Eucalyptus urophylla S.T.Blake is an important commercial tropical plantation species worldwide. In China, a breeding program for this species has progressed through three cycles but genetic diversity and structure in the breeding populations are uncertain. A sampling of field trials from these populations was carried out to evaluate their genetic diversity and structure using 16 microsatellite loci. Significant deviations from Hardy-Weinberg equilibrium were recorded at all 16 loci in the populations. Overall expected and observed heterozygosity (H_e and H_o) estimates of 0.87 and 0.59 respectively for the first cycle population, and 0.88 and 0.60 respectively for the third cycle population, revealed reasonably high levels of genetic diversity. The genetic differentiation coefficient (F_st) revealed low differentiation among pairs of provenances (from the species’ native range) comprising the first cycle population (range: 0.012–0.108), and AMOVA results showed that the majority of molecular genetic variation existed among individuals rather than among provenances for the first cycle population and among individuals rather than among field trial sources in the third cycle population. Levels of genetic diversity appeared to remain unchanged from the first to third cycle populations, and the results indicate prospects for maintaining if not increasing diversity through recurrent breeding. Likely effects of artificial directional selection, prior to sampling, on both populations examined are discussed along with implications for future E. urophylla breeding.

The CesA proteins are key players in cellulose synthesis. Eucalyptus is well-known for cellulose synthesis, although the mechanism of cellulose synthesis is largely unknown. In this study, 11 CcCesA genes were identified by comprehensive bioinformatics analysis in Corymbia citriodora, a eucalypt species. CcCesA genes were generally conserved in each subfamily. Among them, four genes lacked a transmembrane domain in the N-terminal. We showed that genes of CesA2, 4, 5, and 8 may regulate the synthesis of secondary cell walls (SCWs) through phylogenetic trees analysis. Two pairs of segmental duplication and one pair of tandem duplication were detected in CcCesAs. Analysis of Ka/Ks ratios revealed that the duplicated genes were under negative or purifying selection. Numerous cis-acting elements related to plant hormones and light reactions were identified in the promoters of CcCesAs. Expression analysis confirmed that genes of CesA2, 4, and 8 regulate the synthesis of SCWs, and CesA8, in particular, had the highest expression compared with other genes. The results will help us understand the complexity of the CcCesAs in different diameters at breast height and provide valuable information for future functional characterization of specific genes in C. citriodora.

Understanding the rules of genetic recombination in controlled pollination directly related to the selection of parental genotypes and the utilization of heterosis, and genotype identification is a primary study of the genetic rules. The aims of this study were to investigate the ability of near infrared (NIR) spectroscopy to accurately and efficiently discriminate pure species and hybrids within the genus of Eucalyptus, to evaluate the transmission of genetic pedigree in control pollination, and reveal the genetic variation within the genotypes studied. NIR spectra were collected both from fresh leaves and dried, milled leaves of seedlings from pure species E. urophylla and E. grandis, and their F1 hybrids. Principal component analysis (PCA) scores plots of NIR spectra from fresh leaves and dry, milled powder from pure species showed clear segregation, although the species clusters were scattered, suggesting different base genetics and high genetic variation within families of the two pure species. Classification using soft independent modelling of class analogy of the NIR spectra of dried leaves was significantly better than using spectra acquired on fresh leaves, meaning the water content had an effect on the analysis. The projections and orthogonal distance between hybrids and parents, as calculated using PCA models, demonstrated the visualized spectral distance between each hybrid and the parents was very different. Clouds of individuals within a hybrid clusters varied from tightly packed to scattered, which reflected the genetic additive effects inherited from female and male parents were different, and their genetic variation was also different after genetic recombination. The varying response values for partial least squares discriminant analysis prediction verified the conclusions shown by projections and orthogonal distance. The results of this study demonstrate the potential of using NIR spectroscopy to rapidly discriminate taxon. The application of NIR spectroscopy to non-destructively confirm taxonomic identity will greatly facilitate the evaluation of the genetic basis and genetic variation available within breeding populations and for accessing the levels of contamination by non-target pollen in control pollination.

