Haplotype-resolved genome assembly provides insights into the evolutionary origin of waterlogging-tolerant Actinidia valvata hexaploid

Abstract

Kiwifruit plants are much damaged by several days of waterlogging stress. The effect can be a serious problem for the growers in the lowlands or plain areas where floods cannot be drained timely. Actinidia valvata is a polyploid species that has been widely used as waterlogging resistant rootstock in kiwifruit cultivation. Here we report haplotype-resolved chromosome-scale assemblies of an A. valvata male plant ‘DE’, defining two subgenomes, a diploid closely related to Actinidia polygama and an autotetraploid closely related to Actinidia macrosperma as their ancestral contributors, respectively, together to form an allohexaploid. Genome-wide comparisons of the assembled 174 pseudochromosomes with nine distinct Actinidia species revealed the genomic structure, phylogeny and duplication history of ‘DE’ genome. Evolutionary analyses suggest that it was formed ∼0.44 to 0.88 Mya and evolved by a recent alloploid addition to an autotetraploid ancestor. Annotation of sex determining genes ( SyGl and FrBy ) on Y chromosome unraveled that the chromosomal location and organization of sex determining region (SDR) are conserved to and share an identical lineage with A. polygama , the diploid ancestor. Comprehensive transcriptome analysis indicates that its enhanced waterlogging tolerance is due to the restricted activation of anaerobic survival genes and the consequence with prolonged carbohydrate supply to keep the root system quiescently alive under hypoxia. Our study provides valuable genomic resources and offers insights into the evolution and the underlying mechanism of enhanced waterlogging tolerance of A. valvata hexaploid.

Consent for publication

All authors hereby consent to publication of the work.

Acknowledgements

We acknowledge valuable discussions in Liu’s groups. This work was supported by funds from the National Natural Science Foundation of China (Grant nos. U23A20204, 31972474 and 90717110).

Author contributions

Y.Liu, P.Z., J.Y., and Y.Wang designed the research. F.Z., Y.Lin, Y.Wang, P.Z., B.L., H.W., Y.Wu, Y.Z., X.Z., W.R., L.W., Y.Y., and S.W. conducted experiments and analyzed data. F.Z., Y.Lin, Y.Wang, and Y.Liu wrote the manuscript. Y.Liu revised the manuscript.

Data availability

All data generated or analyzed during this study are included in this published article. The raw Hi-Fi, Hi-C and RNA-seq reads, assembled genome, and annotations generated in this study have been deposited in the NGDC database (https://ngdc.cncb.ac.cn/) with the accession number PRJCA030394.

Conflicts of interest statement

The authors declare no competing interests.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Bailey-Serres J, Fukao T, Gibbs DJ, et al. Making sense of low oxygen sensing. Trends Plant Sci. 2012; 17: 129-38.

[2] Lin C-C, Chao Y-T, Chen W-C, et al. Regulatory cascade involving transcriptional and N-end rule pathways in rice under submergence. Proc Natl Acad Sci USA. 2019; 116: 3300-9.

[3] Gibbs J, Greenway H . Mechanisms of anoxia tolerance in plants. I. Growth, survival and anaerobic catabolism. Funct Plant Biol. 2003; 30: 1-47.

[4] Voesenek LACJ, Bailey-Serres J . Flood adaptive traits and processes: an overview. New Phytol. 2015; 206: 57-73.

[5] Drew MC . Oxygen deficiency and root metabolism: injury and acclimation under hypoxia and anoxia. Annu Rev Plant Physiol Plant Mol Biol. 1997; 48: 223-50.

[6] Bailey-Serres J , Voesenek LACJ . Flooding stress: acclimations and genetic diversity. Annu Rev Plant Biol. 2008; 59: 313-39.

[7] Reynoso MA, Kajala K, Bajic M, et al. Evolutionary flexibility in flooding response circuitry in angiosperms. Science. 2019; 365: 1291-5.

[8] Borrego-Benjumea A, Carter A, Tucker JR, et al. Genome-wide analysis of gene expression provides new insights into waterlogging responses in barley (Hordeum vulgare L.). Plants. 2020; 9: 240.

