Liquidambar formosana (Hamamelidaceae) is a relatively fast-growing deciduous tree of high ornamental value that is indigenous to China. However, few molecular markers are available for the species or its close relatives; this has hindered genomic and genetic studies. Here, we develop a series of transferable expressed sequence tag-simple sequence repeats (EST-SSRs) for genomic analysis of L. formosana. We downloaded the sequence of the L. formosana transcriptome from the National Center of Biotechnology Information Database and identified SSR loci in the Unigene library. We found 3284 EST-SSRs by mining 34,491 assembled unigenes. We synthesized 100 random primer pairs for validation of eight L. formosana individuals; of the 100 pairs, 32 were polymorphic. We successfully transferred 12 EST-SSR markers across three related Liquidambar species; the markers exhibited excellent cross-species transferability and will facilitate genetic studies and breeding of Liquidambar. A total of 72 clones of three Liquidambar species were uniquely divided into three main clusters; principal coordinate analysis (PCoA) supported this division. Additionally, a set of 20 SSR markers that did not exhibit nonspecific amplification were used to genotype more than 53 L. formosana trees. The mean number of alleles (Na) was 5.75 and the average polymorphism information content (PIC) was 0.578, which was higher than that of the natural L. formosana population (0.390). In other words, the genetic diversity of the plus L. formosana population increased, but excellent phenotypic features were maintained. The primers will be valuable for genomic mapping, germplasm characterization, gene tagging, and further genetic studies. Analyses of genetic diversity in L. formosana will provide a basis for efficient application of genetic materials and rational management of L. formosana breeding programs.

Small heat shock proteins (HSP20s) are a significant factor in plant growth and development in response to abiotic stress. In this study, we investigated the role of HSP20s’ response to the heat stress of Sorbus pohuashanensis introduced into low-altitude areas. The HSP20 gene family was identified based on the genome-wide data of S. pohuashanensis, and the expression patterns of tissue specificity and the response to abiotic stresses were evaluated. Finally, we identified 38 HSP20 genes that were distributed on 16 chromosomes. Phylogenetic analysis of HSP20s showed that the closest genetic relationship to S. pohuashanensis (SpHSP20s) is Malus domestica, followed by Populus trichocarpa and Arabidopsis thaliana. According to phylogenetic analysis and subcellular localization prediction, the 38 SpHSP20s belonged to 10 subfamilies. Analysis of the gene structure and conserved motifs indicated that HSP20 gene family members are relatively conserved. Synteny analysis showed that the expansion of the SpHSP20 gene family was mainly caused by segmental duplication. In addition, many cis-acting elements connected with growth and development, hormones, and stress responsiveness were found in the SpHSP20 promoter region. Analysis of expression patterns showed that these genes were closely related to high temperature, drought, salt, growth, and developmental processes. These results provide information and a theoretical basis for the exploration of HSP20 gene family resources, as well as the domestication and genetic improvement of S. pohuashanensis.

Liquidambar is an important forestry species used to generate many commercial wood products, such as plywood. Inducing artificial polyploidy is an effective method to encourage genetic enhancements in forestry breeding. This report presents the first in vitro protocol for the induction of genus Liquidambar tetraploids based on the established in vitro regeneration system of hybrid sweetgum (Liquidambar styraciflua × Liquidambar formosana). The leaves and petioles from three genotypes were pre-cultured in woody plant medium (WPM) supplemented with 0.1 mg/L thidiazuron (TDZ), 0.8 mg/L benzyladenine (BA), and 0.1 mg/L α-naphthalene acetic acid (NAA) for a variable number of days (4, 6 or 8 days), and exposed to varying concentrations of colchicine (120, 160, 200 mg/L) for 3, 4 or 5 days; the four factors were investigated using an orthogonal experimental design. Adventitious shoots were rooted in 1/2 WPM medium supplemented with 2.0 mg/L indole butyric acid (IBA) and 0.1 mg/L NAA. The ploidy level was assessed using flow cytometry and chromosome counting. Four tetraploids and nine mixoploids were obtained from the leaves. Pre-treatment of the leaves for 8 days and exposure to 200 mg/L colchicine for 3 days led to the most efficient tetraploid induction. Producing 11 tetraploids and five mixoploids from petioles, the best tetraploid induction treatment for petioles was almost the same as that with the leaves, except that pre-culturing was required for only 6 days. In total, 15 tetraploids were obtained with these treatments. This study described a technique for the induction of tetraploid sweetgum from the leaves or petioles of parental material. Based on the success of polyploid breeding in other tree species, the production of hybrid sweetgum allotetraploids constitutes a promising strategy for the promotion of future forestry breeding.

