Abstract
Haplotype-level allelic characterization facilitates research on the functional, evolutionary and breeding-related features of extremely large and complex plant genomes. We report a 21.7-Gb chromosome-level haplotype-resolved assembly in Pinus densiflora. We found genome rearrangements involving translocations and inversions between chromosomes 1 and 3 of Pinus species and a proliferation of specific long terminal repeat (LTR) retrotransposons (LTR-RTs) in P. densiflora. Evolutionary analyses illustrated that tandem and LTR-RT-mediated duplications led to an increment of transcription factor (TF) genes in P. densiflora. The haplotype sequence comparison showed allelic imbalances, including presence–absence variations of genes (PAV genes) and their functional contributions to flowering and abiotic stress-related traits in P. densiflora. Allele-aware resequencing analysis revealed PAV gene diversity across P. densiflora accessions. Our study provides insights into key mechanisms underlying the evolution of genome structure, LTR-RTs and TFs within the Pinus lineage as well as allelic imbalances and diversity across P. densiflora.
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Data availability
The genome assembly and annotation data for the haplotypes of P. densiflora and the genotype information generated from resequencing analysis of P. densiflora accessions have been deposited in figshare+ (https://doi.org/10.25452/figshare.plus.25546534)105. Resequencing data have been deposited at the NCBI SRA under BioProject accession number PRJNA1089250.
Code availability
The related code has been deposited in GitHub (https://github.com/minjeongjj/pinus_densiflora_haplotype_genome) and Zenodo (https://doi.org/10.5281/zenodo.12791823)106. All software used in this study is publicly available as described in Methods and Reporting Summary.
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Acknowledgements
This study was supported by the Basic Science Research Program through a National Research Foundation of Korea grant funded by the Korean government (NRF-2022R1C1C1004918) to S.K., by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry through the Digital Breeding Transformation Technology Development Program funded by the Ministry of Agriculture, Food, and Rural Affairs (322075-3) to S.K., by a grant from the Korea Forest Service of the Korean government through the R&D Program for Forestry Technology (2014071H10-2022-AA04) to S.K. and E.-J.P. and by a National Institute of Forest Science grant (Forest Science Research project number FG0603-2021-01-2022) to E.-J.P. This research was supported in part by the Plant Biotic Interaction program of the National Science Foundation of the USA (NSF-IOS-2126256) to E.P. and J.W. and the intramural research program of the US Department of Agriculture, National Institute of Food and Agriculture Hatch Capacity (7000762) to E.P. We appreciate S. J. Lee of DNA Link, who provided support for genome sequencing analyses during this project.
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S.K. designed and organized the study as a lead contact. S.K. and E.-J.P. initiated the project. M.-J.J., H.J.C., Y.-S.P., S.-J.K., J.-W.C., G.Y.C., J.-Y.G. and S.K. performed data generation and/or bioinformatics analysis. M.-J.J., G.Y.C. and Y.-M.K. performed de novo genome assembly and annotation. H.J.C., Y.-S.P. and J.-W.C. performed TF gene analysis. M.-J.J. and H.J.C. performed haplotype variation and ortholog-based analysis. J.-W.C., J.-Y.G. and J.-H.K. performed the transcriptome analysis. M.-J.J., Y.-S.P., H.J.C. and M.-S.K. performed the resequencing analysis. D.Y.K., H.J.C., M.-J.J., S.-J.K. and S.-H.K. performed PAV and ASE validation. H.J.C. and E.-K.B. performed protein structure and abiotic stress analyses. E.-K.B., M.-J.K., H.L., K.-S.C., I.S.K., K.-S.K. and E.-J.P. prepared plant material, DNA and RNA samples and raw resequencing data. H.-Y.L., S.J., H.J., J.W., E.P. and D.C. performed ectopic expression and subcellular localization analyses. H.-Y.L. and E.P. designed, organized and drafted the initial manuscript of the ectopic expression and subcellular localization analyses. M.-J.J. and H.J.C. wrote the initial draft manuscript. S.K., M.-J.J. and H.J.C. reviewed and edited the manuscript. All authors approved the final version of the manuscript.
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Extended data
Extended Data Fig. 1 The k-mer analysis for genome size estimation and polymorphism of P. densiflora.
a, 21 k-mer depth distribution for genome size estimation. The x-axis indicates the k-mer depth and the y-axis indicates the frequency of k-mers. The dotted line represents the peak value. b, Haplotype-specific k-mer assembly spectrum (spectra-asm) plot from Merqury results. The graph shows k-mer proportion of haplotype-specific (red and blue), shared (green), and read-only (grey). The red and blue lines represent the evenly bisected the haplotype specific portion of k-mers, respectively.
Extended Data Fig. 2 Hi-C contact map for each of the 12 chromosomes in the P. densiflora genome.
a, Haplotype A (HA). b, Haplotype B (HB). The color scale from white to red indicates low to high contact probability.