[9] Javed T, Shabbir R, Ali A, et al. Transcription factors in plant stress responses: challenges and potential for sugarcane improvement. Plants. 2020; 9: 491.

[10] Salvatierra A, Toro G, Mateluna P, et al. Keep calm and survive: adaptation strategies to energy crisis in fruit trees under root hypoxia. Plants. 2020; 9: 1108.

[11] Gandar PW, Hughes KA . Kiwifruit root systems 1. Root-length densities. N Z J Exp Agric. 1988; 16: 35-46.

[12] Bai D, Li Z, Qi X, et al. Physiological responses and tolerance evaluation of four species of Actinidia to waterlogging stress. J Fruit Sci. 2019; 36: 163-73.

[13] Li Z, Bai D, Zhong Y, et al. Physiological responses of two contrasting kiwifruit (Actinidia spp.) rootstocks against waterlogging stress. Plants. 2021; 10: 2586.

[14] Bai D, Li Z, Gu S, et al. Effects of kiwifruit rootstocks with opposite tolerance on physiological responses of grafting combinations under waterlogging stress. Plants. 2022; 11: 2098.

[15] Mustroph A, Zanetti ME, Jang CJ, et al. Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis. Proc Natl Acad Sci USA. 2009; 106: 18843-8.

[16] Li Z, Bai D, Zhong Y, et al. Full-length transcriptome and RNA-seq analyses reveal the mechanisms underlying waterlogging tolerance in kiwifruit (Actinidia valvata). Int J Mol Sci. 2022; 23: 3237.

[17] Liu J, Chen Y, Wang W-Q, et al. Transcription factors AcERF74/75 respond to waterlogging stress and trigger alcoholic fermentation-related genes in kiwifruit. Plant Sci. 2022; 314: 111115.

[18] Wurms KV, Reglinski T, Buissink P, et al. Effects of drought and flooding on phytohormones and abscisic acid gene expression in kiwifruit. Int J Mol Sci. 2023; 24: 7580.

[19] Zhang J-Y, Huang S-N, Wang G, et al. Overexpression of Actinidia deliciosa pyruvate decarboxylase 1 gene enhances waterlogging stress in transgenic Arabidopsis thaliana. Plant Physiol Biochem. 2016; 106: 244-52.

[20] Zhang J-Y, Huang S-N, Chen Y-H, et al. Identification and characterization of two waterlogging responsive alcohol dehydrogenase genes (AdADH1 and AdADH2) in Actinidia deliciosa. Mol Breeding. 2017; 37: 52.

[21] Pan DL, Wang G, Wang T, et al. AdRAP2.3, a novel ethylene response factor VII from Actinidia deliciosa, enhances waterlogging resistance in transgenic tobacco through improving expression levels of PDC and ADH genes. Int J Mol Sci. 2019; 20: 1189.

[22] Chen Y, Su WY, Ren CJ, et al. Restricted responses of AcMYB68 and AcERF74/75 enhanced waterlogging tolerance in kiwifruit. Plant J. 2024; 119: 1059-72.

[23] Zhong Y, Qi, Chen J-Y, et al. Growth and physiological responses of four kiwifruit genotypes to salt stress and resistance evaluation. J Integr Agric. 2019; 18: 83-95.

[24] Cheng H, Concepcion GT, Feng X, et al. Haplotype-resolved de novo assembly using phased assembly graphs with Hifi-asm. Nat Methods. 2021; 18: 170-5.

[25] Zhang X, Zhang S, Zhao Q, et al. Assembly of allele-aware, chromosomal-scale autopolyploid genomes based on hi-C data. Nat Plants. 2019; 5: 833-45.

[26] Mapleson D, Garcia Accinelli G , Kettleborough G, et al. KAT: a K-mer analysis toolkit to quality control NGS datasets and genome assemblies. Bioinformatics. 2017; 33: 574-6.

[27] Yue J, Chen Q, Wang Y, et al. Telomere-to-telomere and gap-free reference genome assembly of the kiwifruit Actinidia chinensis. Hortic Res. 2023; 10: 264.

[28] Manni M, Berkeley MR, Seppey M, et al. BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol Biol Evol. 2021; 38: 4647-54.

