Reichhart D. A P450-centric view of plant evolution. Plant J. 2011; 66:194-211

[19] Nelson DR, Koymans L, Kamataki T. et al. P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics. 1996; 6:1-42

[20] Hamberger B, Bak S. Plant P450s as versatile drivers for evolution of species-specific chemical diversity. Philos Trans R Soc Lond Ser BBiolSci. 2013; 368:20120426

[21] De Bruyn C, Ruttink T, Lacchini E. et al. Identification and characterization of CYP 71 subclade cytochrome P450 enzymes involved in the biosynthesis of bitterness compounds in Cicho-rium intybus. Front Plant Sci. 2023; 14:1200253

[22] Ralston L, Kwon ST, Schoenbeck M. et al. Cloning, het-erologous expression, and functional characterization of 5-epi-aristolochene-1,3-dihydroxylase from tobacco (Nicotiana tabacum). Arch Biochem Biophys. 2001; 393:222-35

[23] Drew DP, Dueholm B, Weitzel C. et al. Transcriptome analy-sis of Thapsia laciniata Rouy provides insights into terpenoid biosynthesis and diversity in Apiaceae. Int J Mol Sci. 2013; 14: 9080-98

[24] Höfer R, Boachon B, Renault H. et al. Dual function of the cytochrome P450 CYP76 family from Arabidopsis thaliana in the metabolism of monoterpenols and phenylurea herbicides. Plant Physiol. 2014; 166:1149-61

[25] Wang Q, Hillwig ML, Wu Y. et al. CYP701A8: a rice ent-kaurene oxidase paralog diverted to more specialized diter-penoid metabolism. Plant Physiol. 2012; 158:1418-25

[26] Pandey RP, Parajuli P, Koffas MAG. et al. Microbial production of natural and non-natural flavonoids: pathway engineering, directed evolution and systems/synthetic biology. Biotechnol Adv. 2016; 34:634-62

[27] Werck-Reichhart D. Cytochromes P450 in phenylpropanoid metabolism. Drug Metabol Drug Interact. 1995; 12:221-44

[28] Liu X, Cheng J, Zhang G. et al. Engineering yeast for the produc-tion of breviscapine by genomic analysis and synthetic biology approaches. Nat Commun. 2018; 9:448

[29] Zhao Q, Cui MY, Levsh O. et al. Two CYP82D enzymes function as flavone hydroxylases in the biosynthesis of root-specific 4’-Deoxyflavones in Scutellaria baicalensis. Mol Plant. 2018; 11: 135-48

[30] Pauli HH, Kutchan TM. Molecular cloning and functional heterologous expression of two alleles encoding (S)-N-methylcoclaurine 3’-hydroxylase (CYP80B1), a new methyl jasmonate-inducible cytochrome P-450-dependent mono-oxygenase of benzylisoquinoline alkaloid biosynthesis. Plant J. 1998; 13:793-801

[31] Hori K, Yamada Y, Purwanto R. et al. Mining of the uncharacter-ized cytochrome P450 genes involved in alkaloid biosynthesis in California poppy using a draft genome sequence. Plant Cell Physiol. 2018; 59:222-33

[32] Dang TT, Facchini PJ. Cyp82Y1 is N-methylcanadine 1-hydroxylase, a key noscapine biosynthetic enzyme in opium poppy. JBiolChem. 2014; 289:2013-26

[33] Beaudoin GA, Facchini PJ. Isolation and characterization of a cDNA encoding (S)-cis-N-methylstylopine 14-hydroxylase from opium poppy, a key enzyme in sanguinarine biosynthesis. Biochem Biophys Res Commun. 2013; 431:597-603

[34] Schoch G, Goepfert S, Morant M. et al. CYP98A3 from Arabidopsis thaliana is a 3’-hydroxylase of phenolic esters, a missing link in the phenylpropanoid pathway. JBiolChem. 2001; 276:36566-74

[35] Alber AV, Renault H, Basilio-Lopes A. et al. Evolution of coumaroyl conjugate 3-hydroxylases in land plants: lignin biosynthesis and defense. Plant J. 2019; 99:924-36

[36] Hannemann F, Bichet A, Ewen KM. et al. Cytochrome P450 systems-biological variations of electron transport chains. Biochim Biophys Acta. 2007; 1770:330-44

[37] Neve EP, Ingelman-Sundberg M.Cytochrome P450 proteins: retention and distribution from the endoplasmic reticulum. Curr Opin Drug Discov Devel. 2010; 13:78-85