Extended Data Fig. 3 Comparison of genomic analysis between P. densiflora and other Pinus species.
a, Genome-to-genome alignment of P. densiflora HA and other Pinus species. The color gradient from white to red represents gene-poor and gene-rich chromosome regions. The color gradient from white to green represents the increase in matched alignment length in each Pinus genome by the P. densiflora genome, while the height of the bar represents the alignment length in the P. densiflora genome by other Pinus genomes. The blue dotted line indicates the total proportion of matched genome sequences in the P. densiflora genome from other Pinus species. Asterisks (*) denote the top three highly duplicated regions in each chromosome of P. tabuliformis. b, Synteny comparison between P. densiflora HA and P. tabuliformis.
Extended Data Fig. 4 Genome rearrangements between chromosomes 1 and 3 in Pinus species.
Physical and genetic maps of Pinus species are depicted. The blocks (with a window size of 60 Mb) in the P. densiflora and P. tabuliformis genomes, mapped by representative markers of a, P. thunbergii, b, P. densiflora, and c, P. taeda are illustrated in grey color. LG, linkage group. In accordance with P. densiflora, the red and blue color for line, background, and marker represent chromosomes 1 and 3, respectively. Asterisks (*) denote the markers shown in Fig. 1c.
Extended Data Fig. 5 The correlation between the number of genes and LTR-RTs in the gypsy and copia subgroups on chromosomes of P. densiflora HA.
For each chromosome: a, upper correlation plots show LTR-RT subgroups that positively, negatively, or neutrally correlated with gene density and b, only neutrally correlated with gene density. The line colors indicate each LTR-RT subgroup. The number of genes (top) and LTR-RTs (bottom) were plotted as density within 30 Mb intervals. The color gradient from white to red, blue, and green represents increased number of genes, negatively correlated LTR-RTs with genes, and positively correlated LTR-RTs with genes, respectively.
Extended Data Fig. 6 Evolutionary analysis of Pinus and other plant species.
a, Evolution of gene families in 20 plant species. The numbers in blue and orange, separated by a slash, indicate the expanded and contracted gene families and rapidly evolved gene families, respectively (a two-sided P < 0.01) (left). On the right, the size and color of the circles indicate the number of rapid or not gene family expansion/contraction and gene gain/loss in each species. b, Domain repertoire of rapidly evolved genes in 4 Pinus species. The red, blue, orange, and green diamonds indicate 4, 3, 2, and individual Pinus species, respectively. c, Domain repertoire of genes in repeat regions of P. densiflora. d, Domain repertoire of rapidly evolved genes in individual Pinus species.
Extended Data Fig. 7 Enrichment analysis and phylogenetic relationships of TFs in Pinus, gymnosperm, and angiosperm.
a, The average number of TFs in Pinus (n = 4), gymnosperm (n = 7), and angiosperm (n = 9). Asterisks (***) denote a significance level of P < 0.001 based on a one-sided Fisher’s test. The individual data points are listed in Supplementary Table 3. Error bars indicate the standard error (SE). b, The phylogenetic relationships of TFs in 20 plant species. Dot colors on the nodes represents species, while the ring indicates subgroups.
Extended Data Fig. 8 Allelic imbalance of genic regions.
a, Genomic variations in genes including 2 kb upstream and downstream between P. densiflora HA and HB. The bar graphs show the total number of variations and overall number of genes containing SNPs, indels, and SVs in each genic region. b, Allelic gene categorization of P. densiflora. c, Domain repertoire of PAV and ASE genes. d, GO descriptions in biological process of PAV and ASE genes.
Extended Data Fig. 9 Validation of PAVs and ASEs in P. densiflora and characterization of Arabidopsis functional orthologous genes (FOGs).
a, Sequence validation of haplotype-specific presence of PAVs. b, Tissue abundant and haplotype unbalanced expression of ASEs in leaf, stem, and root. Asterisks (**) denote a significance level of P < 0.01 based on a one-sided unpaired Student’s t-test. At least two biological replicates are used. Error bars indicate the SE. The brown boxes and black lines indicate exons and introns, respectively. The red and blue backgrounds of amino acids indicate presence and absence, respectively. The hashtags (#) and asterisks (*) indicate frameshift mutation and stop codon, respectively. c, Arabidopsis FOGs annotated in P. densiflora HA and HB. d, Domain repertoire (left) and GO descriptions in biological process (right) of allele, PAV, and ASE genes. The pink, blue, and orange bars indicate allele, PAV, and ASE genes, respectively.
Extended Data Fig. 10 Genome-wide distribution for allele and PAV genes of P. densiflora and 30 wild accessions.
The outer track represents 12 chromosomes. The inner tracks represent gene density and PAV density for each accession.
Supplementary information
Supplementary Tables
Supplementary Tables 1–10.
Supplementary Data 1
Supplementary Data 1.
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Jang, MJ., Cho, H.J., Park, YS. et al. Haplotype-resolved genome assembly and resequencing analysis provide insights into genome evolution and allelic imbalance in Pinus densiflora. Nat Genet 56, 2551–2561 (2024). https://doi.org/10.1038/s41588-024-01944-y
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DOI: https://doi.org/10.1038/s41588-024-01944-y
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