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

[30] Cantalapiedra CP, Hernández-Plaza A, Letunic I, et al. eggNOG-mapper v2: functional annotation, orthology assignments, and domain prediction at the metagenomic scale. Mol Biol Evol. 2021; 38: 5825-9.

[31] Lin Y, Ye C, Li X, et al. quarTeT: a telomere-to-telomere toolkit for gap-free genome assembly and centromeric repeat identification. Hortic Res. 2023; 10: 127.

[32] Ma J, Wing RA, Bennetzen JL, et al. Plant centromere organization: a dynamic structure with conserved functions. Trends Genet. 2007; 23: 134-9.

[33] Perumal S, Koh CS, Jin L, et al. A high-contiguity brassica nigra genome localizes active centromeres and defines the ancestral brassica genome. Nat Plants. 2020; 6: 929-41.

[34] Wang Y, Dong M, Wu Y, et al. Telomere-to-telomere and haplotype-resolved genome of the kiwifruit Actinidia eriantha. Mol Hortic. 2023; 3: 4.

[35] Zhang F, Wang Y, Lin Y, et al. Haplotype-resolved genome assembly provides insights into evolutionary history of the Actinidia arguta tetraploid. Mol Hortic. 2024; 4: 4.

[36] Chen J, Wang Z, Tan K, et al. A complete telomere-to-telomere assembly of the maize genome. Nat Genet. 2023; 55: 1221-31.

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

[38] Sanderson MJ . r8s: inferring absolute rates of molecular evolution and divergence times in the absence of a molecular clock. Bioinformatics. 2003; 19: 301-2.

[39] Akagi T, Varkonyi-Gasic E, Shirasawa K, et al. Recurrent neo-sex chromosome evolution in kiwifruit. Nat Plants. 2023; 9: 393-402.

[40] Xia H, Deng H, Li M, et al. Chromosome-scale genome assembly of a natural diploid kiwifruit (Actinidia chinensis var. deliciosa). Sci Data. 2023; 10: 92.

[41] Qi X, Xie X, Yang M, et al. Characterization of the complete chloroplast genome of Actinidia hemsleyana. Mitochondrial DNA Part B Resour. 2021; 6: 3259-60.

[42] Han X, Zhang Y, Zhang Q, et al. Two haplotype-resolved, gap-free genome assemblies for Actinidia latifolia and Actinidia chinensis shed light on the regulatory mechanisms of vitamin C and sucrose metabolism in kiwifruit. Mol Plant. 2023; 16: 452-70.

[43] Li X, Huo L, Li X, et al. Genomes of diverse Actinidia species provide insights into cis-regulatory motifs and genes associated with critical traits. BMC Biol. 2024; 22: 200.

[44] Zhang X, Chen S, Shi L, et al. Haplotype-resolved genome assembly provides insights into evolutionary history of the tea plant Camellia sinensis. Nat Genet. 2021; 53: 1250-9.

[45] Zhou Y, Zhang Z, Bao Z, et al. Graph pangenome captures missing heritability and empowers tomato breeding. Nature. 2022; 606: 527-34.

[46] Jaillon O, Aury JM, Noel B, et al. The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature. 2007; 449: 463-7.

[47] Lamesch P, Berardini TZ, Li D, et al. The Arabidopsis information resource (TAIR): improved gene annotation and new tools. Nucleic Acids Res. 2012; 40: D1202-10.

[48] Ouyang S, Zhu W, Hamilton J, et al. The TIGR rice genome annotation resource: improvements and new features. Nucleic Acids Res. 2007; 35: D883-7.

[49] Camacho C, Boratyn GM, Joukov V, et al. ElasticBLAST: accelerating sequence search via cloud computing. BMC Bioinformatics. 2023; 24: 117.

[50] Yue J, Chen Q, Zhang S, et al. Origin and evolution of the kiwifruit Y chromosome. Plant Biotechnol J. 2024; 22: 287-9.

[51] Geng J, Shi G, Li X, et al. Combining physiology and transcriptome data screening for key genes in Actinidia arguta response to waterlogging stress. Agronomy. 2024; 14: 2391.

