Céline Lalanne

Comparative genome and QTL mapping between maritime and loblolly pines

Genetic markers developed from expressed sequence tags (ESTs) were used as orthologous loci for comparative genome studies in the genus Pinus. A total of 309 ESTs derived from conifer gene sequences were tested for amplification and polymorphism in maritime pine (Pinus pinaster Ait.). Electrophoresis-based techniques made it possible to map 50 expressed sequence tag polymorphisms (ESTPs). The map positions of 32 markers were compared to putative orthologous loci on the loblolly pine (Pinus taeda L.) linkage map, which is the reference map of the conifer genetic mapping community. Overall, synteny was maintained between the two species. This report agrees with other pairwise genome comparisons in pine and supports the cytogenetic evidence that chromosome evolution in the genus is conservative. The alignment of homologous linkage groups allowed, for the first time in conifers, the comparison of QTL location. The position of two QTLs controlling wood density and cell wall components were found to be conserved between the two species.

Decoding the oak genome: public release of sequence data, assembly, annotation and publication strategies

The 1.5 Gbp/2C genome of pedunculate oak (Quercus robur) has been sequenced. A strategy was established for dealing with the challenges imposed by the sequencing of such a large, complex and highly heterozygous genome by a whole‐genome shotgun (WGS) approach, without the use of costly and time‐consuming methods, such as fosmid or BAC clone‐based hierarchical sequencing methods. The sequencing strategy combined short and long reads. Over 49 million reads provided by Roche 454 GS‐FLX technology were assembled into contigs and combined with shorter Illumina sequence reads from paired‐end and mate‐pair libraries of different insert sizes, to build scaffolds. Errors were corrected and gaps filled with Illumina paired‐end reads and contaminants detected, resulting in a total of 17 910 scaffolds (>2 kb) corresponding to 1.34 Gb. Fifty per cent of the assembly was accounted for by 1468 scaffolds (N50 of 260 kb). Initial comparison with the phylogenetically related Prunus persica gene model indicated that genes for 84.6% of the proteins present in peach (mean protein coverage of 90.5%) were present in our assembly. The second and third steps in this project are genome annotation and the assignment of scaffolds to the oak genetic linkage map. In accordance with the Bermuda and Fort Lauderdale agreements and the more recent Toronto Statement, the oak genome data have been released into public sequence repositories in advance of publication. In this presubmission paper, the oak genome consortium describes its principal lines of work and future directions for analyses of the nature, function and evolution of the oak genome.

High-density linkage mapping in a pine tree reveals a genomic region associated with inbreeding depression and provides clues to the extent and distribution of meiotic recombination

BackgroundThe availability of a large expressed sequence tags (EST) resource and recent advances in high-throughput genotyping technology have made it possible to develop highly multiplexed SNP arrays for multi-objective genetic applications, including the construction of meiotic maps. Such approaches are particularly useful in species with a large genome size, precluding the use of whole-genome shotgun assembly with current technologies.ResultsIn this study, a 12 k-SNP genotyping array was developed for maritime pine from an extensive EST resource assembled into a unigene set. The offspring of three-generation outbred and inbred mapping pedigrees were then genotyped. The inbred pedigree consisted of a classical F2 population resulting from the selfing of a single inter-provenance (Landes x Corsica) hybrid tree, whereas the outbred pedigree (G2) resulted from a controlled cross of two intra-provenance (Landes x Landes) hybrid trees. This resulted in the generation of three linkage maps based on SNP markers: one from the parental genotype of the F2 population (1,131 markers in 1,708 centimorgan (cM)), and one for each parent of the G2 population (1,015 and 1,110 markers in 1,447 and 1,425 cM for the female and male parents, respectively). A comparison of segregation patterns in the progeny obtained from the two types of mating (inbreeding and outbreeding) led to the identification of a chromosomal region carrying an embryo viability locus with a semi-lethal allele. Following selfing and segregation, zygote mortality resulted in a deficit of Corsican homozygous genotypes in the F2 population. This dataset was also used to study the extent and distribution of meiotic recombination along the length of the chromosomes and the effect of sex and/or genetic background on recombination. The genetic background of trees in which meiotic recombination occurred was found to have a significant effect on the frequency of recombination. Furthermore, only a small proportion of the recombination hot- and cold-spots were common to all three genotypes, suggesting that the spatial pattern of recombination was genetically variable.ConclusionThis study led to the development of classical genomic tools for this ecologically and economically important species. It also identified a chromosomal region bearing a semi-lethal recessive allele and demonstrated the genetic variability of recombination rate over the genome.

