Haplotype-resolved and chromosome-level reference genome assembly of Diospyros deyangensis provides insights into the evolution and juvenile growth of persimmon

Abstract

The Diospyros genus, which includes both wild and cultivated species such as Diospyros lotus and Diospyros kaki, represents a diverse genetic pool with significant agricultural value. In this study, we present a high-quality, haplotype-resolved, chromosome-level genome assembly for Diospyros deyangensis (hereinafter referred to as ‘Deyangshi’), an autotetraploid wild species notable for its short juvenile phase, by integrating high-fidelity single-molecule, nanopore sequencing, and high-throughput chromosome conformation capture techniques. The assembled genome size is ~3.01 Gb, anchored onto 60 pseudochromosomes. Comparative genomic analysis revealed that the D. deyangensis genome underwent an additional whole-genome duplication (WGD) event following the eudicots shared ancient hexaploidy event. Resequencing and clustering on 63 samples representing 11 geographically diverse Diospyros accessions revealed significant genetic differentiation between D. deyangensis and D. kaki, as well as between D. kaki and other Diospyros species using population genomic analyses, suggesting that D. kaki followed an independent evolutionary pathway. Additionally, we identified DdELF4 (EARLY FLOWERING 4) from the ‘Deyangshi’ backcross population using bulked segregant RNA sequencing (BSR-seq) with 50 early-flowering and 50 non-early-flowering individuals. Overexpression of DdELF4 in Arabidopsis resulted in delayed flowering and downregulation of FT gene expression, indicating its role as a flowering repressor. This high-quality genome assembly of ‘Deyangshi’ provides an essential genomic resource for the Diospyros genus, particularly for breeding programs focused on developing early-flowering persimmon varieties.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (32272672), the Key Research and Development Program of Shaanxi Province, China (2022NY-11,2), and the Innovation Capability Support Program of Shaanxi Province, China (2022PT-25).

Author contributions

T.Z., and C.F.G. coordinated the project, and conceived and designed the experiments. C.F.G., Y.X.L., Z.X.L., Q.G.Z., Y.X.Z., Z.G.L., J.F., and J.Y.L. analyzed all the data. C.F.G. provided experiment materials. C.F.G., Y.X.L., Z.X.L., and T.Z. wrote the manuscript. P.X.Z., X.X.S., Q.L.Z., Q.M.G., Z.R.L., and Y.Y. revised the manuscript. All the authors have read and approved the paper.

Data availability

The raw sequence reads of the ‘Deyangshi’ genome and the whole-genome resequenced reads of the 63 Diospyros samples have been deposited in NCBI SRA, with the accession number PRJNA861725. The genome assembly, annotation data, and pangenome graph have been deposited at Zenodo (https://doi.org/10.5281/zenodo.14501941).

Conflict of interest statement

No conflicts of interest were declared.

Supplementary Data

Supplementary data is available at Horticulture Research online.

References

[1] Luo ZR, Wang RZ. Persimmon in China: domestication and traditional utilizations of genetic resources. Adv Hortic Sci. 2008; 22: 239-43

[2] Tang DL, Zhang QL, Xu LQ. et al. Number of species and geo-graphical distribution of Diospyros L. (Ebenaceae) in China. Hortic Plant J. 2019; 2:59-69

[3] Guan CF, Duan XY, Zhang QL. et al. DkPK genes promote natural deastringency in C-PCNA persimmon by up-regulating DkPDC and DkADH expression. Front Plant Sci. 2017; 8:149

[4] Matheus JRV, de Andrade CJ, Miyahira RF. et al. Persimmon (Diospyros kaki L.): chemical properties, bioactive compounds and potential use in the development of new products-a review. Food Rev Int. 2022; 38:384-401

[5] Zhuang D, Kitajima A, Ishida M. et al. Chromosome number of Diospyros kaki cultivars. J Jpn Soc Hortic Sci. 1990; 59:289-97

[6] Kanzaki S. The origin and cultivar development of Japanese per-simmon (Diospyros kaki Thunb.). Nippon Shokuhin Kagaku Kogaku Kaishi. 2016; 63:328-30

[7] Zhang YF, Yang Y, Guo J. et al. Taxonomic status of “Deyangshi” based on chromosome number and SRAP markers. Sci Hortic. 2016; 207:57-64

[8] Guan CF, Liu SY, Wang MK. et al. Comparative transcriptomic analysis reveals genetic divergence and domestication genes in Diospyros. BMC Plant Biol. 2019; 19:227

[9] Fang J, Long H, Wang Z. et al. Genome-wide analysis of the early flowering 4 (ELF4) gene family in short-juvenile persim-mon “Deyangshi” (Diospyros deyangensis) and its role of DdELF4-8 during flowering control. Sci Hortic. 2023; 310:111736

[10] Chen H, Zeng Y, Yang Y. et al. Allele-aware chromosome-level genome assembly and efficient transgene-free genome editing for the autotetraploid cultivated alfalfa. Nat Commun. 2020; 11:2494

