Phylogeny-driven pangenome analysis uncovers the genomic landscape of domesticated and wild Armeniaca species

Abstract

Long-read sequencing and pangenomics are revolutionizing crop research by providing more complete genome information and revealing crucial structural variations (SVs) linked to important agricultural traits. Building on recent advances in intraspecific pangenome construction, this study addresses the challenge of creating broader, cross-taxon pangenomes, using the Armeniaca taxonomic section as a model. Leveraging a diverse panel of genome assemblies as well as completing it with seven more genome assemblies generated for this study, we constructed a pangenome graph and cataloged the associated genetic variation, identifying approximately 25 million single nucleotide polymorphisms and over 537 000 structural variants. We characterized the diversity of these variants and assessed the extent to which different taxa contribute to overall pangenome expansion. Additionally, we evaluated the performance of low-depth sample mapping to the graph-based reference, highlighting key technical limitations that may affect the quality of downstream analyses. We further identified specific subsets of SVs that exhibit associations with particular classes of transposable elements (TEs). We showed that TEs are a major driver of SV, particularly insertions and deletions, with distinct size and distribution patterns (peaking in the 200- to 400-bp indel bin). They are also nonrandomly positioned in the genome, showing a tight concentration near coding genes, which suggests a role in gene regulation. As a case study illustrating the potential functional relevance of graph-derived SVs, we examined the genomic configuration of the Dormancy-Associated MADS box locus within the Armeniaca pangenome. These findings provide a framework to investigate adaptation in perennial fruit trees of the Armeniaca section.

References

[1] Li W, Liu J, Zhang H, et al. Plant pan-genomics: recent advances, new challenges, and roads ahead. J Genet Genomics. 2022a; 49: 833-46.

[2] Lye ZN, Purugganan MD . Copy number variation in domestication. Trends Plant Sci. 2019; 24: 352-65.

[3] Dolatabadian A, Bayer PE, Tirnaz S, et al. Characterization of disease resistance genes in the Brassica napus pangenome reveals significant structural variation. Plant Biotechnol J. 2020; 18: 969-82.

[4] Schreiber M, Jayakodi M, Stein N, et al. Plant pangenomes for crop improvement, biodiversity and evolution. Nat Rev Genet. 2024; 25: 563-77.

[5] Gong Y, Li Y, Liu X, et al. A review of the pangenome: how it affects our understanding of genomic variation, selection and breeding in domestic animals? J Anim Sci Biotechnol. 2023; 14: 73.

[6] Li L, Bartholomew B . Armeniaca. In: Wu Z, Raven PH, eds. Flora of China.Beijing and St. Science Press and Missouri Botanical Garden Press: Louis, 2003, 396-401.

[7] Decroocq S, Cornille A, Tricon D, et al. New insights into the history of domesticated and wild apricots and its contribution to plum pox virus resistance. Mol Ecol. 2016; 25: 4712-29.

[8] Groppi A, Liu S, Cornille A, et al. Population genomics of apricots unravels domestication history and adaptive events. Nat Commun. 2021; 12: 3956.

[9] Rieger M. Introduction to Fruit Crops. Boca Raton: CRC Press; 2006.

[10] Golicz AA, Bayer PE, Bhalla PL, et al. Pangenomics comes of age: from bacteria to plant and animal applications. Trends Genet. 2020; 36: 132-45.

[11] Li N, He Q, Wang J, et al. Super-pangenome analyses highlight genomic diversity and structural variation across wild and cultivated tomato species. Nat Genet. 2023; 55: 852-60.

[12] Liao WW, Asri M, Ebler J, et al. A draft human pangenome reference. Nature. 2023; 617: 312-24.

[13] Bayer PE, Golicz AA, Scheben A, et al. Plant pan-genomes are the new reference. Nat Plants. 2020; 6: 914-20.

[14] Cochetel N, Minio A, Guarracino A, et al. A super-pangenome of the North American wild grape species. Genome Biol. 2023; 24: 290.

[15] Chen W, Xie Q, Fu J, et al. Graph pangenome reveals the regulation of malate content in blood-fleshed peach by NAC transcription factors. Genome Biol. 2025; 26: 7.