Molecular and phenotypic profiling from the base to the crown in maritime pine wood‐forming tissue

Environmental, developmental and genetic factors affect variation in wood properties at the chemical, anatomical and physical levels. Here, the phenotypic variation observed along the tree stem was explored and the hypothesis tested that this variation could be the result of the differential expression of genes/proteins during wood formation. Differentiating xylem samples of maritime pine (Pinus pinaster) were collected from the top (crown wood, CW) to the bottom (base wood, BW) of adult trees. These samples were characterized by Fourier transform infrared spectroscopy (FTIR) and analytical pyrolysis. Two main groups of samples, corresponding to CW and BW, could be distinguished from cell wall chemical composition. A genomic approach, combining large-scale production of expressed sequence tags (ESTs), gene expression profiling and quantitative proteomics analysis, allowed identification of 262 unigenes (out of 3512) and 231 proteins (out of 1372 spots) that were differentially expressed along the stem. A good relationship was found between functional categories from transcriptomic and proteomic data. A good fit between the molecular mechanisms involved in CW-BW formation and these two types of wood phenotypic differences was also observed. This work provides a list of candidate genes for wood properties that will be tested in forward genetics.

Heat induced changes in protein expression profiles of Norway spruce (Picea abies) ecotypes from different elevations.

Although tree species typically exhibit low genetic differentiation between populations, ecotypes adapted to different environmental conditions can vary in their capacity to withstand and recover from environmental stresses like heat stress. Two month old seedlings of a Picea abies ecotype adapted to high elevation showed lower level of thermotolerance and higher level of tolerance to oxidative stress relative to a low elevation ecotype. Protein expression patterns following exposure to severe heat stress of the two ecotypes were compared by means of 2‐DE. Several proteins exhibiting ecotype and tissue specific expression were identified by MS/MS. Among them, small heat shock proteins of the HSP 20 family and proteins involved in protection from oxidative stress displayed qualitative and quantitative differences in expression between the ecotypes correlated with the observed phenotypic differences. On the basis of these results, it can be speculated that the observed interpopulation polymorphism of protein regulation in response to heat stress could underlie their different capacities to withstand and recover from heat stress. These local adaptations are potentially relevant for the species adaptation to the conditions predicted by the current models for climate change.

Soil water stress affects both cuticular wax content and cuticle-related gene expression in young saplings of maritime pine (Pinus pinaster Ait)

BackgroundThe cuticle is a hydrophobic barrier located at the aerial surface of all terrestrial plants. Recent studies performed on model plants, such as Arabidopsis thaliana, have suggested that the cuticle may be involved in drought stress adaptation, preventing non-stomatal water loss. Although forest trees will face more intense drought stresses (in duration and intensity) with global warming, very few studies on the role of the cuticle in drought stress adaptation in these long-lived organisms have been so far reported.ResultsThis aspect was investigated in a conifer, maritime pine (Pinus pinaster Ait.), in a factorial design with two genetic units (two half-sib families with different growth rates) and two treatments (irrigated vs non-irrigated), in field conditions. Saplings were grown in an open-sided greenhouse and half were irrigated three times per week for two growing seasons. Needles were sampled three times per year for cuticular wax (composition and content) and transcriptome (of 11 genes involved in cuticle biosynthesis) analysis. Non-irrigated saplings (i) had a higher cuticular wax content than irrigated saplings and (ii) overexpressed most of the genes studied. Both these trends were more marked in the faster growing family.ConclusionsThe higher cuticular wax content observed in the non-irrigated treatment associated with strong modifications in products from the decarbonylation pathway suggest that cuticular wax may be involved in drought stress adaptation in maritime pine. This study provides also a set of promising candidate genes for future forward genetic studies in conifers.

(Not) Keeping the stem straight: a proteomic analysis of maritime pine seedlings undergoing phototropism and gravitropism

BackgroundPlants are subjected to continuous stimuli from the environment and have evolved an ability to respond through various growth and development processes. Phototropism and gravitropism responses enable the plant to reorient with regard to light and gravity.ResultsWe quantified the speed of maritime pine seedlings to reorient with regard to light and gravity over 22 days. Seedlings were inclined at 15, 30 and 45 degrees with vertical plants as controls. A lateral light source illuminated the plants and stem movement over time was recorded. Depending on the initial angle of stem lean, the apical response to the lateral light source differed. In control and 15° inclined plants, the apex turned directly towards the light source after only 2 h. In plants inclined at 30° and 45°, the apex first reoriented in the vertical plane after 2 h, then turned towards the light source after 24 h. Two-dimensional gel electrophoresis coupled with mass spectrometry was then used to describe the molecular response of stem bending involved in photo- and gravi-tropism after 22 hr and 8 days of treatment. A total of 486 spots were quantitatively analyzed using image analysis software. Significant changes were determined in the protein accumulation of 68 protein spots. Early response gravitropic associated proteins were identified, which are known to function in energy related and primary metabolism. A group of thirty eight proteins were found to be involved in primary metabolism and energy related metabolic pathways. Degradation of Rubisco was implicated in some protein shifts.ConclusionsOur study demonstrates a rapid gravitropic response in apices of maritime pine seedlings inclined >30°. Little or no response was observed at the stem bases of the same plants. The primary gravitropic response is concomitant with a modification of the proteome, consisting of an over accumulation of energy and metabolism associated proteins, which may allow the stem to reorient rapidly after bending.

