Relationship between inbreeding depression and inbreeding coefficient in maritime pine (Pinus pinaster)

The relationship between inbreeding depression and inbreeding coefficient (F) for several important traits was investigated in an 11-year trial of maritime pine (Pinus pinaster). Five levels of inbreeding (F=0; 0.125; 0.25; 0.5; 0.75) were obtained in a mating design involving ten plus-trees, or their progenies, as parents (total of 51 families). For F=0.75, the mean inbreeding depressions were 27% for height, 37% for circumference at breast height (63% for bole volume), 23% for basal straightness (better straightness of the inbred trees), and 89% for female fertility (number of cones). Large differences were observed among inbred families for the same level of inbreeding. The evolution of depression with F was more or less linear, depending on the traits. Significant differences among F-levels appeared very early for height (from 5-years of age). Inbreeding depression was much more expressed during unfavorable years than during favorable years for yearly height growth. When compared with other Pinus species, maritime pine appears to be less affected by inbreeding, especially for the percentage of filled seeds and general vigor. A reduced genetic load in maritime pine may result from the evolutionary history of the species and its scattered distribution.     

Plasticity of maritime pine (Pinus pinaster) wood-forming tissues during a growing season

The seasonal effect is the most significant external source of variation affecting vascular cambial activity and the development of newly divided cells, and hence wood properties. Here, the effect of edapho-climatic conditions on the phenotypic and molecular plasticity of differentiating secondary xylem during a growing season was investigated. Wood-forming tissues of maritime pine (Pinus pinaster) were collected from the beginning to the end of the growing season in 2003. Data from examination of fibre morphology, Fourier-transform infrared spectroscopy (FTIR), analytical pyrolysis, and gas chromatography/mass spectrometry (GC/MS) were combined to characterize the samples. Strong variation was observed in response to changes in edapho-climatic conditions. A genomic approach was used to identify genes differentially expressed during this growing season. Out of 3512 studied genes, 19% showed a significant seasonal effect. These genes were clustered into five distinct groups, the largest two representing genes over-expressed in the early- or late-wood-forming tissues, respectively. The other three clusters were characterized by responses to specific edapho-climatic conditions. This work provides new insights into the plasticity of the molecular machinery involved in wood formation, and reveals candidate genes potentially responsible for the phenotypic differences found between early- and late-wood.     

An estimation of the genome length of maritime pine (Pinus pinaster Ait.)

The genome length, in units of Morgans or centimorgans, is a fundamental feature of a species. It can be calculated from a complete linkage map. However, the genome size can be estimated with partial linkage data. Using linkage data obtained by the analysis of a two-dimensional electrophoresis of the proteins contained in an haploid organ, the megagametophyte, we suggest an estimation and a confidence interval of the genome length of a gymnosperm, the maritime pine (Pinus pinaster Ait.). The results indicate an important gap between the physical and the genetic maps.     

Genetic variation and migration pathways of maritime pine (Pinus pinaster Ait) in the Iberian peninsula

The genetic variability and migration pathways of Pinus pinaster after glaciation in the Iberian peninsula was studied by means of 18 loci from 12 natural populations of the species. The analysis showed the existence of three groups of populations with different levels of diversity and patterns of recolonization. The southern Iberian group displays a high level of diversity, with a stepping-stone model of variation. The presence of rare alleles in this group and their position in the phylogenetic tree suggest the existence of refugia during glaciations in this zone. The eastern Iberian group also has high levels of diversity but is clearly separate from the first group based on their genetic distances. The Atlantic group displays a low level of diversity that could be interpreted as a rapid recolonization of the entire area by the Eastern group that has not yet developed to a divergence in this area. The southern Iberian range is indicated to be the dispersal centre of the species after the last glaciation. Received: 15 February 1999 / Accepted: 30 April 1999     

Variation in chloroplast single-sequence repeats in Portuguese maritime pine (Pinus pinaster Ait.)

Genetic variation in 12 Pinus pinaster (maritime pine) populations spanning most of the distribution range of the species in Portugal was evaluated using six polymorphic chloroplast microsatellite (cpSSR) loci. Thirty-two haplotypes were found. There were indications of very weak differentiation among populations (Weir’s θcoefficient, 0.023), and the R _ST value, derived from the stepwise mutation model (SMM), was not significantly different from zero. The pattern, in which similarities in allele size, in base pairs, do not contribute to the genetic structure, may be due to the recent mixing of genetic material from different stands through plantations. Overall, a high level of haplotypic variation within populations was detected. Using the SMM estimator (mean genetic distance of individuals within populations, D ² _sh –––) we divided the populations into two groups, with above and below average values. The first group contained 5 populations, mainly from the central part of the country, which possess, in general, high levels of haplotypic diversity. Among them, 2 populations were divergent from the others based on the pair-wise Nei’s distance. The results indicate that there is no discernible geographic genetic pattern for the Portuguese populations of P. pinaster investigated. The history of expansion of the species range in Portugal during the twentieth century (mainly due to human activity) and extensive gene flow among populations associated with the expansion could explain this finding. Received: 15 February 2000 / Accepted: 14 April 2000     

