Luc E. Pâques

Application of AFLP, RAPD and ISSR markers to genetic mapping of European and Japanese larch.

Abstract Genetic linkage maps have been increasingly developed for a wide variety of plants, using segregating populations such as F2s or backcrosses between inbred lines. These pedigrees are rarely available in outbred species like forest trees which have long generation times. Thus genetic mapping studies have to use peculiar pedigrees and markers in appropriate configurations. We constructed single-tree genetic linkage maps of European larch (Larix decidua Mill.) and Japanese larch [Larix kaempferi (Lamb.) Carr.] using segregation data from 112 progeny individuals of an hybrid family. A total of 266 markers (114 AFLP, 149 RAPD and 3 ISSR loci) showing a testcross configuration, i.e.heterozygous in one parent and null in the other parent, were grouped at LOD 4.0, θ=0.3. The maternal parent map (L. decidua)consisted of 117 markers partitioned within 17 linkage groups (1152 cM) and the paternal parent map (L. kaempferi) had 125 markers assembled into 21 linkage groups (1206 cM). The map distance covered by markers was determined by adding a 34.7-cM independence distance at the end of each group and unlinked marker. It reached 2537 cM and 2997 cM respectively for European larch and Japanese larch, and represented respectively a 79.6% and 80.8% coverage of the overall genome. A few 3:1 segregating markers were used to identify homologous linkage groups between the European larch and the Japanese larch genetic maps. The PCR-based molecular markers allowed the construction of genetic maps, thus ensuring a good coverage of the larch genome for further QTL detection and mapping studies.

Colour of larch heartwood and relationships to extractives and brown-rot decay resistance

Larch heartwood is appreciated for its good mechanical properties, its colour and its texture, and it is often used outdoors because of its natural durability (decay resistance). In this study the colour of larch heartwood was studied in relation to extractives and decay resistance, with the aim to estimate durability of larch heartwood from its colour. On a total of 293 trees colour in the CIE L*a*b* space (L* lightness, a* red/green axis, b* yellow/blue axis), extractives content (acetone and hot-water extractives, amount of phenolics) and the brown-rot decay resistance were determined. For calculating the relative decay resistance ( x), mass loss after inoculation for 16 weeks with two fungi [ Coniophora puteana (Schum.ex.Fr.) Karst., Poria placenta (Fr.) Cke, European standard EN 113] of larch heartwood samples was compared to Scots pine ( Pinus sylvestris L) sapwood reference samples (EN 350-1). Different species [Japanese larch ( Larix kaempferi Lamb.), Hybrid larch (Larix deciduax L. kaempferi) and European larch ( L. decidua Mill.)], provenances and age classes (38-year, >150-year) were included. Japanese larch heartwood turned out to be significantly more reddish (higher a*-values) compared to the European larch provenances. Reddishness of the hybrids was intermediate. The red hue (+a*) was strongly correlated with the amount of phenols ( r =0.84) and decay resistance ( r =0.63) and therefore suitable for prediction of both parameters. The results suggest that colour measurements of larch heartwood could be of benefit in tree breeding programs and for an optimised utilization of larch timber.

Somatic embryogenesis in forestry with a focus on Europe: state-of-the-art, benefits, challenges and future direction

Vegetative propagation of forest trees offers advantages to both tree breeders and the forest industry. This review will describe benefits, type of vegetative propagation, and its integration into breeding programmes. Of all of the different methods for vegetative propagation, only rooted cuttings and somatic embryogenesis (and the combined use of both) offer any practical methods for large-scale commercial use. However, it is very difficult to fully appreciate the overall level of activity of the research and application of somatic embryogenesis of forest trees. Publications and reports only highlight a small fraction of the ongoing work. To this end, a survey was conducted across Europe (under EU Research Infrastructure Concerted Action “Treebreedex”) to document the species involved, the state-of-the-art of somatic embryogenesis, its stage of development and its application in tree improvement programmes and to commercial forestry. The results of this survey are presented and discussed. In addition, this review presents the challenges (biological, economic, public acceptance and regulatory) and their relationships to European forestry. Finally, a strategy to promote the use of this technology is proposed.