[38] Zhou Z, Feng J, Huo J. et al. Versatile CYP98A enzymes catalyse meta-hydroxylation reveals diversity of salvianolic acids biosyn-thesis. Plant Biotechnol J. 2024; 22:1536-48

[39] Karamat F, Olry A, Doerper S. et al. Cyp98a22, a phenolic ester 3’-hydroxylase specialized in the synthesis of chlorogenic acid, as a new tool for enhancing the furanocoumarin concentration in Ruta graveolens. BMC Plant Biol. 2012; 12:152

[40] Durst F, Nelson DR. Diversity and evolution of plant P450 and P450-reductases. Drug Metabol Drug Interact. 1995; 12:189-206

[41] Bak S, Beisson F, Bishop G. et al. Cytochromes P450. The Arabidop-sis Book/American Society of Plant Biologists. 2011; 9:e0144

[42] Paquette SM, Bak S, Feyereisen R. Intron-exon organization and phylogeny in a large superfamily, the paralogous cytochrome P450 genes of Arabidopsis thaliana. DNA Cell Biol. 2000; 19:307-17

[43] Petersen M, Abdullah Y, Benner J. et al. Evolution of rosmarinic acid biosynthesis. Phytochemistry. 2009; 70:1663-79

[44] Franke R, Hemm MR, Denault JW. et al. Changes in secondary metabolism and deposition of an unusual lignin in the ref8 mutant of Arabidopsis. Plant J. 2002; 30:47-59

[45] Renault H, Alber A, Horst NA. et al. A phenol-enriched cuticle is ancestral to lignin evolution in land plants. Nat Commun. 2017; 8:14713

[46] Comino C, Hehn A, Moglia A. et al. The isolation and mapping of a novel hydroxycinnamoyltransferase in the globe artichoke chlorogenic acid pathway. BMC Plant Biol. 2009; 9:30

[47] Niggeweg R, Michael AJ, Martin C. Engineering plants with increased levels of the antioxidant chlorogenic acid. Nat Biotech-nol. 2004; 22:746-54

[48] Andreotti C, Ravaglia D, Ragaini A. et al. Phenolic compounds in peach (Prunus persica) cultivars at harvest and during fruit maturation. Ann Appl Biol. 2008; 153:11-23

[49] Chen X, Li W, Lu X. et al. Research progress on the biosynthesis, metabolic engineering, and pharmacology of bioactive com-pounds from the ∗lonicera∗ genus. Medic Plant Biol. 2024; 3:0-0

[50] Gang DR, Beuerle T, Ullmann P. et al. Differential production of meta hydroxylated phenylpropanoids in sweet basil peltate glandular trichomes and leaves is controlled by the activi-ties of specific acyltransferases and hydroxylases. Plant Physiol. 2002; 130:1536-44

[51] Moglia A, Comino C, Portis E. et al. Isolation and mapping of a C3’H gene (CYP98A49) from globe artichoke, and its expression upon UV-C stress. Plant Cell Rep. 2009; 28:963-74

[52] Mahesh V, Million-Rousseau R, Ullmann P. et al. Functional characterization of two p-coumaroyl ester 3’-hydroxylase genes from coffee tree: evidence of a candidate for chlorogenic acid biosynthesis. Plant Mol Biol. 2007; 64:145-59

[53] Pu G, Wang P, Zhou B. et al. Cloning and characterization of Lonicera japonicap-coumaroyl ester 3-hydroxylase which is involved in the biosynthesis of chlorogenic acid. Biosci Biotechnol Biochem. 2014; 77:1403-9

[54] Sullivan ML, Zarnowski R. Red clover coumarate 3’-hydroxylase (CYP98A44) is capable of hydroxylating p-coumaroyl-shikimate but not p-coumaroyl-malate: implications for the biosynthesis of phaselic acid. Planta. 2009; 231:319-28

[55] Levsh O, Pluskal T, Carballo V. et al. Independent evolution of rosmarinic acid biosynthesis in two sister families under the lamiids clade of flowering plants. JBiolChem. 2019; 294: 15193-205

[56] Morant M, Schoch GA, Ullmann P. et al. Catalytic activity, dupli-cation and evolution of the CYP98 cytochrome P450 family in wheat. Plant Mol Biol. 2006; 63:1-19