[16] Campoy JA, Sun H, Goel M, et al. Gamete binning: chromosome-level and haplotype-resolved genome assembly enabled by high-throughput single-cell sequencing of gamete genomes. Genome Biol. 2020; 21: 306.

[17] Jalil M, Quddos F, Anwer F, et al. Comparative pan-genomic analysis revealed an improved multi-locus sequence typing scheme for Staphylococcus aureus. Genes. 2022; 13: 2160.

[18] Meguerditchian C, Ergun A, Decroocq V, et al. Pipeline to detect the positional and directional relationship between transposable elements and adjacent genes in host genome. Peer Commun J. 2021; 1: e48.

[19] Crysnanto D, Pausch H . Bovine breed-specific augmented reference graphs facilitate accurate sequence read mapping and unbiased variant discovery. Genome Biol. 2020; 21: 184.

[20] Balogh E, Halász J, Soltész A, et al. Identification, structural and functional characterization of dormancy regulator genes in apricot (Prunus armeniaca L.) . Front Plant Sci. 2019; 10: 402.

[21] Quesada-Traver C, Lloret A, Carretero-Paulet L, et al. Evolutionary origin and functional specialization of Dormancy-Associated MADS box (DAM) proteins in perennial crops. BMC Plant Biol. 2022; 22: 473.

[22] Michael TP, VanBuren R . Building near-complete plant genomes. Curr Opin Plant Biol. 2020; 54: 26-33.

[23] Zhou Y, Chebotarov D, Kudrna D, et al. A platinum standard pan-genome resource that represents the population structure of Asian rice. Sci Data. 2020; 7: 113.

[24] Kreplav J, Madoui MA, Cápal P, et al. A reference genome for pea provides insight into legume genome evolution. Nat Genet. 2019; 51: 1411-22.

[25] Yang T, Liu R, Luo Y, et al. Improved pea reference genome and pan-genome highlight genomic features and evolutionary characteristics. Nat Genet. 2022; 54: 1553-63.

[26] Beaulieu C, Libourel C, Mbadinga Zamar DL, et al. The Marchantia polymorpha pangenome reveals ancient mechanisms of plant adaptation to the environment. Nat Genet. 2025; 57: 729-40.

[27] He Q, Tang S, Zhi H, et al. A graph-based genome and pan-genome variation of the model plant Setaria . Nat Genet. 2023; 55: 1232-42.

[28] Tettelin H, Masignani V, Cieslewicz MJ, et al. Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: implications for the microbial “pan-genome”. Proc Natl Acad Sci USA. 2005; 102: 13950-5.

[29] Liu C, Feng C, Peng W, et al. Chromosome-level draft genome of a diploid plum (Prunus salicina) . GigaScience. 2020; 9: giaa130.

[30] Wang W, Mauleon R, Hu Z, et al. Genomic variation in 3,010 diverse accessions of Asian cultivated rice. Nature. 2018; 557: 43-9.

[31] Jiang F, Zhang J, Wang S, et al. The apricot (Prunus armeniaca L.) genome elucidates Rosaceae evolution and beta-carotenoid synthesis . Hortic Res. 2019; 6: 128.

[32] Bao F, Ding A, Zhang T, et al. Expansion of PmBEAT genes in the Prunus mume genome induces characteristic floral scent production. Hortic Res. 2019; 6: 24.

[33] Hübner S, Bercovich N, Todesco M, et al. Sunflower pan-genome analysis shows that hybridization altered gene content and disease resistance. Nat Plants. 2018; 5: 54-62.

[34] Jayakodi M, Lu Q, Pidon H, et al. Structural variation in the pangenome of wild and domesticated barley. Nature. 2024; 636: 654-62.

[35] Tan Q, Li S, Zhang Y, et al. Chromosome-level genome assemblies of five Prunus species and genome-wide association studies for key agronomic traits in peach. Hortic Res. 2021; 8: 213.

[36] Jauregui B, de Vicente MC, Messeguer R, et al. A reciprocal translocation between ‘Garfi’ almond and ‘Nemared’ peach. Theor Appl Genet. 2001; 102: 1169-76.