[11] Zaman QU, Hui L, Nazir MF. et al. Chromosome-level genome assembly of autotetraploid Selenicereus megalanthus and gain-ing genomic insights into the evolution of trait pattern-ing in diploid and polyploid pitaya species. 2024; bioRxiv 2024.2006.2023.600268

[12] Wang F, Wang Y, Zeng X. et al. MIKE: an ultrafast, assembly-, and alignment-free approach for phylogenetic tree construction. Bioinformatics. 2024; 40:4

[13] Guo M, Bi G, Wang H. et al. Genomes of autotetraploid wild and cultivated Ziziphus mauritiana reveal polyploid evolution and crop domestication. Plant Physiol. 2024; 196:2701-20

[14] Jia KH, Wang ZX, Wang L. et al. SubPhaser: a robust allopolyploid subgenome phasing method based on subgenome-specific k-mers. New Phytol. 2022; 235:801-9

[15] Lu XM, Yu XF, Li GQ. et al. Genome assembly of autote-traploid Actinidia arguta highlights adaptive evolution and enables dissection of important economic traits. Plant Commun. 2024; 5:100856

[16] Akagi T, Shirasawa K, Nagasaki H. et al. The persimmon genome reveals clues to the evolution of a lineage-specific sex determi-nation system in plants. PLoS Genet. 2020; 16:e1008566

[17] Horiuchi A, Masuda K, Shirasawa K. et al. Ongoing rapid evolu-tion of a post-Y region revealed by chromosome-scale genome assembly of a hexaploid monoecious persimmon (Diospyros kaki). Mol Biol Evol. 2023; 40:7

[18] Mao W, Yao G, Wang S. et al. Chromosome-level genomes of seeded and seedless date plum based on third-generation DNA sequencing and Hi-C analysis. Forestry Research. 2021; 1:0

[19] Suo Y, Sun P, Cheng H. et al. A high-quality chromoso-mal genome assembly of Diospyros oleifera Cheng. GigaScience. 2020; 9:1

[20] Zhu QG, Xu Y, Yang Y. et al. The persimmon (Diospyros oleifera Cheng) genome provides new insights into the inheritance of astringency and ancestral evolution. Hortic Res. 2019; 6:138

[21] Li WQ, Liu YL, Yang Y. et al. Interspecific chloroplast genome sequence diversity and genomic resources in Diospyros. BMC Plant Biol. 2018; 18:210

[22] Liu H, Guo S, Lu M. et al. Biosynthesis of DHGA(12) and its roles in Arabidopsis seedling establishment. Nat Commun. 2019; 10:1768

[23] Chang S, Puryear J, Cairney J. A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Report. 1993; 11:113-6

[24] Jiao F, Luo RS, Dai XL. et al. Chromosome-level reference genome and population genomic analysis provide insights into the evo-lution and improvement of domesticated mulberry (Morus alba). Mol Plant. 2020; 13:1001-12

[25] Rao SS, 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

[26] Kokot M, Dlugosz M, Deorowicz S. KMC 3: counting and manip-ulating k-mer statistics. Bioinformatics. 2017; 33:2759-61

[27] Ranallo-Benavidez TR, Jaron KS, Schatz MC. GenomeScope 2.0 and Smudgeplot for reference-free profiling of polyploid genomes. Nat Commun. 2020; 11:1432

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

[29] Cheng H, Jarvis ED, Fedrigo O. et al. Haplotype-resolved assem-bly of diploid genomes without parental data. Nat Biotechnol. 2022; 40:1332-5

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

[31] Zhou C, McCarthy SA, Durbin R. YaHS: yet another Hi-C scaf-folding tool. Bioinformatics. 2023; 39:1

[32] 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

[33] Lin Y, Ye C, Li X. et al. quarTeT: a telomere-to-telomere toolkit for gap-free genome assembly and centromeric repeat identifi-cation. Hortic Res. 2023; 10:uhad127

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

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

[36] Ou SJ, Su WJ, Liao Y. et al. Benchmarking transposable element annotation methods for creation of a streamlined, comprehen-sive pipeline. Genome Biol. 2019; 20:275

[37] Tarailo-Graovac M, Chen N. Using RepeatMasker to identify repetitive elements in genomic sequences. Curr Protoc Bioinfor-matics. 2009; 25:4.10.11-14.10.14

[38] Ellinghaus D, Kurtz S, Willhoeft U. LTRharvest, an efficient and flexible software for de novo detection of LTR retrotransposons. BMC Bioinformatics. 2008; 9:18

[39] Xu Z, Wang H. LTR_FINDER: an efficient tool for the pre-diction of full-length LTR retrotransposons. Nucleic Acids Res. 2007; 35:W265-8

[40] Holt C, Yandell M. MAKER2: an annotation pipeline and genome-database management tool for second-generation genome projects. BMC Bioinformatics. 2011; 12:491

