Volume 67 (2018): Issue 1 (February 2018)

The cinnamyl alcohol dehydrogenase (CAD) enzyme catalyzes the last step of monolignols synthesis in the lignin pathway. Tectona grandis (teak) is a tropical tree with high valuable tim­ber. As there is few genetic information about lignin formation in teak, the purpose of this study is to characterize members of CAD family in this species. As methodology, PCR amplification using cDNA samples, vector cloning, sequencing, bioinforma­tics analyses and gene expression studies using real time RT-qPCR were performed. As results, four members (TgCAD1- TgCAD4) were obtained. Comparative analyses showed that all of them have conserved residues for catalytic zinc action, structural zinc ligation, NADPH binding and substrate specifici­ty, consistent with the mechanism of alcohol dehydrogenases. Phylogenetic analysis showed that TgCADs are present in three main classes and seven groups. Expression analyses revealed that TgCAD1 was highly expressed in leaves and could be rela­ted with pathogen defense. TgCAD2 was more expressed in branches and roots. Differently, TgCAD3 and TgCAD4 were highly expressed in juvenile and mature sapwood, suggesting a crucial role in wood development and lignin biosynthesis, with tissue-specialized expression profiles. Furthermore, TgCAD4 could be related with teak maturation for being more expressed in sapwood of mature teak trees. As conclusion, this work is the first to characterize genes of CAD family in Tectona grandis. These genes could be interesting to develop transge­nic plants for basic research and field applications.

First results of new trembling aspen (Populus tremula L.) special factorial crosses are presented. At first, productive parent trees were selected: five maternal and five paternal trees without symptoms of heart rot and one tree with fruiting bodies of this fungal attack. The results of phenotypic and genotypic analyses of parent trees, their compatibility, and growth performance are presented. The analysis of the hybrid seedlings’ survival and their growth performance on open ground are shown. Estimates of general and specific combining abilities of the parent trees were carried out and potentially best hybrid families and seedlings were selected.

Koa (Acacia koa) and sub-species koaia (A. koaia) are two of more than 1,200 species from the genus Acacia within the Leguminosae (also designated Fabaceae) family. In the past, koa and koaia forests were found throughout the Hawaiian archipelago but populations have dramatically decreased. Comprehensive analyses of simple sequence repeats (SSRs) have not been published previously. Here we use genome sequencing and bioinformatics tools to report development of 100,000+ nuclear SSR (nuSSR) markers for use in koa and koaia genome studies with transcriptome SSR information was inclu­ded for comparison. Over 10,000 high-value SSRs (40-60 % GC content) were isolated with 3,600+ further validated by ePCR. SSRs generated in this work can assist current efforts to sustai­nably increase in koa and koaia populations.

Provenance experiments traditionally provide information on genetic variation within tree species in adaptation ability and other traits important for commercial forestry. In this study we investigated variation in growth among 20 populations of Nor­way spruce (Picea abies (L.) H. Karst) at four common-garden sites of the IUFRO 1972 provenance experimental series at the age close to half of rotation. Because stand density varied among sites, we analyzed stand density-adjusted basal area (BA) and quadratic mean diameter (Dq). The examined prove­nances varied significantly in both analyzed traits. We identi­fied provenances that performed consistently better or worse than average across all four sites. Among the well-growing and possibly adaptive seed sources were those from the uplands of the eastern and central Poland, Sudety Mts, and from the regi­on of Istebna in Beskid Mts. Performance of the other populati­ons from Beskid Mountains was average to poor, and all high-altitude populations were poor-growing. The results of this study help to verify the knowledge of genetic variation pattern among Norway spruce populations in Poland, and to guide management decisions regarding spruce planting material.