[57] LiuC, ZhouG, QinH. et al. Metabolomics combined with physiology and transcriptomics reveal key metabolic pathway responses in apple plants exposure to different selenium con-centrations. J Hazard Mater. 2024; 464:132953

[58] Zhang L, Zhou L, Meng J. et al. Comparative transcriptome analysis of the resistance mechanism of Hemerocallis citrina Baroni to Puccinia hemerocallidis infection. J Plant Interact. 2023; 18: 2260410

[59] Matsuno M, Nagatsu A, Ogihara Y. et al. Cyp98a6 from Lithosper-mum erythrorhizon encodes 4-coumaroyl-4’-hydroxyphenyllactic acid 3-hydroxylase involved in rosmarinic acid biosynthesis. FEBS Lett. 2002; 514:219-24

[60] Su Z, Jia H, Sun M. et al. Integrative analysis of the metabolome and transcriptome reveals the molecular mechanism of chloro-genic acid synthesis in peach fruit. Front Nutr. 2022; 9:9

[61] Wu Y, Xu S, Tian XY. The effect of salvianolic acid on vascular protection and possible mechanisms. Oxidative Med Cell Longev. 2020; 2020:5472096

[62] Qin T, Rasul A, Sarfraz A. et al. Salvianolic acid a & B: potential cytotoxic polyphenols in battle against cancer via targeting multiple signaling pathways. Int J Biol Sci. 2019; 15:2256-64

[63] Yang CK, Pan QQ, Tian Z. et al. Protective mechanism of salviano-lic acid B on blood vessels. Zhongguo Zhong Yao Za Zhi. 2023; 48: 1176-85

[64] Nair RB, Xia Q, Kartha CJ. et al. Arabidopsis CYP98A3 mediat-ing aromatic 3-hydroxylation. Developmental regulation of the gene, and expression in yeast. Plant Physiol. 2002; 130:210-20

[65] Barros J, Escamilla-Trevino L, Song L. et al. 4-coumarate 3-hydroxylase in the lignin biosynthesis pathway is a cytosolic ascorbate peroxidase. Nat Commun. 2019; 10:1994

[66] Karimzadegan V, Koirala M, Sobhanverdi S. et al. Characteri-zation of cinnamate 4-hydroxylase (CYP73A) and p-coumaroyl 3’-hydroxylase (CYP98A) from Leucojum aestivum,a source of Amaryllidaceae alkaloids. Plant Physiol Biochem. 2024; 210: 108612

[67] Zhang B, Lewis JA, Vermerris W. et al. A sorghum ascorbate peroxidase with four binding sites has activity against ascorbate and phenylpropanoids. Plant Physiol. 2023; 192:102-18

[68] Eberle D, Ullmann P, Werck-Reichhart D. et al. Cdna cloning and functional characterisation of CYP98A14 and NADPH:cytochrome P450 reductase from ∗Coleus blumei∗ involved in rosmarinic acid biosynthesis. Plant Mol Biol. 2008; 69: 239-53

[69] Wang B, Sun W, Li Q. et al. Genome-wide identification of phenolic acid biosynthetic genes in ∗Salvia miltiorrhiza∗. Planta. 2014; 241:711-25

[70] Feng D, Zhang A, Yang Y. et al. Coumarin-containing hybrids and their antibacterial activities. Arch Pharm (Weinheim). 2020; 353:e1900380

[71] Graña E, Costas-Gil A, Longueira S. et al. Auxin-like effects of the natural coumarin scopoletin on Arabidopsis cell structure and morphology. J Plant Physiol. 2017; 218:45-55

[72] Vanholme R, Sundin L, Seetso KC. et al. COSY catalyses trans-cis isomerization and lactonization in the biosynthesis of coumarins. Nat Plants. 2019; 5:1066-75

[73] Vogt T. Phenylpropanoid biosynthesis. Mol Plant. 2010; 3:2-20

[74] Kai K, Mizutani M, Kawamura N. et al. Scopoletin is biosyn-thesized via ortho-hydroxylation of feruloyl CoA by a 2-oxoglutarate-dependent dioxygenase in Arabidopsis thaliana. Plant J. 2008; 55:989-99

[75] Kai K, Shimizu BI, Mizutani M. et al. Accumulation of coumarins in Arabidopsis thaliana. Phytochemistry. 2006; 67:379-86