[41] Keilwagen J, Hartung F, Grau J. GeMoMa: homology-based gene prediction utilizing intron position conservation and RNA-seq data. Methods Mol Biol. 2019; 1962:161-77

[42] Stanke M, Keller O, Gunduz I. et al. AUGUSTUS: ab initio pre-diction of alternative transcripts. Nucleic Acids Res. 2006; 34: W435-9

[43] Haas BJ, Salzberg SL, Zhu W. et al. Automated eukaryotic gene structure annotation using evidence modeler and the program to assemble spliced alignments. Genome Biol. 2008; 9:R7

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

[45] Goel M, Sun H, Jiao WB. et al. SyRI: finding genomic rear-rangements and local sequence differences from whole-genome assemblies. Genome Biol. 2019; 20:277

[46] Goel M, Schneeberger K. Plotsr: visualizing structural similar-ities and rearrangements between multiple genomes. Bioinfor-matics. 2022; 38:2922-6

[47] Emms DM, Kelly S. OrthoFinder: phylogenetic orthology infer-ence for comparative genomics. Genome Biol. 2019; 20:1-14

[48] Bu D, Luo H, Huo P. et al. KOBAS-i: intelligent prioritization and exploratory visualization of biological functions for gene enrichment analysis. Nucleic Acids Res. 2021; 49:W317-w325

[49] Nguyen LT, Schmidt HA, Haeseler A. et al. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015; 32:268-74

[50] Yang Z. PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol. 2007; 24:1586-91

[51] Zhao T, Zwaenepoel A, Xue JY. et al. Whole-genome microsynteny-based phylogeny of angiosperms. Nat Commun. 2021; 12:3498

[52] Han MV, Thomas GWC, Lugo-Martinez J. et al. Estimating gene gain and loss rates in the presence of error in genome assembly and annotation using CAFE 3. MolBiolEvol. 2013; 30:1987-97

[53] Wang D, Zhang Y, Zhang Z. et al. KaKs_Calculator 2.0: a toolkit incorporating gamma-series methods and sliding window strategies. Genomics Proteomics Bioinformatics. 2010; 8:77-80

[54] Zhao T, Holmer R, et al. Phylogenomic syn-teny network analysis of MADS-box transcription factor genes reveals lineage-specific transpositions, ancient tandem dupli-cations, and deep positional conservation. Plant Cell. 2017; 29: 1278-92

[55] He W, Yang J, Jing Y. et al. NGenomeSyn: an easy-to-use and flexible tool for publication-ready visualization of syntenic relationships across multiple genomes. Bioinformatics. 2023; 39:3

[56] Hickey G, Monlong J, Ebler J. et al. Pangenome graph construction from genome alignments with Minigraph-Cactus. Nat Biotechnol. 2024; 42:663-73

[57] Ebler J, Ebert P, Clarke WE. et al. Pangenome-based genome inference allows efficient and accurate genotyping across a wide spectrum of variant classes. Nat Genet. 2022; 54:518-25

[58] Danecek P, Bonfield JK, Liddle J. et al. Twelve years of SAMtools and BCFtools. GigaScience. 2021; 10:2

[59] Purcell S, Neale B, Todd-Brown K. et al. PLINK: a tool set for whole-genome association and population-based linkage anal-yses. Am J Hum Genet. 2007; 81:559-75

[60] Alexander DH, Lange K. Enhancements to the ADMIXTURE algorithm for individual ancestry estimation. BMC Bioinformatics. 2011; 12:246

[61] Francis RM. Pophelper: an R package and web app to analyse and visualize population structure. Mol Ecol Resour. 2017; 17:27-32

[62] Katz LS, Griswold T, Morrison SS. et al. Mashtree: a rapid com-parison of whole genome sequence files. J Open Source Softw. 2019; 4:44

[63] Letunic I, Bork P. Interactive tree of life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021; 49:W293-6

[64] Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015; 12:357-60

[65] Zhang L, Duan Y, Zhang Z. et al. OcBSA: an NGS-based bulk seg-regant analysis tool for outcross populations. Mol Plant. 2024; 17: 648-57

[66] Zhang XR, Henriques R, Lin SS. et al. Agrobacterim-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc. 2006; 1:641-6

[67] Zhang PX, Li XL, Wang YF. et al. PRMT6 physically associates with nuclear factor Y to regulate photoperiodic flowering in Arabidopsis. aBIOTECH. 2021; 2:403-14

[68] Chen P, Li Z, Zhang D. et al. Insights into the effect of human civ-ilization on Malus evolution and domestication. Plant Biotechnol J. 2021a; 19:2206-20

[69] Chen WX, Zheng QY, Li JW. et al. DkMYB14 is a bifunc-tional transcription factor that regulates the accumulation of proanthocyanidin in persimmon fruit. Plant J. 2021b; 106: 1708-27