Our aim was to verify the effect on gain and genetic diversity through the restriction of the number of trees per family in selection, in order to compose an elite population of Eucalyp­tus urophylla in two trials under distinct management levels. We studied 166 open-pollinated families of E. urophylla in Anhembi, Sao Paulo State, Brazil under commercial practices, and the same families in Selvíria, Mato Grosso do Sul State, Bra­zil under lower management level (mainly no mineral fertiliza­tion). Mortality, height and diameter at breast height (DBH) were measured. DBH was analyzed by the REML/BLUP to select the best 25 trees, with four levels of tree restriction per family (no restriction; 1; 2 and 3 per family). We evaluated heritability; genetic gain and effective size of number of total and private alleles; observed and expected heterozygosity; coancestry and fixation index. A large difference in survival (48 and 83 %) and productivity (MAI of 26 and 44 m³ha^-1y^-1) was observed bet­ween trials due to the different levels of management applied. The highest restriction in number of individuals per family caused a small decrease in gain, corresponding to 7 % in the more productive trial and 3 % in the less productive one. Observed and expected heterozygosity, coancestry and fixati­on index were not significantly (lower than 5 %) affected by the restriction in both sites. The restriction of one tree per family allowed different alleles to be kept in the selected population and higher effective population size in order to insure variation for the next generations.

In the present work, a new estimator to be known as r_IBD is pro­posed. The proposed estimator indirectly quantifies the relati­ve role of gene flow mediated by pollen in relation to the gene flow mediated by seeds in hermaphrodite angiosperm species when an isolation by distance model is assumed. The proposed estimator complements the well-known estimator proposed by Ennos, which is appropriate for studies under the island model. In the present work, the proposed r_IBD index was used to analyze microsatellite data from uni- and biparentally inherited genomes generated by simulations, as well as for the analysis of an empirical data set obtained from public databases of forest tree species. The differences in median values of the pro­posed r_IBD index for simulated data using the Stepping Stone Model and Truncated Pareto Distribution Model coincided with the magnitude expected in terms of differences between levels of pollen and seed dispersion previously established in the simulations. In empirical data, the proposed r_IBD index shows lower levels of gene flow by seed versus gene flow by pollen, the ratio between them being three times lower than the ratio obtained by the Ennos index estimated under the island model. From the analyses carried out, it is feasible to consider the r_IBD index as a suitable estimator of the role of gene flow by seeds in relation to gene flow by pollen under iso­lation by distance.

One of the determinant factors in the success of breeding pro­grams that aim to select genotypes for different geographical regions is understanding the interaction between genotypes and environments (GxE). The objectives of this study were to evaluate GxE interaction, stability, and adaptability, and deter­mine the need for environmental stratification of open pollina­ted progenies of Eucalyptus urophylla. Five progeny tests were established in study areas with different environmental condi­tions in southeast and mid-west Brazil. We used a complete randomized block experimental design with 138 to 167 proge­nies, and variations in the numbers of replicates and plants per plot. The trait measured was diameter at breast height (DBH) at two years of age and the AMMI method was used to determine patterns of GxE interaction. Significant effects were detected for genotypes, environments, and for GxE interaction. The effect of environment was responsible for the greatest propor­tion of the phenotypic variation, followed by the effect of genotypes and GxE interaction. Some progenies with greater productivity and stability were identified, although stability is not associated with productivity. The stratification of the selec­tion in three specific environments is necessary due to the occurrence of a complex GxE interaction.

To assess the genetic and environmental components of gene-expression variation among trees we used RNA-seq technology and Eucalyptus urophylla x grandis hybrid clones tested in field conditions. Leaf and xylem transcriptomes of three 20 month old clones differing in terms of growth, repeated in two blocks, were investigated. Transcriptomes were very similar between ramets. The number of expressed genes was significantly (P<0.05) higher in leaf (25,665±634) than in xylem (23,637±1,241). A pairwise clone comparisons approach showed that 4.5 to 14 % of the genes were diffe­rentially expressed (false discovery rate [FDR]<0.05) in leaf and 7.1 to 16 % in xylem. An assessment of among clone variance components revealed significant results in leaf and xylem in 3431 (248) genes (at FDR<0.2) and 160 (3) (at FDR<0.05), respectively. These two complementary approa­ches displayed correlated results. A focus on the phenylpro­panoid, cellulose and xylan pathways revealed a large majo­rity of low expressed genes and a few highly expressed ones, with RPKM values ranging from nearly 0 to 600 in leaf and 10,000 in xylem. Out of the 115 genes of these pathways, 45 showed differential expression for at least one pair of geno­type, five of which displaying also clone variance compo­nents. These preliminary results are promising in evaluating whether gene expression can serve as possible ‘intermediate phenotypes’ that could improve the accuracy of selection of grossly observable traits.