[76] Ma Q, Xu J, Feng Y. et al. Knockdown of p-coumaroyl shiki-mate/quinate 3’-hydroxylase delays the occurrence of post-harvest physiological deterioration in cassava storage roots. Int JMol Sci. 2022; 23:9231

[77] Siwinska J, Kadzinski L, Banasiuk R. et al. Identification of QTLs affecting scopolin and scopoletin biosynthesis in Arabidopsis thaliana. BMC Plant Biol. 2014; 14:280

[78] Stringlis IA, de Jonge R, Pieterse CMJ. The age of coumarins in plant-microbe interactions. Plant Cell Physiol. 2019; 60:1405-19

[79] Ahuja I, Kissen R, Bones AM. Phytoalexins in defense against pathogens. Trends Plant Sci. 2012; 17:73-90

[80] Annunziata F, Pinna C, Dallavalle S. et al. An overview of coumarin as a versatile and readily accessible scaffold with broad-ranging biological activities. Int J Mol Sci. 2020; 21:4618

[81] Pei T, Zhu S, Liao W. et al. Gap-free genome assembly and CYP450 gene family analysis reveal the biosynthesis of anthocyanins in Scutellaria baicalensis. Hortic Res. 2023;10:uhad235

[82] Zheng M, Fang Y, Zhao Q. Comparative analysis of flavones from six commonly used ∗Scutellaria∗ species. Medic Plant Biol. 2023; 2:12

[83] Wu Z, Li R, Sun M. et al. Current advances of Carthamus tincto-rius L.: a review of its application and molecular regulation of flavonoid biosynthesis. Medic Plant Biol. 2024; 3:0-0

[84] Chen Y, Zhang T, Chen C. et al. Transcriptomics explores the potential of flavonoid in non-medicinal parts of Saposhnikovia divaricata (Turcz.) Schischk. Schischk. Front Plant Sci. 2023; 14:14

[85] Li W-F, Mao J, Yang SJ. et al. Anthocyanin accumulation corre-lates with hormones in the fruit skin of ‘red delicious’ and its four generation bud sport mutants. BMC Plant Biol. 2018; 18:363

[86] Liu Z, Tavares R, et al. Forsythe ES. Evolutionary interplay between sister cytochrome P450 genes shapes plasticity in plant metabolism. Nat Commun. 2016; 7:13026

[87] Wu L, du G, Bao R. et al. De novo assembly and discovery of genes involved in the response of Solanum sisymbriifolium to Verticillium dahlia. Physiol Mol Biol Plants. 2019; 25:1009-27

[88] Wang Z, Wang W, Wu W. et al. Integrated analysis of transcrip-tome, metabolome, and histochemistry reveals the response mechanisms of different ages Panax notoginseng to root-knot nematode infection. Front Plant Sci. 2023; 14:1258316

[89] Bonawitz ND, Chapple C. The genetics of lignin biosynthesis: connecting genotype to phenotype. Annu Rev Genet. 2010; 44: 337-63

[90] Abdulrazzak N, Pollet B, Ehlting J. et al. A coumaroyl-ester-3-hydroxylase insertion mutant reveals the existence of nonre-dundant meta-hydroxylation pathways and essential roles for phenolic precursors in cell expansion and plant growth. Plant Physiol. 2006; 140:30-48

[91] Gabriac B, Werck-Reichhart D, Teutsch H. et al. Purification and immunocharacterization of a plant cytochrome P450: the cinnamic acid 4-hydroxylase. Arch Biochem Biophys. 1991; 288: 302-9

[92] Poulos TL, Finzel BC, Gunsalus IC. et al. The 2.6-A crystal structure of Pseudomonas putida cytochrome P-450. JBiolChem. 1985; 260:16122-30

[93] Gu M, Wang M, Guo J. et al. Crystal structure of CYP76AH1 in 4-PI-bound state from Salvia miltiorrhiza. Biochem Biophys Res Commun. 2019; 511:813-9

[94] Niu G, Guo Q, Wang J. et al. Structural basis for plant lutein biosynthesis from α-carotene. Proc Natl Acad Sci. 2020; 117: 14150-7

[95] Zhang B, Lewis KM, Abril A. et al. Structure and func-tion of the cytochrome P450 monooxygenase cinnamate 4-hydroxylase from Sorghum bicolor. Plant Physiol. 2020; 183: 957-73

[96] Fujiyama K, Hino T, Kanadani M. et al. Structural insights into a key step of brassinosteroid biosynthesis and its inhibition. Nat Plants. 2019; 5:589-94