[37] Sun S, Meng J, Zhang W, et al. A translocation between chromosome 6 and 8 influences lncRNA_MYB114 and PpRPP13 expression and underpins red leaf trait and powdery mildew resistance in peach. New Phytol. 2025; 246: 1198-216.

[38] Feschotte C . The contribution of transposable elements to the evolution of regulatory networks. Nat Rev Genet. 2008; 9: 397-405.

[39] Oliver KR, Greene WK . Transposable elements: powerful facilitators of evolution. BioEssays. 2009; 31: 703-14.

[40] Morales-Díaz N, Sushko S, Campos-Dominguez L, et al. Tandem LTR-retrotransposon structures are common and highly polymorphic in plant genomes. Mobile DNA. 2025; 16: 10.

[41] Su W, Gu X, Peterson T . TIR-learner, a new ensemble method for TIR transposable element annotation, provides evidence for abundant new transposable elements in the maize genome. Mol Plant. 2019; 12: 447-60.

[42] Cho J, Paszkowski J . Regulation of rice root development by a retrotransposon acting as a microRNA sponge. eLife. 2017; 6: e30038.

[43] Feschotte C, Pritham EJ . DNA transposons and the evolution of eukaryotic genomes. Annu Rev Genet. 2007; 41: 331-68.

[44] Hassan AH, Mokhtar MM, El Allali A . Transposable elements: multifunctional players in the plant genome. Front Plant Sci. 2024; 14: 1330127.

[45] Zheng PX, Ko CY, Ou JY, et al. Transposable elements drive evolution and perturb gene expression in Brassica rapa and B. oleracea . Plant J. 2025; 123: e70452.

[46] Alioto T, Alexiou KG, Bardil A, et al. Transposons played a major role in the diversification between the closely related almond and peach genomes: results from the almond genome sequence. Plant J. 2020; 101: 455-72.

[47] Yu Z, Li J, Wang H, et al. Transposable elements in Rosaceae: insights into genome evolution, expression dynamics, and syntenic gene regulation. Hortic Res. 2024; 11: uhae118.

[48] Li H, Wang S, Chai S, et al. Graph-based pan-genome reveals structural and sequence variations related to agronomic traits and domestication in cucumber. Nat Commun. 2022b; 13: 682.

[49] Alonge M, Wang X, Benoit M, et al. Major impacts of widespread structural variation on gene expression and crop improvement in tomato. Cell. 2020; 182: 145-61.e23.

[50] Hufford MB, Seetharam AS, Woodhouse MR, et al. De novo assembly, annotation, and comparative analysis of 26 diverse maize genomes. Science. 2021; 373: 655-662.

[51] Straub SCK, Parks M, Weitemier K, et al. Navigating the tip of the genomic iceberg: next-generation sequencing for plant systematics. Am J Bot. 2012; 99: 349-64.

[52] 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.

[53] Garrison E, Guarracino A, Heumos S, et al. Building pangenome graphs. Nat Methods. 2024; 21: 2008-12.

[54] Li H, Handsaker B, Wysoker A, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009; 25: 2078-9.

[55] Ondov BD, Treangen TJ, Melsted P, et al. Mash: fast genome and metagenome distance estimation using MinHash. Genome Biol. 2016; 17: 132.

[56] R Core Team . R: A Language and Environment for Statistical Computing . Vienna, Austria: R Foundation for Statistical Computing; 2023.

[57] Cabanettes F, Klopp C . D-GENIES: dot plot large genomes in an interactive, efficient and simple way. PeerJ. 2018; 6: e4958.

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

[59] Goel M, Schneeberger K . Plotsr: visualizing structural similarities and rearrangements between multiple genomes. Bioinformatics. 2022; 38: 2922-6.

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

[61] Garrison E, Guarracino A . Unbiased pangenome graphs. Bioinformatics. 2023; 39: btac743.

[62] Jonkheer EM, van Workum DJM, Sheikhizadeh Anari S, et al. PanTools v3: functional annotation, classification and phylogenomics. Bioinformatics. 2022; 38: 4403-5.