Sorbus caloneura has high ornamental and medicinal value but is endangered, and significant effort is required to preserve this natural resource. In this study, the stem and sterilized leaves of S. caloneura were used to explore the effects of different plant hormones basic medium type, initial callus quality, initial liquid volume, and ratio of old liquid culture medium to new on stem proliferation, regeneration and callus suspension culture. Naphthylacetic acid (NAA) and 6-benzyladenine (6-BA) significantly affected the proliferation of stem tissue. Murashige and Skoog (MS) basal medium supplemented with 1.75 mg/L 6-BA, 0.25 mg/L NAA, and 0.25 mg/L indole butyric acid (IBA) yielded a proliferation rate of 100%, with an average number of adventitious shoots per stem of 4.9. The best callus induction was observed with MS basal medium containing 0.5 mg/L 6-BA, 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.2 mg/L thidiazuron (TDZ). The adventitious shoots were directly induced by MS basal medium containing 5.0 mg/L 6-BA, 0.5 mg/L NAA, and 1.0 mg/L kinetin (KT) and indirectly induced by medium containing 3.0 mg/L 6-BA, 0.3 mg/L NAA, and 0.1 mg/L TDZ. NAA significantly affected rooting rate, with ideal conditions found to be medium supplemented with 0.2 mg/L NAA and 1.5 mg/L IBA. MS basal medium with 4.5 g of calli and 120 mL of liquid culture medium without retention of the original culture medium yielded the best suspension effect for callus proliferation. Taken together, the results of this study lay a foundation for the breeding, preservation of germplasm resources, and genetic transformation of S. caloneura.

Sorbus pohuashanensis (Hance) Hedl. is an important forestry species valued for its ornamental, medicinal, and ecological properties. Polyploidy breeding is an important method of germplasm innovation; however, polyploidy induction and phenotypic variation caused by chromosome doubling in S. pohuashanensis are poorly understood. In this study, S. pohuashanensis seeds were used to explore the effects of different colchicine concentrations, cold stratification times, and seeds from different sources on polyploidy induction. Ploidy levels of the regenerated plants were determined by flow cytometry. The results showed that the tetraploid induction effect of S. pohuashanensis seeds was significantly affected by colchicine concentration, and the highest tetraploid induction rate of 24.75% was achieved by immersion in 0.2% (w/v) colchicine for 48 h. After 2 years of induction, 77 tetraploid plants were obtained. Compared to diploids, tetraploid plants showed significant variations in plant height, leaf morphology (apical leaflet width, middle leaflet width), and diameter of the middle petiole. The stomatal size and chloroplast number increased with chromosome doubling whereas the stomatal number and density decreased. In addition, significant differences in the percentage of sunburn associated with ploidy changes were observed. This study provides a technique for tetraploid induction of S. pohuashanensis seeds, showing the variation in traits caused by polyploidization and the effect of chromosome doubling on sunburn resistance. Tetraploidy induction provides a new direction for S. pohuashanensis germplasm innovation.

A high-density genetic linkage map is essential for plant genetics and genomics research. However, due to the deficiency of genomic data and high-quality molecular markers, no genetic map has been published for Prince Rupprecht’s larch (Larix principis-rupprechtii Mayr), a conifer species with high ecological and commercial value in northern China. In this study, 145 F1 progeny individuals from an intraspecific cross between two elite clones of L. principis-rupprechtii and their parents were employed to construct the first genetic map in this important tree species using specific-locus amplified fragment sequencing (SLAF-seq). After preprocessing, the procedure yielded 300.20 Gb of raw data containing 1501.22 M pair-end reads. A total of 324,352 SNP markers were detected and 122,785 of them were polymorphic, with a polymorphism rate of 37.86%. Ultimately, 6099 SNPs were organized into a genetic map containing 12 linkage groups, consistent with the haploid chromosome number of larch and most other species in the Pinaceae family. The linkage map spanned 2415.58 cM and covered 99.6% of the L. principis-rupprechtii genome with an average of 0.4 cM between adjacent markers. To the best of our knowledge, this map is the first reference map for L. principis-rupprechtii, as well as the densest one obtained in larch species thus far. The genome-wide SNPs and the high-resolution genetic map will provide a foundation for future quantitative trait loci mapping, map-based cloning, marker-assisted selection, comparative genomics, and genome sequence assembly for larch trees.