Effective gene dispersal and female reproductive success in Mediterranean maritime pine (Pinus pinaster Aiton)

Understanding population-scale processes that affect allele frequency changes across generations is a long-standing interest in genetic, ecological and evolutionary research. In particular, individual differences in female reproductive success and the spatial scale of gene flow considerably affect evolutionary change and patterns of local selection. In this study, a recently developed maximum-likelihood (ML) method based on established offspring, the Seedling Neighbourhood Model , was applied and exponentially shaped dispersal kernels were fitted to both genetic and ecological data in a widespread Mediterranean pine, Pinus pinaster Aiton. The distribution of female reproductive success in P. pinaster was very skewed (about 10% of trees mothered 50% of offspring) and significant positive female selection gradients for diameter (γ = 0.7293) and cone crop (γ = 0.4524) were found. The selective advantage of offspring mothered by bigger trees could be due to better-quality seeds. These seeds may show more resilience to severe summer droughts and microsite variation related to water and nutrient availability. Both approaches, ecological and of parentage, consistently showed a long-distance dispersal component in saplings that was not found in dispersal kernels based on seed shadows, highlighting the importance of Janzen-Connell effects and microenvironmental variation for survival at early stages of establishment in this Mediterranean key forest tree.     

Clonal variation for shoot ontogenetic heteroblasty in maritime pine (Pinus pinaster Ait.)

Pine seedling shoots undergo sharp heteroblastic changes during the early ontogenetic stages. The rate of these changes has been seen to vary between species and provenances within species, but there is a marked lack of information about its genetic control at the lower hierarchical levels. We used clonal replicates of maritime pine to determine broad-sense heritability of shoot ontogenetic heteroblasty and its correlation to rooting ability. We applied a simple ontogenetic index based on the proportion of basal nodes with secondary needles in rooted cuttings of 15 clones from 9 environmentally contrasting origins. We found a high clonal heritability for shoot ontogenetic index and a moderately high heritability for rooting ability, but both genetic and phenotypic correlations between these two traits were weak and non-significant. These results indicate that both developmental phenomena are genetically controlled, but not strictly associated in this species.     

Analysis of genetic stability at SSR loci during somatic embryogenesis in maritime pine (Pinus pinaster)

Somatic embryogenesis (SE) is a propagation tool of particular interest for accelerating the deployment of new high-performance planting stock in multivarietal forestry. However, genetic conformity in in vitro propagated plants should be assessed as early as possible, especially in long-living trees such as conifers. The main objective of this work was to study such conformity based on genetic stability at simple sequence repeat (SSR) loci during somatic embryogenesis in maritime pine (Pinus pinaster Ait.). Embryogenic cell lines (ECLs) subjected to tissue proliferation during 6, 14 or 22 months, as well as emblings regenerated from several ECLs, were analyzed. Genetic variation at seven SSR loci was detected in ECLs under proliferation conditions for all time points, and in 5 out of 52 emblings recovered from somatic embryos. Three of these five emblings showed an abnormal phenotype consisting mainly of plagiotropism and loss of apical dominance. Despite the variation found in somatic embryogenesis-derived plant material, no correlation was established between genetic stability at the analyzed loci and abnormal embling phenotype, present in 64% of the emblings. The use of microsatellites in this work was efficient for monitoring mutation events during the somatic embryogenesis in P. pinaster. These molecular markers should be useful in the implementation of new breeding and deployment strategies for improved trees using SE.     

Effects of wounds on the terpene content of twigs of maritime pine (Pinus pinaster Ait.)

Summary The effect of wounds on the volatile terpene composition of the living bark of Pinus pinaster Ait. (maritime pine) twigs was investigated with respect to the processes of mono- and sesquiterpene hydrocarbon biosynthesis. The large increase in the amounts of - and -pinene is a characteristic feature after a mechanical injury, whereas the quantities of the other terpenes are only slightly increased. This is due to the reactivation of the resin duct secretory cells of primary origin located in cortical tissues. The effect of wounding is observed over a long period and the terpene profiles are very different at the end of the experiments as compared with the initial profiles of the same tissues. The traumatic essential oil (obtained after mechanical traumatism) resembles an oleoresin extracted from tissues of secondary origin. Statistical analysis underlines the effects of the between-tree variations and of the dates of application of the wound.     

Effects of wounding on the terpene content of twigs of maritime pine (Pinus pinaster Ait.)

Summary After wounding the cortical tissues of twigs of Pinus pinaster, we investigated changes in diterpene resin acid content and the ultrastructural modification of secretory structures. There was an increase in total resin acid content in the cortical and woody tissues located near the wound. Not all resin acids responded in the same way, but in wounded tissues the amounts of isopimaric acid and of the group of dehydroabietic, levopimaric and palustric acids significantly increased. The composition of cortical tissues becomes closely related to that of woody tissues. The resin acid enrichment of cortical tissues is correlated with the reactivation of the epithelial cells of the resin ducts and the de novo synthesis of resin acids demonstrated by labelling with ₁₄CO².     

