Restriction site associated DNA sequencing (RAD-seq), a next-generation sequencing technology, has greatly facilitated genetic linkage mapping studies in outbred species. RAD-seq is capable of discovering thousands of genetic markers for linkage mapping across many individuals, and can be applied in species with or without a reference genome. Although several analytical tools are available for RAD-seq data, alternative strategies are necessary for improving the marker quality and hence the genetic mapping accuracy. We demonstrate a strategy for constructing dense genetic linkage maps in hybrid forest trees by combining RAD-seq and whole-genome sequencing technologies. We performed RAD-seq of 150 progeny and whole-genome sequencing of the two parents in an F1 hybrid population of Populus deltoides × P. simonii. Two rough references were assembled from the whole-genome sequencing reads of the two parents separately. Based on the parental reference sequences, 3442 high-quality single nucleotide polymorphisms (SNPs) were identified that segregate in the ratio of 1:1. The maternal linkage map of P. deltoides was constructed with 2012 SNPs, containing 19 linkage groups and spanning 4067.16 cM of the genome with an average distance of 2.04 cM between adjacent markers, while the male map of P. simonii consisted of 1430 SNPs and the same number of linkage groups with a total length of 4356.04 cM and an average interval distance of 3.09 cM. Collinearity between the parental linkage maps and the reference genome of P. trichocarpa was also investigated. Compared with the result on the basis of the existing reference genome, our strategy identified more high-quality SNPs and generated parental linkage groups that nicely match the karyotype of Populus. The strategy of simultaneously using RAD and whole-genome sequencing technologies can be applied to constructing high-density genetic maps in forest trees regardless of whether a reference genome exists. The two parental linkage maps constructed here provide more accurate genetic resources for unraveling quantitative trait loci and accelerating molecular breeding programs, as well as for comparative genomics in Populus.

Populus simonii is an important tree in the genus Populus, widely distributed in the Northern Hemisphere and having a long cultivation history. Although this species has ecologically and economically important values, its genome sequence is currently not available, hindering the development of new varieties with wider adaptive and commercial traits. Here, we report a chromosome-level genome assembly of P. simonii using PacBio long-read sequencing data aided by Illumina paired-end reads and related genetic linkage maps. The assembly is 441.38 Mb in length and contain 686 contigs with a contig N50 of 1.94 Mb. With the linkage maps, 336 contigs were successfully anchored into 19 pseudochromosomes, accounting for 90.2% of the assembled genome size. Genomic integrity assessment showed that 1,347 (97.9%) of the 1,375 genes conserved among all embryophytes can be found in the P. simonii assembly. Genomic repeat analysis revealed that 41.47% of the P. simonii genome is composed of repetitive elements, of which 40.17% contained interspersed repeats. A total of 45,459 genes were predicted from the P. simonii genome sequence and 39,833 (87.6%) of the genes were annotated with one or more related functions. Phylogenetic analysis indicated that P. simonii and Populus trichocarpa should be placed in different sections, contrary to the previous classification according to morphology. The genome assembly not only provides an important genetic resource for the comparative and functional genomics of different Populus species, but also furnishes one of the closest reference sequences for identifying genomic variants in an F₁ hybrid population derived by crossing P. simonii with other Populus species.

Populus deltoides has important ecological and economic values, widely used in poplar breeding programs due to its superior characteristics such as rapid growth and resistance to disease. Although the genome sequence of P. deltoides WV94 is available, the assembly is fragmented. Here, we reported an improved chromosome-level assembly of the P. deltoides cultivar I-69 by combining Nanopore sequencing and chromosome conformation capture (Hi-C) technologies. The assembly was 429.3 Mb in size and contained 657 contigs with a contig N50 length of 2.62 Mb. Hi-C scaffolding of the contigs generated 19 chromosome-level sequences, which covered 97.4% (418 Mb) of the total assembly size. Moreover, repetitive sequences annotation showed that 39.28% of the P. deltoides genome was composed of interspersed elements, including retroelements (23.66%), DNA transposons (6.83%), and unclassified elements (8.79%). We also identified a total of 44 362 protein-coding genes in the current P. deltoides assembly. Compared with the previous genome assembly of P. deltoides WV94, the current assembly had some significantly improved qualities: the contig N50 increased 3.5-fold and the proportion of gaps decreased from 3.2% to 0.08%. This high-quality, well-annotated genome assembly provides a reliable genomic resource for identifying genome variants among individuals, mining candidate genes that control growth and wood quality traits, and facilitating further application of genomics-assisted breeding in populations related to P. deltoides.