SUMMARY To improve our understanding of genetic mechanisms underlying complex traits in plants, a comprehensive analysis of gene variants is required. Eucalyptus is an important forest plantation genus that is highly outbred. Trait dissection and molecular breeding in eucalypts currently relies on biallelic single‐nucleotide polymorphism (SNP) markers. These markers fail to capture the large amount of haplotype diversity in these species, and thus multi‐allelic markers are required. We aimed to develop a gene‐based haplotype mining panel for Eucalyptus species. We generated 17 999 oligonucleotide probe sets for targeted sequencing of selected regions of 6293 genes implicated in growth and wood properties, pest and disease resistance, and abiotic stress responses. We identified and phased 195 834 SNPs using a read‐based phasing approach to reveal SNP‐based haplotypes. A total of 8915 target regions (at 4637 gene loci) passed tests for Mendelian inheritance. We evaluated the haplotype panel in four Eucalyptus species (E. grandis, E. urophylla, E. dunnii and E. nitens) to determine its ability to capture diversity across eucalypt species. This revealed an average of 3.13–4.52 haplotypes per target region in each species, and 33.36% of the identified haplotypes were shared by at least two species. This haplotype mining panel will enable the analysis of haplotype diversity within and between species, and provide multi‐allelic markers that can be used for genome‐wide association studies and gene‐based breeding approaches.

SummaryDe novo phased (haplo)genome assembly using long-read DNA sequencing data has improved the detection and characterization of structural variants (SVs) in plant and animal genomes. Able to span across haplotypes, long reads allow phased, haplogenome assembly in highly outbred organisms such as forest trees. Eucalyptus tree species and interspecific hybrids are the most widely planted hardwood trees with F1 hybrids of Eucalyptus grandis and E. urophylla forming the bulk of fast-growing pulpwood plantations in subtropical regions. The extent of structural variation and its effect on interspecific hybridization is unknown in these trees. As a first step towards elucidating the extent of structural variation between the genomes of E. grandis and E. urophylla, we sequenced and assembled the haplogenomes contained in an F1 hybrid of the two species. Using Nanopore sequencing and a trio-binning approach, we assembled the separate haplogenomes (567 Mb and 545 Mb) to 98.8% BUSCO completion. High-density SNP genetic linkage maps of both parents allowed scaffolding of 88% of the haplogenome contigs into 11 pseudo-chromosomes (scaffold N50 of 43.82 Mb and 42.45 Mb for the E. grandis and E. urophylla haplogenomes, respectively). We identify 48,729 SVs between the two haplogenomes providing the first detailed insight into genome structural rearrangement in these species. The two haplogenomes have similar gene content, 35,572 and 33,915 functionally annotated genes, of which 34% are contained in genome rearrangements. Knowledge of SV and haplotype diversity in the two species will form the basis for understanding the genetic basis of hybrid superiority in these trees.Significance statementWe have produced phased, haplogenome assemblies of an interspecific F1 hybrid using a trio-binning approach and performed the first genome-wide analysis of genome synteny between a subtropical Eucalyptus tree species, E. grandis, and a tropical eucalypt, E. urophylla. This revealed a large number of previously undescribed genome structural variants as a step towards understanding genome structural evolution in this iconic genus of fast-growing woody perennials.

To improve our understanding of genetic mechanisms underlying complex traits in plants, a comprehensive analysis of gene variants is required. Eucalyptus is an important forest plantation genus that is highly outbred. Trait dissection and molecular breeding in eucalypts currently relies on biallelic SNP markers. These markers fail to capture the large amount of haplotype diversity in these species and thus multi-allelic markers are required. We aimed to develop a gene-based haplotype mining panel for Eucalyptus species. We generated 17 999 oligonucleotide probe sets for targeted sequencing of selected regions of 6 293 genes implicated in growth and wood properties, pest and disease resistance and abiotic stress responses. We identified and phased 195 834 SNPs using a read-based phasing approach to reveal SNP-based haplotypes. A total of 8 915 target regions (at 4 637 gene loci) passed tests for Mendelian inheritance. We evaluated the haplotype panel in four Eucalyptus species (E. grandis, E. urophylla, E. dunnii and E. nitens) to determine its ability to capture diversity across eucalypt species. This revealed an average of 3.13 to 4.52 haplotypes per target region in each species and 33.36% of the identified haplotypes were shared by at least two species. This haplotype mining panel will enable the analysis of haplotype diversity within and between species and provide multi-allelic markers that can be used for genome-wide association studies and gene-based breeding approaches.