[52] Luo J, Yuan L-Y . Responses of physiological activities and lateral root anatomical structure of Cercis glabra to waterlogging stress. Not Bot Horti Agrobot Cluj-Na. 2020; 48: 1573-84.

[53] Yang X, Xie H, Weng Q, et al. Rice OsCASP1 orchestrates Casparian strip formation and suberin deposition in small lateral roots to maintain nutrient homeostasis. Front Plant Sci. 2022; 13: 1007300.

[54] Li J, Zhang Y, Chen Y, et al. The roles of cell wall polysaccharides in response to waterlogging stress in Brassica napus L. root. BMC Biol. 2024; 22: 191.

[55] Bashar KK, Tareq MZ, Islam MS . Unlocking the mystery of plants’ survival capability under waterlogging stress. Plant Sci Today. 2020; 7: 142-53.

[56] Katz A, Orell O . Protein synthesis and the stress response. In: Biyani M, ed. Cell-free Protein Synthesis. InTech, 2012; 7: 111-34.

[57] Yu L, Zhou C, Fan J, et al. Mechanisms and functions of membrane lipid remodeling in plants. Plant J. 2021; 107: 37-53.

[58] Liu Y, Zhou Y, Cheng F, et al. Chromosome-level genome of putative autohexaploid Actinidia deliciosa provides insights into polyploidisation and evolution. Plant J. 2024; 118: 73-89.

[59] Weits DA, Van Dongen JT, Licausi F . Molecular oxygen as a signaling component in plant development. New Phytol. 2021; 229: 24-35.

[60] Lee H-J, Park J-S, Shin SY, et al. Submergence deactivates wound-induced plant defence against herbivores. Commun Biol. 2020; 3: 651.

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

[62] Huang H-W, Gong J-J, Wang S-M, et al. Genetic diversity in the genus Actinidia. Biodivers Sci. 2000; 08: 1-12.

[63] Zhou C, Brown M, Blaxter M, et al. Oatk: a de novo assembly tool for complex plant organelle genomes. Genome Biol. 2025; 26: 235.

[64] Fan T, Song S, Bian N, et al. Engineering saline-alkali-tolerant apple rootstocks by overexpressing MdHYL1 in M9-T337. Mol Breed. 2025; 45: 58.

[65] Edger PP, Poorten TJ, VanBuren R, et al. Origin and evolution of the octoploid strawberry genome. Nat Genet. 2019; 51: 541-7.

[66] Hovav R, Chaudhary B, Udall JA, et al. Parallel domestication, convergent evolution and duplicated gene recruitment in allopolyploid cotton. Genetics. 2008; 179: 1725-33.

[67] Feldman M, Levy AA, Fahima T, et al. Genomic asymmetry in allopolyploid plants: wheat as a model. J Exp Bot. 2012; 63: 5045-59.

[68] Kim SH, Kim HS, Bahk S, et al. Phosphorylation of the transcriptional repressor MYB15 by mitogen-activated protein kinase 6 is required for freezing tolerance in Arabidopsis. Nucleic Acids Res. 2017; 45: 6613-27.

[69] Podwyszyńska M, Markiewicz M, Broniarek-Niemiec A, et al. Apple autotetraploids with enhanced resistance to apple scab (Venturia inaequalis) due to genome duplication-phenotypic and genetic evaluation. Int J Mol Sci. 2021; 22: 527.

[70] Oustric J, Morillon R, Luro F, et al. Tetraploid Carrizo citrange rootstock (Citrus sinensis Osb.×Poncirus trifoliata L. Raf.) enhances natural chilling stress tolerance of common clementine (citrus Clementina Hort. Ex Tan). J Plant Physiol. 2017; 214: 108-15.

[71] Allario T, Brumos J, Colmenero-Flores JM, et al. Tetraploid rangpur lime rootstock increases drought tolerance via enhanced constitutive root abscisic acid production. Plant Cell Environ. 2013; 36: 856-68.

[72] Wei T, Wang Y, Xie Z, et al. Enhanced ROS scavenging and sugar accumulation contribute to drought tolerance of naturally occurring autotetraploids in Poncirus trifoliata. Plant Biotechnol J. 2019; 17: 1394-407.

[73] Tu Y, Jiang A, Gan L, et al. Genome duplication improves rice root resistance to salt stress. Rice. 2014; 7: 15.