Pinus sibirica and P. pumila are Asian five-needle pines with vast geographic distributions that are partially overlapping. Natural hybrids with intermediate morphology have been found previ­ously, but there is a lack of evidence of ongoing introgression. The goal of our study was to elucidate the genetic structure of P. sibirica and P. pumila populations growing in the north-east of their sympatry zone (Aldan plateau) using cytoplasmic DNA markers. All studied P. sibirica and P. pumila trees had usual spe­cies-specific growth habits. Using nad1 intron2 of mitochond­rial DNA (mtDNA) and trnV of chloroplast DNA (cpDNA) we found that trees morphologically identified as P. sibirica had pumila-specific mtDNA. Moreover, some of them also had pumila-specific cpDNA. P. pumila trees were typical and had pumila-type cytoplasmic DNA markers. These results suggest that interspecific hybridization took place long ago and lead to introgression and cryptic hybrids with P. sibirica appearance and P. pumila mtDNA

The composition of secondary cell wall determines the indust­rially relevant wood properties in tree species. Hence, its bioge­nesis is one of the most extensively studied developmental processes during wood formation. Presently, systems genetics approach is being applied to understand the biological net­works and their interactions operational during secondary development. Genome-scale analyses of secondary cell wall formation were documented and gene regulatory networks were reported in Arabidopsis, poplar, pine, spruce, rice and sug­arcane. In the present study, the expression patterns of 2651 transcripts representing different pathways governing secon­dary development was documented across four genotypes of E. tereticornis. A co-expression network was constructed with 330 nodes and 4512 edges and the degree ranged from 11 to 53. The network documented 75 (22 %) transcription factors with high degree of interaction. Secondary wall associated NAC domain transcription factor (SND2) was identified as the top hub transcript with 53 interactions. The present study revealed that functional homologs regulating secondary cell wall formation are conserved among angiosperms and gym­nosperms.

Remnant riparian woodlands have an important landscape function, due to their ability to act as ecological corridors. In this study we used molecular markers to assess the genetic variation occurring within and between spontaneous white willow (Salix alba L.) riparian woodlands. Our main goal was to evaluate the extent to which the fragmentation of a woodland corridor along a heavily impacted river in northeastern Italy and stand structural conditions may have affected the population genetics. Although having different structures, the three examined white willow stands showed high estimates of genetic similarity, as well as low genetic differentiation between them, indicating that they shared a similar gene pool and that the stands could result from a common set of individual genotypes, and should be regarded as metapopulations. The magnitude of genetic diversity within each of the stands and genetic differentiation between them, despite their high sexual reproductive capacity associated with a highly marked gene flow, suggest that these stands are dynamic and capable of adaptive responses to possible changes in their fluvial environment. However, the factors influencing genetic diversity should be interpreted from a long-term perspective. Fluvial geomorphic patterns in regulated rivers may be modified to a degree that could lead to changes in dispersal processes, sexu­al reproduction vs. asexual propagation, and hence genetic diversity.

Leaf shape analysis of three Aquilaria species was undertaken using Elliptical Fourier descriptors (EFD) within a research plan­ting in tropical Australia. This approach was taken to develop a method for discriminating the species in the absence of repro­ductive traits or genetic analysis and as a morphological scale of leaf variation. Leaf outlines were analysed to distinguish variability between species. Principal components analysis and canonical discriminant analysis were applied to group the spe­cies based on 25 Fourier descriptors. Four components were required to explain 78 % of the variance and the first and second canonical functions discriminated three groups of spe­cies therefore EFD was a useful approach for analysing leaf out­line variability and distinguish species in the genus Aquilaria. This approach provides a rapid method that could assist in the species identification in Aquilaria where reproductive traits are absent.