Heartwood extractives and lignin content of different larch species (Larix sp.) and relationships to brown-rot decay-resistance

The extractive content of lignin and the brown-rot decay-resistance against Coniophora puteana and Poria placenta were studied in larch heartwood from different species and origin (Larix decidua var. decidua, L. decidua var. sudetica, L. kaempferi , L. × eurolepis). The study material consisted of 106 trees from a 39-year old provenance trial in France. The hot-water-soluble extractives were very variable (from5.66% to 20.50% of dry weight), but there was no significant variation between the investigated species and origins. In contrast, acetone extractives, the total amount of phenolics and lignin showed significant differences. The concentration of phenolics and lignin was significantly higher in L. kaempferi and in L. × eurolepis than in L. decidua. The total phenolics content was strongly correlated with decay-resistance in all investigated larch origins. A higher concentration of phenolics goes hand in hand with higher decay resistance and phenolics might therefore be a promising parameter to rapidly evaluate the level of decay-resistance in larch.

Species-diagnostic markers in Larix spp. based on RAPDs and nuclear, cpDNA, and mtDNA gene sequences, and their phylogenetic implications

Genetic markers from the nuclear, chloroplast, and mitochondrial genomes were developed to distinguish unambiguously among four larch species [Larix laricina (Du Roi) K. Koch, Larix decidua (Mill.), Larix kaempferi (Lamb.) Sarg., and Larix sibirica (Ledeb.)] used in intensive forestry in eastern North America. Nine random amplified polymorphic DNA (RAPD) fragments had good diagnostic value, and 3 out of 12 nuclear genes were found to harbor fixed interspecific polymorphisms implicating a total of 17 single nucleotide polymorphisms (SNPs) and 2 indels. The sequencing of five mtDNA introns (cox1-intron1, matR-intron1, nad1-intron b/c, nad3-intron1, and nad5-intron1) and four cpDNA regions (matK, trnL-intron, trnT–trnL and trnL–trnF intergenic spacers) resulted in the identification of 14 sites with fixed interspecific differences among the four species. Including the ten Larix species, one polymorphic site per 47 nucleotide sites sampled was observed for nuclear genes, one per 283 sites for cpDNA, and one per 374 sites for mtDNA. The phylogeny of the genus could be estimated from variation among the ten species detected in two cpDNA intergenic regions and four mtDNA introns. There was congruence between cpDNA and mtDNA phylogenies with three large groups delineated: the North American, North Eurasian, and South Asian taxa. The position of L. sibirica differed between organelle genomes. It was regrouped with South Asian species on the cpDNA tree, but with its North Eurasian congenerics on the mtDNA tree. To simplify the detection of diagnostic DNA sequence polymorphisms among the four main Larix species, cleaved amplified polymorphic sequence (CAPS) assays were developed from the polymorphisms identified in the various genomes. Seventeen primer–enzyme combinations were tested, and six were selected for their high level of informativeness. These new species-specific diagnostic markers should be useful for the certification of larch breeding materials and hybrid stocks used in intensive forestry in the northern hemisphere.

Simplified and improved somatic embryogenesis of hybrid larches (Larix × eurolepis and Larix × marschlinsii). Perspectives for breeding