Differential accumulation of soluble proteins in roots of metallicolous and nonmetallicolous populations of Agrostis capillaris L. exposed to Cu

Differential expression of soluble proteins was explored in roots of metallicolous (M) and non‐M (NM) plants of Agrostis capillaris L. exposed to increasing Cu to partially identify molecular mechanisms underlying higher Cu tolerance in M plants. Plants were cultivated for 2 months on perlite with a CuSO4 (1–30 μM) spiked‐nutrient solution. Soluble proteins extracted by the trichloroacetic acid/acetone procedure were separated with 2DE (linear 4–7 pH gradient). After Coomassie Blue staining and image analysis, 19 proteins differentially expressed were identified using LC‐MS/MS and Expressed Sequence Tag (ESTs) databases. At supra‐optimal Cu exposure (15–30 μM), glycolysis was likely altered in NM roots with increased production of glycerone‐P and methylglyoxal based on overexpression of triosephosphate isomerase and fructose bisphosphate aldolase. Changes in tubulins and higher expressions of 5‐methyltetrahydropteroyltriglutamatehomocysteine methyltransferase and S‐adenosylmethionine synthase underpinned impacts on the cytoskeleton and stimulation of ethylene metabolism. Increased l‐methionine and S‐adenosylmethionine amounts may also facilitate production of nicotianamine, which complexes Cu, and of l‐cysteine, needed for metallothioneins and GSH. In M roots, the increase of [Cu/Zn] superoxide dismutase suggested a better detoxification of superoxide, when Cu exposure rose. Higher Cu‐tolerance of M plants would rather result from simultaneous cooperation of various processes than from a specific mechanism.

Analysis of the distribution of marker classes in a genetic linkage map: a case study in Norway spruce (Picea abies karst)

In order to analyze the large-scale structure of the genome of Norway spruce (Picea abies Karst.), a pseudo-testcross genetic linkage map was built using markers of six different types, belonging to the low (amplified fragment length polymorphisms, simple sequence repeats) or high (sequence-specific amplified polymorphisms, inter-retrotransposon amplified polymorphisms) copy-number fraction of the genome, and including expressed region-derived markers (expressed sequence tag polymorphisms). Twenty seven and 23 linkage groups of at least four markers were obtained for the female and the male parent maps, respectively. A subset of these linkage groups coalesced into 13 bi-parental linkage groups through markers shared between the two maps. This map was used to investigate the frequency of each marker type over chromosomes and the distribution of marker types relative to each other, using autocorrelation techniques. Our results show that, while the composition of chromosomes is homogeneous, low- and high-copy-number markers tend to occupy separate regions of the linkage groups, and that expressed sequences are preferentially associated with microsatellites and separated from retrotransposons. These results indicate that the spatial structure of Norway spruce chromosomes is not homogeneous.

Protein Polymorphism between 2 Picea abies Populations Revealed by 2-Dimensional Gel Electrophoresis and Tandem Mass Spectrometry

In species with high gene flow and consequent low interpopulation differentiation over wide geographic ranges, differential gene expression along ecological gradients often reveals adaptive significance. We investigated potential differences in protein expression between Picea abies ecotypes adapted to contrasting altitude conditions. Protein expression patterns were compared between needles and roots of 2-month-old P. abies seedlings by means of 2-dimensional electrophoresis. Proteins exhibiting differential expression between the 2 ecotypes were analyzed by tandem mass spectrometry. A total of 19 proteins exhibited qualitative or quantitative polymorphism between the 2 populations. These proteins exhibited organ-specific expression, and the level of interpopulation protein polymorphism was organ dependent. Among differentially expressed proteins, we identified proteins involved in photosynthesis, photorespiration, root tracheary element differentiation, and transmitochondrial membrane transport. Our results show that P. abies seedlings from locally adapted ecotypes exhibit consistent differences in protein expression. The expression polymorphism of some of these proteins has potential adaptive significance.