Mytilaria laosensis is a fast-growing tropical broadleaf tree that is extensively used for wood production and has significant ecological benefits. To investigate the genetic diversity and population structure of M. laosensis, eight major natural popu­lations in China were analyzed by using simple sequence repeat (SSR) markers. A total of 88 microsatellite-containing fragments were obtained by the method of magnetic bead enrichment, among which 26 pairs of SSR primers were scree­ned out and used to generate a total of 190 alleles among 152 individuals. The average of observed number of alleles, Shannon’s information index and polymorphism information content per locus were 18.3, 1.1577 and 0.7759, respectively, implying a high level of genetic diversity in M. laosensis popu­lations. The variation within populations accounted for 81.74 % of total variation based on analysis of molecular variance. Clus­ter analysis divided the eight populations into four groups, among which five populations from the southern parts of Guangxi province were classified as one major group. Mantel test showed that there was highly significant correlation bet­ween Euclidean genetic distance and geographic distance, suggesting that geographic isolation contribute to the high genetic diversity of M. laosensis. Together, these could provide support for the feasibility of exploration and utilization of M. laosensis in subtropical areas of East Asia including Jiangxi, Hunan and Fujian province of China.

Pinus kesiya (Khasi pine) is the principal pine species in northeast India having high commercial value. Chloroplast microsatellites (cpSSR) were used to study the genetic diversity and population genetic structure of 10 populations of P. kesiya covering entire natural range of distribution in India. A total of 33 primer pairs (cpSSRs) of P. thunberghii and P. sylvestris were tested in P. kesiya for their transferability, out of which 18 chlo­roplast primers showed positive amplification and 10 were found polymorphic. A total of 250 individuals from 10 different populations were genotyped using the selected 10 cpSSRs. When alleles at each of the 10 loci were jointly analysed a total of 36 size variants were discovered, which combined to desig­nate 90 haplotypes among 250 individuals. None of the haplo­type was found common among the populations as they were population specific. The cpSSR indicated that P. kesiya popula­tions have maintained a moderately high genetic diversity (HT=0.638) which is typical in most coniferous species. Howe­ver, the inter-population genetic diversity was higher than the intra population diversity and the genetic differentiation bet­ween populations was also found to be very high (FST=0.47). A Bayesian cluster analysis separated the populations into six clusters where most of the individuals were found in single population clusters with minor admixtures. The distribution of genetic diversity and sub structuring of P. kesiya reflect week pollen mediated gene flow due to geographic isolation and genetic drift. The study has revealed useful cpSSR markers for P. kesiya, which were lacking earlier and also added an insight into the state of Khasi pine forest in the region, which can be useful for the better management and future conservation programs.

Reproductive isolation between related oak species within one taxonomic section is incomplete. Even though pre- and post­zygotic isolation mechanisms have been described for interfer­tile oak species, natural hybridization is common in contact zones between related oaks. The apparent restriction of inter­specific hybrids between ecologically divergent species to intermediate environments in contact zones suggests postzy­gotic isolation via environmental selection against hybrids in parental environments. Overrepresentation of hybrids in seeds as compared to adult trees provides additional indirect evi­dence for selection against hybrids. Here, we used genetic assignment analyses in progeny obtained from a sympatric stand of Quercus rubra and Quercus ellipsoidalis, two interfertile species with different adaptations to drought, to characterize the number of hybrids and “pure” species in the non-germinated acorns and in seedlings. The occurrence of 43.6 % F1 hybrids and introgressive forms among the non-germinated acorns and their very low frequency in the seedlings (9.3 %) is to our knowledge the first direct evidence for early selection against hybrids in oaks possibly as result of genetic incompatibilities. Additionally, we found a signature of positive selection on EST-SSR PIE200 in Q. rubra which needs further confirmation. These results contribute to our under­standing of reproductive isolation and divergence between interfertile oak species with different ecological adaptations.

We grafted scions of Dahurian larch (Larix gmelinii var. japonica) onto Japanese larch (L. kaempferi) and F1 hybrid larch (L. gmeli­nii var. japonica × L. kaempferi) rootstocks and examined root­stock-scion compatibility by assessing the survival rate (SR) in two independent experiments. Scion overgrowth on the root­stock was not observed. SR was not significantly different among rootstocks due to large interquartile ranges (IQR) among clones within a rootstock type. Results suggested that the SR was more dependent on the clonal characteristics of the scion than on the growth vigor of the rootstock. Shoot elonga­tion of grafts on F1 hybrid rootstock was superior to that of grafts on Japanese larch rootstock. Selection of an appropriate combination of scion and rootstock may improve the SR of grafted Dahurian larch and shorten the cultivation period.

