Comparison of ddRADseq and EUChip60K SNP genotyping systems for population genetics and genomic selection in Eucalyptus dunnii (Maiden).

Eucalyptus dunnii is one of the most important Eucalyptus species for short-fiber pulp production in regions where other species of the genus are affected by poor soil and climatic conditions. In this context, E. dunnii holds promise as a resource to address and adapt to the challenges of climate change. Despite its rapid growth and favorable wood properties for solid wood products, the advancement of its improvement remains in its early stages. In this work, we evaluated the performance of two single nucleotide polymorphism, (SNP), genotyping methods for population genetics analysis and Genomic Selection in E. dunnii. Double digest restriction-site associated DNA sequencing (ddRADseq) was compared with the EUChip60K array in 308 individuals from a provenance-progeny trial. The compared SNP set included 8,011 and 19,008 informative SNPs distributed along the 11 chromosomes, respectively. Although the two datasets differed in the percentage of missing data, genome coverage, minor allele frequency and estimated genetic diversity parameters, they revealed a similar genetic structure, showing two subpopulations with little differentiation between them, and low linkage disequilibrium. GS analyses were performed for eleven traits using Genomic Best Linear Unbiased Prediction (GBLUP) and a conventional pedigree-based model (ABLUP). Regardless of the SNP dataset, the predictive ability (PA) of GBLUP was better than that of ABLUP for six traits (Cellulose content, Total and Ethanolic extractives, Total and Klason lignin content and Syringyl and Guaiacyl lignin monomer ratio). When contrasting the SNP datasets used to estimate PAs, the GBLUP-EUChip60K model gave higher and significant PA values for six traits, meanwhile, the values estimated using ddRADseq gave higher values for three other traits. The PAs correlated positively with narrow sense heritabilities, with the highest correlations shown by the ABLUP and GBLUP-EUChip60K. The two genotyping methods, ddRADseq and EUChip60K, are generally comparable for population genetics and genomic prediction, demonstrating the utility of the former when subjected to rigorous SNP filtering. The results of this study provide a basis for future whole-genome studies using ddRADseq in non-model forest species for which SNP arrays have not yet been developed.

Harder, better, faster, stronger: Frost tolerance of Eucalyptus benthamii under cold acclimation.

Eucalypts are the most planted hardwood trees worldwide because of their very rapid growth, exceptional wood quality and adaptability. However, most commercial species and derived hybrids are sensitive to frost, which remains as the largest obstacle to their introduction in warm/temperate climates. As evergreen species, Eucalypts have developed the ability to tolerate frost events based on physiological and molecular responses triggered by previous exposure to cold temperatures, globally named cold acclimation. To characterize the acclimation process in two species with different tolerance to frost, E. grandis (Eg) and E. benthamii (Eb), seedlings were exposed for different times to low temperatures. Frost tolerance was estimated in leaves by an electrolyte leakage assay, and metabolome and morpho-physiological changes studied and correlated to the observed acclimation responses. Eb showed higher basal frost tolerance and an earlier and stronger acclimation response to cold temperatures than in the frost sensitive Eg. Eb was able to modify several morpho-physiological parameters, with a restriction in plant height, leaf area and leaf fresh weight during acclimation. Metabolome characterization allowed us to differentiate species and strengthen our understanding of their acclimation response dynamics. Interestingly, Eb displayed an early phase of sugar accumulation followed by a rise of different metabolites with possible roles as osmolytes and antioxidants, that correlated to frost tolerance and may explain Eb higher capacity to acclimate. This novel approach has helped us to point to the main metabolic processes underlying the cold tolerance acquisition process in two relevant Eucalyptus species.

Single-step genomic prediction of Eucalyptus dunnii using different identity-by-descent and identity-by-state relationship matrices.

Genomic selection based on the single-step genomic best linear unbiased prediction (ssGBLUP) approach is becoming an important tool in forest tree breeding. The quality of the variance components and the predictive ability of the estimated breeding values (GEBV) depends on how well marker-based genomic relationships describe the actual genetic relationships at unobserved causal loci. We investigated the performance of GEBV obtained when fitting models with genomic covariance matrices based on two identity-by-descent (IBD) and two identity-by-state (IBS) relationship measures. Multiple-trait multiple-site ssGBLUP models were fitted to diameter and stem straightness in five open-pollinated progeny trials of Eucalyptus dunnii, genotyped using the EUChip60K. We also fitted the conventional ABLUP model with a pedigree-based covariance matrix. Estimated relationships from the IBD estimators displayed consistently lower standard deviations than those from the IBS approaches. Although ssGBLUP based in IBS estimators resulted in higher trait-site heritabilities, the gain in accuracy of the relationships using IBD estimators has resulted in higher predictive ability and lower bias of GEBV, especially for low-heritability trait-site. ssGBLUP based on IBS and IBD approaches performed considerably better than the traditional ABLUP. In summary, our results advocate the use of the ssGBLUP approach jointly with the IBD relationship matrix in open-pollinated forest tree evaluation.

Cold acclimation and freezing tolerance in three Eucalyptus species: A metabolomic and proteomic approach.