[74] Zhu H, Zhao S, Lu X, et al. Genome duplication improves the resistance of watermelon root to salt stress. Plant Physiol Biochem. 2018; 133: 11-21.

[75] Li Z, Zhong Y, Bai D, et al. Comparative transcriptome analysis of two contrasting kiwifruit (Actinidia) genotypes under waterlogging stress. Preprints. 2020; 105: 2020070395.

[76] Bai D-F, Li Z, Hu C-G, et al. Transcriptome-wide identification and expression analysis of ERF family genes in Actinidia valvata during waterlogging stress. Sci Hortic. 2021; 281: 109994.

[77] Gao Y, Jiang Z, Shi M, et al. Comparative transcriptome provides insight into responding mechanism of waterlogging stress in Actinidia valvata Dunn. Gene. 2022; 845: 146843.

[78] Bai D, Gu S, Qi X, et al. The ERF-VII transcription factor AvERF75 positively regulates the waterlogging tolerance by interacting with AvLOB41 in kiwifruit (Actinidia valvata). Environ Exp Bot. 2023; 212: 105401.

[79] Bai D, Zhong Y, Gu S, et al. AvERF73 positively regulates waterlogging tolerance in kiwifruit by participating in hypoxia response and mevalonate pathway. Hortic Plant J. 2025; 11: 162-74.

[80] Nguyen T-N, Tuan PA, Mukherjee S, et al. Hormonal regulation in adventitious roots and during their emergence under waterlogged conditions in wheat. J Exp Bot. 2018; 69: 4065-82.

[81] Ou L-J, Liu Z-B, Zhang Y-P, et al. Effects of exogenous Ca ²⁺ on photosynthetic characteristics and fruit quality of pepper under waterlogging stress . Chil J Agric Res. 2017; 77: 126-33.

[82] Zhang YJ, Song X, Yang G, et al. Physiological and molecular adjustment of cotton to waterlogging at peak-flowering in relation to growth and yield. Field Crop Res. 2015; 179: 164-72.

[83] Yao Y, He RJ, Xie QL, et al. ETHYLENE RESPONSE FACTOR 74 (ERF74) plays an essential role in controlling a respiratory burst oxidase homolog D (RbohD)-dependent mechanism in response to different stresses in Arabidopsis. New Phytol. 2017; 213: 1667-81.

[84] Licausi F, Kosmacz M, Weits DA, et al. Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization. Nature. 2011; 479: 419-22.

[85] Weits DA, Giuntoli B, Kosmacz M, et al. Plant cysteine oxidases control the oxygen-dependent branch of the N-end-rule pathway. Nat Commun. 2014; 5: 3425.

[86] Song L, Valliyodan B, Prince S, et al. Characterization of the XTH gene family: new insight to the roles in soybean flooding tolerance. Int J Mol Sci. 2018; 19: 2705.

[87] Lee K-W, Chen P-W, Lu C-A, et al. Coordinated responses to oxygen and sugar deficiency allow rice seedlings to tolerate flooding. Sci Signal. 2009; 2: ra61.

[88] Park M, Yim H-K, Park H-G, et al. Interference with oxidative phosphorylation enhances anoxic expression of rice alpha-amylase genes through abolishing sugar regulation. J Exp Bot. 2010; 61: 3235-44.

[89] Islam MR, Rahman MM, Akter M, et al. Insights into the role of cytokinin and gibberellic acid in improving waterlogging tolerance of mung bean. Res Sq. 2021; 7: 1-21.

[90] Chen J-H, Jiang HW, Hsieh EJ, et al. Drought and salt stress tolerance of an Arabidopsis glutathione S-transferase U17 knockout mutant are attributed to the combined effect of glutathione and abscisic acid. Plant Physiol. 2012; 158: 340-51.

[91] Santiago J, Rodrigues A, Saez A, et al. Modulation of drought resistance by the abscisic acid receptor PYL5 through inhibition of clade a PP2Cs. Plant J. 2009; 60: 575-88.

[92] Martin T, Frommer WB, Salanoubat M, et al. Expression of an Arabidopsis sucrose synthase gene indicates a role in metabolization of sucrose both during phloem loading and in sink organs. Plant J. 1993; 4: 367-77.