Artificial hybridization remains the most effective method for genetic improvement of eucalypt, though limited research exists on the genetic basis of heterosis for interesting traits in Chinese eucalypt. We attempted to use Python in combined with SAS and SPSS to explore the relationship between parental combining ability and heterosis in controlled pollination populations of eucalypt. Our results indicated that E. urophylla had the highest general combining ability (GCA). The special combining ability (SCA) of U3423 × 3327, 06H16 × LL131, and H0733 × U6 were the highest. H0733 × U6, H0733 × P9060, and (E. urophylla × E. grandis) × 06H241 had the strongest mid-parent heterosis (MPH). U3423 × 3327, U952 × C2232, and (E. urophylla × E. grandis) × 06H241 had the strongest high-parent heterosis (HPH). We found that completely controlled by non-additive effects were (E. tereticornis × E. urophylla) × (E. urophylla), E. urophylla or (E. urophylla × E. grandis) × (open pollination). Fully controlled by additive effects were (E. tereticornis × E. urophylla) × E. camaldulensis or E. grandis, (E. urophylla) × (E. pellita × E. tereticornis), (E. urophylla × E. grandis) × E. pellita or E. tereticornis. All the findings in the present research explained the genetic basis of heterosis in eucalypt growth, and enriched the theory of hybrid breeding in eucalypt.

Selecting high-quality varieties with disease resistance by artificial crossbreeding is the most fundamental way to address the damage caused by Calonectria spp. in eucalypt plantations. However, understanding the mechanism of disease-resistant heterosis occurrence in eucalypts is crucial for successful crossbreeding. Two eucalypt hybrids, the susceptible EC333 (H1522 × unknown) and the resistant EC338 (W1767 × P9060), were screened through infection with Calonectria isolates, a pathogen that causes eucalypt leaf blight. RNA-Seq was performed on the susceptible hybrid, the disease-resistant hybrid, and their parents. The gene differential expression analysis showed that there were 3912 differentially expressed genes between EC333 and EC338, with 1631 up-regulated and 2281 down-regulated genes. The expression trends of the differential gene sets in P9060 and EC338 were similar. However, the expression trend of W1767 was opposite that of EC338. The similarity of the expression and the advantage of stress resistance in E. pellita suggested that genes with significant differences in expression likely relate to disease resistance. A GSEA based on GO annotations revealed that the carbohydrate binding pathway genes were differentially expressed between EC338 and EC333. The gene pathways that were differentially expressed between EC338 and EC333 revealed by the GSEA based on KEGG annotations were the sesquiterpenoid and triterpenoid biosynthesis pathways. The alternative splicing analysis demonstrated that an AS event between EC338 and EC333 occurred in LOC104426602. According to our SNP analysis, EC338 had 626 more high-impact mutation loci than the male parent P9060 and 396 more than the female parent W1767; W1767 had 259 more mutation loci in the downstream region than EC338, while P9060 had 3107 fewer mutation loci in the downstream region than EC338. Additionally, EC338 had 9631 more mutation loci in the exon region than EC333. Modules were found via WGCNA that were strongly and oppositely correlated with EC338 and EC333, such as module MEsaddlebrown, likely associated with leaf blight resistance. The present study provides a detailed explanation of the genetic basis of eucalypt leaf blight resistance, providing the foundation for exploring genes related to this phenomenon.

The fundamental way to solve the problems of a Limited interspecific gene pool, poor stress resistance, and low yield in Chinese eucalypt plantations is to use the Heterosis derived from the eucalypt gene pool to select new varieties with rich genotypes. In order to reveal the genetic mechanism of the formation of Heterosis in eucalypt growth, based on previous research on the relationship between eucalypt heterosis and parental combining ability, we selected two artificial hybrids 18H167 (T15 × U3423) and 19H74 (U3423 × U6) with significant differences in birth length and similar parental relationships as the research objects. Transcriptome analysis using RNA-seq technology revealed correlations between gene expression levels that indicated the male parent had a greater impact on the heterosis of eucalypt growth. Based on GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) annotations, GSEA (Gene Set Enrichment Analysis) enrichment and WGCNA (Weighted Gene Co-expression Network Analysis) analysis identified 6 pathways and 8 Hub genes. The biological functions of these pathways involve resistance ability, mainly summarized as ribosomal subunits, extracellular regulatory mechanisms, and three amino acid synthesis pathways. We believe that one or several genes with altered expression levels in candidate pathways, either individually or in combination, directly regulate the growth of eucalypt or indirectly affect growth through adaptation to environmental stress. AS (Alternative Splicing) analysis showed that the AS events of the two hybrids were significantly higher than those of their parents, with SE (Skipped Exon) events possibly being related to growth disadvantages and RI (Retained Intron) events more likely to be related to growth advantages. This study provides a more in-depth exploration of the formation mechanism of heterosis in eucalypt growth and adds Python code to speed up the process, which are expected to guide the selection of parents in eucalypt hybrid breeding. The discovery of genetic mechanisms of heterosis related to growth provides genetic information for eucalypt genome annotation and molecular marker assisted breeding.