[63] Kokot M, Długosz M, Deorowicz S . KMC 3: counting and manipulating k-mer statistics. Bioinformatics. 2017; 33: 2759-61.

[64] Schliep KP . Phangorn: phylogenetic analysis in R. Bioinformatics. 2011; 27: 592-3.

[65] Paradis E, Schliep K . Ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R. Bioinformatics. 2019; 35: 526-8.

[66] Huson DH, Richter DC, Rausch C, et al. Dendroscope: an interactive viewer for large phylogenetic trees. BMC Bioinf. 2007; 8: 460.

[67] Robinson DF, Foulds LR . Comparison of phylogenetic trees. Math Biosci. 1981; 53: 131-47.

[68] Letunic I, Bork P . Interactive tree of life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics. 2007; 23: 127-8.

[69] Rehder A . Manual of Cultivated Trees and Shrubs Hardy in North America: Exclusive of the Subtropical and Warmer Temperate Regions . Portland, OR: Dioscorides Press; 1986.

[70] Kurtzer GM, Sochat V, Bauer MW . Singularity: scientific containers for mobility of compute. PLoS One. 2017; 12: e0177459.

[71] Garrison E, Sirén J, Novak AM, et al. Variation graph toolkit improves read mapping by representing genetic variation in the reference. Nat Biotechnol. 2018; 36: 875-9.

[72] Zytnicki M . Assessing genome conservation on pangenome graphs with PanSel. Bioinf Adv. 2025; 5: vbaf018.

[73] Danecek P, Auton A, Abecasis G, et al. The variant call format and VCFtools. Bioinformatics. 2011; 27: 2156-8.

[74] Wickham H, François R, Henry L, et al. 2025. dplyr: A Grammar of Data Manipulation. https://cran.r-project.org/web/packages/dplyr.

[75] Wickham H . ggplot2 . Cham: Springer International Publishing; 2016.

[76] Parmigiani L, Garrison E, Stoye J, et al. Panacus: fast and exact pangenome growth and core size estimation. Bioinformatics. 2024; 40: btae720.

[77] Altschul SF, Madden TL, Schäffer AA, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997; 25: 3389-402.

[78] Flutre T, Duprat E, Feuillet C, et al. Considering transposable element diversification in de novo annotation approaches. PLoS One. 2011; 6: e16526.

[79] Ahmed I, Sarazin A, Bowler C, et al. Genome-wide evidence for local DNA methylation spreading from small RNA-targeted sequences in Arabidopsis. Nucleic Acids Res. 2011; 39: 6919-31.

[80] Quesneville H, Bergman CM, Andrieu O, et al. Combined evidence annotation of transposable elements in genome sequences. PLoS Comput Biol. 2005; 1: 166-75.

[81] Jamilloux V, Daron J, Choulet F, et al. Corrections to “De novo annotation of transposable elements: tackling the fat genome issue” [Jamilloux et al., Proc IEEE, vol. 105, no. 3, pp. 474-481, Mar 2017, DOI:10.1109/JPROC.2016.2590833]. Proc IEEE. 2017; 105: 978-8.

[82] Hoede C, Arnoux S, Moisset M, et al. PASTEC: an automatic transposable element classification tool. PLoS One. 2014; 9: e91929.

[83] Barrett T, Dowle M, Srinivasan A, et al. 2025. data.table: Extension of ‘data.frame’. https://cran.r-project.org/web/packages/data.table.

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

[85] Sirén J, Monlong J, Chang X, et al. Pangenomics enables genotyping of known structural variants in 5202 diverse genomes. Science. 2021; 374: abg8871.

[86] Camacho C, Coulouris G, Avagyan V, et al. BLAST+: architecture and applications. BMC Bioinf. 2009; 10: 421.

[87] Wick RR, Schultz MB, Zobel J, et al. Bandage: interactive visualization of de novo genome assemblies. Bioinformatics. 2015; 31: 3350-2.

[88] Jung S, Lee T, Cheng CH, et al. 15 years of GDR: New data and functionality in the Genome Database for Rosaceae. Nucleic Acids Res. 2019; 47: D1137-45.