[93] Huh SU . New function of hypoxia-responsive unknown protein in enhanced resistance to biotic stress. Plant Signal Behav. 2021; 16: 1868131.

[94] Chow C-N, Lee TY, Hung YC, et al. PlantPAN3.0: a new and updated resource for reconstructing transcriptional regulatory networks from ChIP-seq experiments in plants. Nucleic Acids Res. 2019; 47: D1155-63.

[95] Greenfest-Allen E, Cartailler J-P, Magnuson MA, et al. iterativeWGCNA: iterative refinement to improve module detection from WGCNA co-expression networks. Biorxiv. 2017; 12: 234062.

[96] Olsen AN, Ernst HA, Leggio LL, et al. DNA-binding specificity and molecular functions of NAC transcription factors. Plant Sci. 2005; 169: 785-97.

[97] Chow CN, Yang CW, Wu NY, et al. PlantPAN 4.0: updated database for identifying conserved non-coding sequences and exploring dynamic transcriptional regulation in plant promoters. Nucleic Acids Res. 2024; 52: D1569-78.

[98] Wang L, Liu B, Yang Y, et al. The comparative studies of complete chloroplast genomes in Actinidia (actinidiaceae): novel insights into heterogenous variation, clpP gene annotation and phylogenetic relationships. Mol Gen Genomics. 2022; 297: 535-51.

[99] Chat J, Decroocq S, Petit RJ . A one-step organelle capture: gynogenetic kiwifruits with paternal chloroplasts. Proc R Soc Lond B Biol Sci. 2003; 270: 783-9.

[100] Li D, Qi X, Li X, et al. Maternal inheritance of mitochondrial genomes and complex inheritance of chloroplast genomes in Actinidia lind.: evidences from interspecific crosses. Mol Gen Genomics. 2013; 288: 101-10.

[101] Wang W-C, Chen S-Y, Zhang X-Z . Chloroplast genome evolution in actinidiaceae: clpP loss, heterogenous divergence and phylogenomic practice. PLoS One. 2016; 11: e0162324.

[102] Huang HW . Actinidia Classification, Resources, Domestication and Cultivation. Science Press: Beijing, China, 2013.

[103] Liu Y, Li D, Zhang Q, et al. Rapid radiations of both kiwifruit hybrid lineages and their parents shed light on a two-layer mode of species diversification. New Phytol. 2017; 215: 877-90.

[104] Shi T, Huang H, Barker MS . Ancient genome duplications during the evolution of kiwifruit (Actinidia) and related ericales. Ann Bot. 2010; 106: 497-504.

[105] Huang S, Ding J, Deng D, et al. Draft genome of the kiwifruit Actinidia chinensis. Nat Commun. 2013; 4: 2640.

[106] Allen GC, Flores-Vergara MA, Krasynanski S, et al. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat Protoc. 2006; 1: 2320-5.

[107] Rao SSP, Huntley MH, Durand NC, et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell. 2014; 159: 1665-80.

[108] Jo H, Koh G . Faster single-end alignment generation utilizing multi-thread for BWA. Biomed Mater Eng. 2015; 26 Suppl 1: S1791-6.

[109] Danecek P, Bonfield JK, Liddle J, et al. Twelve years of SAMtools and BCFtools. GigaScience. 2021; 10: giab008.

[110] Wu TD, Watanabe CK . GMAP: a genomic mapping and alignment program for mRNA and EST sequences. Bioinformatics. 2005; 21: 1859-75.

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

[112] Durand NC, Robinson JT, Shamim MS, et al. Juicebox provides a visualization system for hi-C contact maps with unlimited zoom. Cell Syst. 2016; 3: 99-101.

[113] Durand NC, Shamim MS, Machol I, et al. Juicer provides a one-click system for analyzing loop-resolution hi-C experiments. Cell Syst. 2016; 3: 95-8.

[114] Pedersen BS, Quinlan AR . Mosdepth: quick coverage calculation for genomes and exomes. Bioinformatics. 2018; 34: 867-8.