Population dynamics of bacteria associated with different strains of the pine wood nematode Bursaphelenchus xylophilus after inoculation in maritime pine (Pinus pinaster)

For a long time it was thought that Bursaphelenchus xylophilus was the only agent of the pine wilt disease. Recently, it was discovered that there are bacteria associated with the nematodes that contribute to the pathogenesis of this disease, mainly through the release of toxins that promote the death of the pines. Among the species most commonly found, are bacteria belonging to the Bacillus, Pantoea, Pseudomonas and Xanthomonas genera.The main objective of this work was to study the effect of inoculation of maritime pine (Pinus pinaster) with four different nematode isolates, in the bacterial population of nematodes and trees, at different stages of disease progression. The monitoring of progression of disease symptoms was also recorded. Also, the identification of bacteria isolated from the xylem of trees and the surface of nematodes was performed by classical identification methods, by the API20E identification system and by sequencing of bacterial DNA.The results showed that for the symptoms progression, the most striking difference was observed for the pines inoculated with the avirulent isolate, C14-5, which led to a slower and less severe aggravation of symptoms than in pines inoculated with the virulent isolates. In general, it was found that bacterial population, inside the tree, increased with disease progression. A superior bacterial quantity was isolated from pines inoculated with the nematode isolates HF and 20, and, comparatively, few bacteria were isolated from pines inoculated with the avirulent isolate. The identification system API20E was insufficient in the identification of bacterial species; Enterobacter cloacae species was identified in 79% of the isolated bacterial colonies and seven of these colonies could not be identified by this method. Molecular identification methods, through bacterial DNA sequencing, allowed a more reliable identification: eleven different bacterial species within the Bacillus, Citrobacter, Enterobacter, Escherichia, Klebsiella, Paenibacillus, Pantoea and Terribacillus genera were identified. General bacterial diversity increased with the progression of the disease. Bacillus spp. were predominant at the earlier stage of disease progression and Klebsiella oxytoca at the later stages. Furthermore, bacterial species isolated from the surface of nematodes were similar to those isolated from the xylem of pines.In the present work new bacterial species were identified which have never been reported before in this type of study and may be associated with their geographical origin (Portugal). P. pinaster, the pine species used in this study, was different from those commonly grown in Japan and China. Furthermore, it was the first time that bacteria were isolated and identified from an avirulent pine wood nematode isolate.     

Susceptibility of embryogenic and organogenic tissues of maritime pine (Pinus pinaster) to antibiotics used in Agrobacterium-mediated genetic transformation

The effects of antibiotics commonly used in Agrobacterium-mediated transformation were studied on Pinus pinaster tissues. Embryogenic tissue growth from three embryogenic lines and adventitious bud induction from cotyledons from three open-pollinated seed families were analysed. Cefotaxizme, carbenicillin and timentin commonly used for Agrobacterium elimination, at concentrations of 200–400 mg l ⁻¹ did not inhibit the embryogenic tissue growth on filter paper nor as clumps. Adventitious bud induction and bud number were significantly reduced for one of the tested families when using 400 mg l⁻¹ cefotaxime or timentin. The selection agent kanamycin significantly inhibited growth of embryogenic tissue on filter paper in all the embryogenic lines␣and concentrations tested (20–50 mg l⁻¹). Kanamycin also inhibited growth of embryogenic clumps after two subcultures at 5–50 mg l⁻¹. In␣cotyledons, kanamycin inhibited adventitious bud␣formation in the three seed families used, regardless of the concentrations tested (5–25 mg l⁻¹). There was a significant effect of the seed family on the bud induction and the number of adventitious buds produced. From the results obtained, we propose the use of timentin to eliminate Agrobacterium in transformation experiments, at concentrations of 400 mg l⁻¹ for embryogenic tissues and of 300 mg l⁻¹ for cotyledons. For selection of transformed tissues carrying the kanamycin resistance gene, kanamycin should be used at 20 mg l⁻¹ for embryogenic tissues on filter paper, at 5 mg l⁻¹ when clumps are in direct contact with the selection medium, and bellow 5 mg l⁻¹ for adventitious bud induction.     

Evidence for an operative glutamine translocator in chloroplasts from maritime pine (Pinus pinaster Ait.) cotyledons

In higher plants, ammonium is assimilated into amino acids through the glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle. This metabolic cycle is distributed in different cellular compartments in conifer seedlings: glutamine synthesis occurs in the cytosol and glutamate synthesis within the chloroplast. A method for preparing intact chloroplasts of pine cotyledons is presented with the aim of identifying a glutamine–glutamate translocator. Glutamine–glutamate exchange has been studied using the double silicone layer system, suggesting the existence of a translocator that imports glutamine into the chloroplast and exports glutamate to the cytoplasm. The translocator identified is specific for glutamine and glutamate, and the kinetic constants for both substrates indicate that it is unsaturated at intracellular concentrations. Thus, the experimental evidence obtained supports the model of the GS/GOGAT cycle in developing pine seedlings that accounts for the stoichiometric balance of metabolites. As a result, the efficient assimilation of free ammonia produced by photorespiration, nitrate reduction, storage protein mobilisation, phenylpropanoid pathway or S‐adenosylmethionine synthesis is guaranteed.