Abstract• Development of clonal propagation method, such as somatic embryogenesis, has numerous applications such as mass-production of genetically improved plants and the amenability of embryogenic cultures to cryogenic storage. Since the 90’s, researchers at INRA have engaged in research on somatic embryogenesis in Larix species (Larix × eurolepis, Larix × marschlinsii).• The aim of this work was to improve and to simplify all steps of somatic embryogenesis and to apply this protocol to the new hybrid variety REVE-VERT.• The somatic embryogenesis initiation frequency from immature zygotic embryos was high (65%) on a medium with reduced plant growth regulator concentrations (2.2 μM of 2.4-dichlorophenoxyacetic acid and 2.3 μM of 6-benzyladenine). Simplified cryopreservation method (no need of programmable freezer) of the embryonal masses resulted in 100% recovery of cryopreserved lines. Maturation of a large number of somatic embryos was greatly improved when embryonal masses were dispersed on filter paper placed on medium containing high concentration of gellan gum (8 g·L−1). Under these conditions, 94% of the lines matured somatic embryos that developed into plantlets. Clearly ageing and cryopreservation did not reduce embryogenic potential of embryonal masses.• Requirements for the effective integration of somatic embryogenesis into the larch breeding programme are discussed.Résumé• Le développement de méthode de multiplication clonale, telle l’embryogenèse somatique, a de nombreuses applications comme la production en masse de plants génétiquement améliorés et la disponibilité des cultures embryogènes en cryoconservation. Depuis les années 1990, l’INRA a engagé des recherches en embryogenèse somatique des mélèzes (Larix × eurolepis, Larix × marschlinsii).• L’objectif du travail a été de simplifier et d’améliorer chaque étape de l’embryogenèse somatique, et d’appliquer ce protocole à la nouvelle variété de mélèze hybride REVE-VERT.• L’initiation de l’embryogenèse somatique à partir d’embryons zygotiques immatures est obtenue à des fréquences élevées (65 %) avec un milieu de culture contenant des concentrations hormonales réduites (2,2 μM d’acide 2.4-dichlorophenoxyacetic et 2,3 μM de 6-benzyladenine). Une méthode simplifiée de congélation (sans l’utilisation d’un congélateur programmable) des masses embryogènes permet la survie de 100 % des lignées congelées. La maturation d’un grand nombre d’embryons somatiques a été fortement améliorée lorsque les masses embryogènes ont été étalées sur filtre papier placé sur milieu de culture à forte concentration en gelrite (8 g·L−1). Sous ces conditions, 94 % des lignées embryogènes régénèrent des embryons somatiques matures qui se développent en plantes. Clairement l’âge et la cryoconservation des lignées embryogènes ne réduisent pas leur potentiel embryogène.• Les besoins pour une intégration efficace de l’embryogenèse somatique au programme d’amélioration du mélèze, sont discutés.

Heterozygosity and hybrid performance in larch.

Random Amplified Polymorphic DNAs (RAPD) were used for estimating genetic distances between 12 European larches (Larix decidua) and 12 Japanese larches (L. kaempferi) that were the parents in a factorial mating design. One hundred and eleven fragments were used for establishing genetic distances based on Jaccards coefficient between parents. Thirteen fragments differentiated the larch species. The genetic distance between individuals of the same species (DJ=0.39 in the Japanese larch and 0.45 in the European larch) was lower than the genetic distance between species (DJ=0.72). A UPGMA dendrogram based on genetic distances clearly clustered each larch species, confirming the speciation at a molecular level. Correlations between genetic distances of the parents and performances of the hybrid families were established for various quantitative traits. Significant values were found for growth characters and branch insertion angle, which suggested an effect of general heterozygosity level on hybrid traits. These correlations also evolved with tree age: the maximal correlation was noticed on 6-year-old trees for height. The lack of correlation between parental genetic distances and hybrid performances for the other quantitative traits suggested that these characters were controlled by fewer genes. The results of this study show that crosses between genetically distant parents produce hybrids with excellent growth performances; this represents a potential selection criterion of the genitors.

Larches (Larix sp.)