[115] Ou S, Su W, Liao Y, et al. Benchmarking transposable element annotation methods for creation of a streamlined, comprehensive pipeline. Genome Biol. 2019; 20: 275.

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

[117] Tarailo-Graovac M, Chen N . Using RepeatMasker to identify repetitive elements in genomic sequences. Curr Protoc Bioinform. 2009; 25: 4.10.1-4.10.14.

[118] Gabriel L, Brůna T, Hoff KJ, et al. BRAKER3: fully automated genome annotation using RNA-seq and protein evidence with GeneMark-ETP, AUGUSTUS and TSEBRA. BioRxiv. 2023; 2: 1-21.

[119] Yu G, Wang LG, Han Y, et al. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012; 16: 284-7.

[120] Marçais G, Delcher AL, Phillippy AM, et al. MUMmer4: a fast and versatile genome alignment system. PLoS Comput Biol. 2018; 14: e1005944.

[121] Goel M, Sun H, Jiao W-B, et al. SyRI: finding genomic rearrangements and local sequence differences from whole-genome assemblies. Genome Biol. 2019; 20: 277.

[122] Chen S . Ultrafast one-pass FASTQ data preprocessing, quality control, and deduplication using fastp. iMeta. 2023; 2: e107.

[123] Guo J, Gao J, Liu Z . HISAT2 parallelization method based on spark cluster. J Phys Conf Ser. 2022; 2179: 12038.

[124] Pertea G, Pertea M . GFF utilities: GffRead and GffCompare. F1000Research. 2020; 9: ISCB-Comm.

[125] Liao Y, Smyth GK, Shi W . featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014; 30: 923- 30.

[126] Grabherr MG, Haas BJ, Yassour M, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011; 29: 644-52.

[127] Chen Y, Lun ATL, McCarthy DJ, Chen L, Ritchie ME, Phipson B . Empirical analysis of digital gene expression data in R. Ref Man, 2023.

[128] Love MI, Huber W, Anders S . Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15: 550.

[129] Tang H, Krishnakumar V, Zeng X, et al. JCVI: a versatile toolkit for comparative genomics analysis. iMeta. 2024; 3: e211.

[130] Hedges SB, Marin J, Suleski M, et al. Tree of life reveals clock-like speciation and diversification. Mol Biol Evol. 2015; 32: 835-45.

[131] Mendes FK, Vanderpool D, Fulton B, et al. CAFE 5 models variation in evolutionary rates among gene families. Bioinformatics. 2021; 36: 5516-8.

[132] Zhang Z, Xiao J, Wu J, et al. ParaAT: a parallel tool for constructing multiple protein-coding DNA alignments. Biochem Biophys Res Commun. 2012; 419: 779-81.

[133] Zhang Z . KaKs_Calculator 3.0: calculating selective pressure on coding and non-coding sequences. Genomics Proteomics Bioinformatics. 2022; 20: 536-40.

[134] Wu H, Ma T, Kang M, et al. A high-quality Actinidia chinensis (kiwifruit) genome. Hortic Res. 2019; 6: 117.

[135] Bi G, Mao Y, Xing Q, et al. HomBlocks: a multiple-alignment construction pipeline for organelle phylogenomics based on locally collinear block searching. Genomics. 2018; 110: 18-22.

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

[137] Langfelder P, Horvath S . WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics. 2008; 9: 559.

[138] Shannon P, Markiel A, Ozier O, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13: 2498-504.

[139] Zheng P, Cao L, Zhang C, et al. MYB43 as a novel substrate for CRL4 PRL1 E3 ligases negatively regulates cadmium tolerance through transcriptional inhibition of HMAs in Arabidopsis . New Phytol. 2022; 234: 884-901.

[140] Zhang S, Zhao Q, Zeng D, et al. RhMYB108, an R2R3-MYB transcription factor, is involved in ethylene- and JA-induced petal senescence in rose plants. Hortic Res. 2019; 6: 131.

[141] Di D, Sun L, Wang M, et al. WRKY46 promotes ammonium tolerance in Arabidopsis by repressing NUDX9 and indole-3-acetic acid-conjugating genes and by inhibiting ammonium efflux in the root elongation zone. New Phytol. 2021; 232: 190-207.