The nucleotide binding site (NBS) domain sequences were iso­lated from genomic DNA in black bamboo, using the degenerate primer designed according to the conserved motifs of the NBS resistance gene. The expected size of the PCR product was about 700 bp. Among 55 positive clones, the amino acid sequence alignment identified 33 black bamboo resistance gene analogs (RGAs) that contain the NBS conserved motifs. All of the 33 RGAs ORFs were constructed in an NJ (Neighbor-joi­ning) tree, and divided into 10 groups. This analysis demonstrated the diversity of the NBS class RGA in black bamboo. The maximum likelihood estimates of various evolutionary models were analyzed; the result showed that 2 groups with a total of 10 sequences and 12 sites demonstrated statistically significant positive selection. Most of the positive selected sites were not located in the NBS conserved motifs. Two groups of gene conversion events had been discovered, which provide a mate­rial basis and research direction in isolating black bamboo R genes.

Japanese flowering cherry (Prunus subgenus Cerasus; Rosa­ceae) cultivars, which are characterized by beautiful flowers, have been generated through hybridization among wild Pru­nus taxa. The analysis of polymorphisms in the maternally inhe­rited chloroplast DNA is an effective complementary approach for tracing the origins of these cultivars. Thus, a set of novel chloroplast simple sequence repeat (cpSSR) markers was deve­loped for the Prunus taxa, and their utility in cross-species amp­lification was evaluated. Five markers were designed from the partial chloroplast genome sequences of thirteen Prunus taxa. In all, we found 19 haplotypes among the 311 individuals tes­ted. The number of haplotypes, haplotype richness and haplo­type diversity in each taxon ranged from 1 to 8, from 1 to 6.613 and from 0 to 0.758, respectively. The major proportion of the total diversity (H_T = 0.831) was accounted for by intraspecific diversity (H_S = 0.559), and the level of species subdivision, G_ST, was 0.327. These five cpSSR markers highly polymorphic, and they will be useful not only for tracing the origins of the culti­vars but also for studying the population structure, diversity, and phylogeography of Prunus.

Due to ongoing climate change, forests are exposed to changing environmental conditions, such as increasing temperatures and lower precipitation, to which trees have to adapt. Successful adaptation to changing and variable environments requires sufficient genetic variation within tree populations. Knowledge of the genetic variation of trees is therefore essential, as it provides information for the long-term conservation, stability and productivity of forests. The genetic variation of a species can be analysed with molecular markers. Despite growing genomic and genetic resources for European beech (Fagus sylvatica L.), which is one of the economically and ecologically most important forest tree species in Central Europe, the number of molecular markers for population genetic analyses is still limited. Therefore, the aim of the work is the development of new EST-SSR markers for this species. A total of 72 DNA samples of European beech from three widely separated regions in Germany were used to test 41 primers for variation and polymorphism, 35 of which were originally developed for American beech (Fagus grandifolia Ehrh.) and 6 for red oak (Quercus rubra L.). Fifteen of the primers were polymorphic, 13 monomorphic and 13 did not amplify. In addition, the transferability of the markers was successfully tested in the related species Castanea dentata Bork., Fagus orientalis Lipsky and Q. rubra. The EST-SSR markers tested in this study will be useful for future population genetic analyses and extend the set of available markers in European beech.

The aim of this study was to determine the genetic variation of growth traits, wood basic density, dry mass of crown, leaf, and stem, and carbon stock within and between two Genipa ameri­cana L. provenances. The provenance and progeny test was installed in the Teaching, Research and Extension Farm, located in Selvíria, Mato Grosso do Sul, Brazil. The test was established using a randomized block design with two provenances and 36 progenies, a spacing of 3 x 2 m, four replicates, and six plants per plot. At 11 years of age, height and diameter at breast height (DBH) were measured. To estimate wood basic density, biomass, and carbon stock, 45 plants were thinned. Growth traits for crown, leaf, and stem, as well as wood basic specific gravity showed significant differences. Both provenances pre­sent high carbon storage, demonstrating the potential of this species to be included in carbon credit programs that help to minimize the effects of global warming. In general, heritability values were low, suggesting limited genetic control of the eva­luated traits. However, the traits presented a high coefficient of genetic variation, indicating adequate sampling among popu­lations; these results are promising for ex situ conservation and for future breeding programs.