European larch (Larix decidua) is a native species from the Alps and from several mountainous ranges in Central Europe. Its early introduction outside its native range, especially in lowlands western and northern Europe has been problematic for ecological (phenology) and sanitary (canker) reasons due to the lack of knowledge of its genetic variability. As a substitute, Japanese larch (Larix kaempferi) and then hybrids between European and Japanese larches have been preferred in these regions together with Siberian larches in northern Scandinavia. Results from intensive provenances testing (as early as in the 1940s) have been the corner stone on which breeding programmes have developed both at the intra- (mostly in Central and Eastern Europe) and inter-specific (mostly in North-Western Europe) levels. Inter-specific hybridization is probably the most emblematic and promising breeding strategy for larch, taking benefit of both species trait complementarity, hybrid vigour (heterosis) and hybrid homeostasis. While seed orchards are now the main source for dissemination of genetic progress for European larch, mass-production of hybrid larch remains problematic either through sexual (hybridization seed orchard) or asexual (vegetative propagation by cutting) ways and still impedes large deployment of hybrids in Europe. While progress is expected from current research on this topic (coupling of somatic embryogenesis with bulk vegetative propagation by cutting, 2nd generation hybridization orchard), other hot issues include decoding of heterosis, evaluation of genetic variability for the most original larch wood properties (heartwood formation and durability), adaptation to climate, etc. Joined efforts at European level are expected for genetic resource conservation, breeding and deployment, and tackling of several scientific and technical bottlenecks.

Identification of European and Japanese Larch and Their Interspecific Hybrid with Morphological Markers: Application to Young Seedlings

Abstract Open-pollinated hybridisation seed orchards of European and Japanese larches produce mixed progenies combining a highly variable proportion of hybrids along with pure parental species. For several reasons, it is desirable to identify and to sort out hybrids from pure species at the seedling stage. Taxa identification of 1-2 yr-old seedlings was attempted using non-destructive assessment of several traits, including morphology, phenology, growth and architecture parameters. Two sets of progenies originating from 10 open-pollinated hybridisation seed orchards were used, relying in a first step on taxa identification of individual seedlings with diagnostic molecular markers. Based on 21 traits assessed, some clear trends in pure species and hybrid features were apparent but due to the large and overlapping ranges of taxa characteristics, no single parameter allowed unambiguous identification of taxa. Combination of traits through linear discriminant analysis made possible correct classification of 90.2% to 98.6% of individuals depending on the orchard although there were a few problematic orchards. Two traits appeared particularly pertinent for discriminating young plants taxa, namely 1st-yr leaf retention (marcescence) and the bark colour of 2nd-year shoot increments. Results were corroborated using progenies from several orchards and over two experimental periods.

Dissecting the Space-Time Structure of Tree-Ring Datasets Using the Partial Triadic Analysis

Tree-ring datasets are used in a variety of circumstances, including archeology, climatology, forest ecology, and wood technology. These data are based on microdensity profiles and consist of a set of tree-ring descriptors, such as ring width or early/latewood density, measured for a set of individual trees. Because successive rings correspond to successive years, the resulting dataset is a ring variables × trees × time datacube. Multivariate statistical analyses, such as principal component analysis, have been widely used for extracting worthwhile information from ring datasets, but they typically address two-way matrices, such as ring variables × trees or ring variables × time. Here, we explore the potential of the partial triadic analysis (PTA), a multivariate method dedicated to the analysis of three-way datasets, to apprehend the space-time structure of tree-ring datasets. We analyzed a set of 11 tree-ring descriptors measured in 149 georeferenced individuals of European larch (Larix decidua Miller) during the period of 1967–2007. The processing of densitometry profiles led to a set of ring descriptors for each tree and for each year from 1967–2007. The resulting three-way data table was subjected to two distinct analyses in order to explore i) the temporal evolution of spatial structures and ii) the spatial structure of temporal dynamics. We report the presence of a spatial structure common to the different years, highlighting the inter-individual variability of the ring descriptors at the stand scale. We found a temporal trajectory common to the trees that could be separated into a high and low frequency signal, corresponding to inter-annual variations possibly related to defoliation events and a long-term trend possibly related to climate change. We conclude that PTA is a powerful tool to unravel and hierarchize the different sources of variation within tree-ring datasets.