The ability of plants to cope with frost events relies on the physiological and molecular responses triggered by cold temperatures. This process, named acclimation, involves reprogramming gene expression in order to adjust metabolism. Planted Eucalyptus species are highly productive but most of them are frost sensitive. However, acclimation process varies among species and environmental conditions, promoting more or less frost damage in young plantations of frost-prone areas. To identify metabolites and proteins responsible for these differences, two acclimation regimes were imposed to seedling of Eucalyptus grandis Hill ex Maiden (Eg), Eucalyptus dunnii Maiden (Ed) and Eucalyptus benthamii Maiden Cambage (Eb), and leaves submitted to biochemical and molecular analyses. Further, seedlings were used for simulated frosts in order to test the acclimation status effect on frost tolerance. Eb showed higher frost tolerance than Ed and Eg under control and acclimation scenarios, possibly due to its higher accumulation of phenolics, anthocyanins and soluble sugars as well as lower levels of photosynthetic pigments and related proteins. Also, a rise in frost tolerance and in osmoprotectants and antioxidants was observed for all the species due to cold acclimation treatment. Interestingly, metabolic profiles differed among species, suggesting different mechanisms to endure frosts and, probably, different requirements for cold acclimation. Shotgun proteomics reinforced differences and commonalities and supported metabolome observations. An in depth understanding of these responses could help to safeguard planted forests productivity through breeding of tolerant genetic material.

Validation of models to estimate the fumigant and larvicidal activity of Eucalyptus essential oils against Aedes aegypti (Diptera: Culicidae).

The aim of this work is to validate the pre-existing models that relate the larvicidal and adulticidal activities of the Eucalyptus essential oils on Aedes aegypti. Previous works at our laboratory described that the larvicidal activity of Eucalyptus essential oils can be estimated from the relative concentration of two main components (p-cymene and 1,8-cineole) and that the adulticidal effectiveness can be explained, to a great extent, by the presence of large amounts of the component 1,8-cineole in it. In general, the results show that the higher adulticidal effect of essential oils the lower their larvicidal activity. Fresh leaves was harvested and distilled. Once the essential oil was obtained, the chemical composition was analysed, evaluating the biological activity of 15 species of the genus Eucalyptus (Eucalyptus badjensis Beuzev and Welch, Eucalyptus badjensis × nitens, Eucalyptus benthamii var Benthamii Maiden and Cambage, Eucalyptus benthamii var dorrigoensis Maiden and Cambage, Eucalyptus botryoides Smith, Eucalyptus dalrympleana Maiden, Eucalyptus fastigata Deane and Maiden, Eucalyptus nobilis L.A.S. Johnson and K.D.Hill, Eucalyptus polybractea R. Baker, Eucalyptus radiata ssp radiata Sieber ex Spreng, Eucalyptus resinifera Smith, Eucalyptus robertsonii Blakely, Eucalyptus robusta Smith, Eucalyptus rubida Deane and Maiden, Eucalyptus smithii R. Baker). Essential oils of these plant species were used for the validation of equations from preexistent models, in which observed and estimated values of the biological activity were compared. The regression analysis showed a strong validation of the models, re-stating the trends previously observed. The models were expressed as follows: A, fumigant activity [KT(50(min)) = 10.65-0.076 × 1,8-cineole (%)](p < 0.01; F, 397; R (2), 0.79); B, larval mortality (%)((40 ppm)) = 103.85 + 0.482 × p-cymene (%) - 0.363 × α-pinene (%) - 1.07 × 1,8-cineole (%) (p < 0.01; F, 300; R (2), 0.90). These results confirmed the importance of the mayor components in the biological activity of Eucalyptus essential oils on A. aegypti. However, it is worth mentioning that two or three species differ in the data estimated by the models, and these biological activity results coincide with the presence of minor differential components in the essential oils. According to what was previously mentioned, it can be inferred that the model is able to estimate very closely the biological activity of essential oils of Eucalyptus on A. aegypti.

Chemical composition and fumigant toxicity of the essential oils from 16 species of Eucalyptus against Haematobia irritans (Diptera: Muscidae) adults.

Oils extracted from various species of Eucalyptus (Eucalyptus badjensis Beuzev & Welch, Eucalyptus badjensis x Eucalyptus nitens, Eucalyptus benthamii variety dorrigoensis Maiden & Cambage, Eucalyptus botryoides Smith, Eucalyptus dalrympleana Maiden, Eucalyptus fastigata Deane & Maiden, Eucalyptus nobilis L.A.S. Johnson & K. D. Hill, Eucalyptus polybractea R. Baker, Eucalyptus radiata ssp. radiata Sieber ex Spreng, Eucalyptus resinifera Smith, Eucalyptus robertsonii Blakely, Eucalyptus rubida Deane & Maiden, Eucalyptus smithii R. Baker, Eucalyptus elata Dehnh, Eucalyptus fraxinoides Deane & Maiden, E. obliqua L'Hér) were obtained by hydrodistillation. The chemical composition of essential oils was determined by gas chromatography coupled to mass spectrometry. Essential oils were mainly composed of 1,8-cineole, alpha-pinene, alpha-terpineol, 4-terpineol, and p-cymene. Vapors from these essential oils and their major components were found to be toxic to Haematobia irritans (L.) (Diptera: Muscidae) adults. An aliquot of each oil was placed in a cylindrical test chamber, and the number of knocked down flies was recorded as a function of time. Knockdown time 50% was then calculated. Results showed that essential oil of E. polybractea had the highest knockdown activity of 3.44 min. A correlation was observed between the content of 1,8-cineole in the Eucalyptus essential oils and the corresponding toxic effect.