New resources for genetic studies in Populus nigra: genome-wide SNP discovery and development of a 12k Infinium array.

Whole genome resequencing of 51 Populus nigra (L.) individuals from across Western Europe was performed using Illumina platforms. A total number of 1 878 727 SNPs distributed along the P. nigra reference sequence were identified. The SNP calling accuracy was validated with Sanger sequencing. SNPs were selected within 14 previously identified QTL regions, 2916 expressional candidate genes related to rust resistance, wood properties, water-use efficiency and bud phenology and 1732 genes randomly spread across the genome. Over 10 000 SNPs were selected for the construction of a 12k Infinium Bead-Chip array dedicated to association mapping. The SNP genotyping assay was performed with 888 P. nigra individuals. The genotyping success rate was 91%. Our high success rate was due to the discovery panel design and the stringent parameters applied for SNP calling and selection. In the same set of P. nigra genotypes, linkage disequilibrium throughout the genome decayed on average within 5-7 kb to half of its maximum value. As an application test, ADMIXTURE analysis was performed with a selection of 600 SNPs spread throughout the genome and 706 individuals collected along 12 river basins. The admixture pattern was consistent with genetic diversity revealed by neutral markers and the geographical distribution of the populations. These newly developed SNP resources and genotyping array provide a valuable tool for population genetic studies and identification of QTLs through natural-population based genetic association studies in P. nigra.

Genetic architecture of qualitative and quantitative Melampsora larici-populina leaf rust resistance in hybrid poplar: genetic mapping and QTL detection.

In order to elucidate the genetic control of resistance to Melampsora larici-populina leaf rust in hybrid poplars, a Populus deltoides x P. trichocarpa F(1) progeny was analysed for qualitative and quantitative rust resistances. This progeny was evaluated for three components of quantitative resistance (latent period, uredinia number and uredinia size) to seven M. larici-populina strains in controlled conditions, and for one component of field susceptibility (rust colonization on the most infected leaf). One qualitative resistance locus inherited from P. deltoides, R(1), was localized on the genetic map. It segregates 1 : 1 in the F(1) progeny and is effective against four of the studied strains. QTL analysis was performed separately on R(1) and r(1) genotype subsets. An additional detection was conducted on the entire F(1) progeny for the three strains able to overcome R(1) and for MAX2. A total of nine QTLs were detected. Two had large, broad-spectrum effects. One (R(US)) is inherited from the P. trichocarpa parent; the other is inherited from P. deltoides and colocalized with R(1). Seven QTLs had only limited and specific effects. Significant interaction effects were detected mainly between the two major QTLs. Implications of these results for durable resistance breeding strategies, and possible benefits from the Populus genome sequence, are discussed.

Genetic analysis of phenotypic variation for ectomycorrhiza formation in an interspecific F1 poplar full-sib family.

A plant's capability to develop ectomycorrhizal symbiosis is under the control of both genetic and environmental factors. In order to determine the roles played by these different factors, we have performed a quantitative genetic analysis of the ability of poplar trees to form ectomycorrhizas. Quantitative genetics were applied to an interspecific family of poplar for which the two parental genetic maps had already been described, and for which data analyses concerning fungal aggressors were obtained. Quantitative trait loci (QTL) related to ectomycorrhiza formation were identified and located in the genetic maps of the two parents. One QTL was located at a linkage group of the genetic map of Populus trichocarpa showing a high concentration of several QTL involved in the pathogenic interaction with the fungus Melampsora larici-populina, the causal agent of leaf rust.

A full saturated linkage map of Picea abies including AFLP, SSR, ESTP, 5S rDNA and morphological markers.

Based on an F(1) progeny of 73 individuals, two parental maps were constructed according to the double pseudo-test cross strategy. The paternal map contained 16 linkage groups for a total genetic length of 1,792 cM. The maternal map covered 1,920 cM, and consisted of 12 linkage groups. These parental maps were then integrated using 66 intercross markers. The resulting consensus map covered 2,035 cM and included 755 markers (661 AFLPs, 74 SSRs, 18 ESTPs, the 5S rDNA and the early cone formation trait) on 12 linkage groups, reflecting the haploid number of chromosomes of Picea abies. The average spacing between two adjacent markers was 2.6 cM. The presence of 39 of the SSR and/or ESTP markers from this consensus map on other published maps of different Picea and Pinus species allowed us to establish partial linkage group homologies across three P. abies maps (up to five common markers per linkage group). This first saturated linkage map of P. abies could be therefore used as a support for developing comparative genome mapping in conifers.

Chloroplast and mitochondrial molecular tests identify European x Japanese larch hybrids.

Hybrids between European and Japanese larches combine the properties of both parental species (drought resistance, canker resistance, stem straightness) and exhibit a fast growth rate. They are produced in seed orchards, generally by natural pollination. Seeds are collected and used for afforestation as interspecific hybrids. However, there are no convenient tests to assess the interspecific hybrid proportion. In the present study, we developed diagnostic molecular markers suitable for the individual identification of hybrids, whatever their developmental stage. Our strategy involved testing a combination of maternally inherited markers from the mitochondrial genome (mtDNA) and paternally inherited markers from the chloroplast genome (cpDNA). Hybrids were then identified by the presence of a mitochondrial sequence inherited from one parental species and a chloroplast sequence inherited from the other parental species. To achieve this aim, markers discriminating both parental species were first sought. Amplifications of mitochondrial and chloroplast sequences were performed using specific PCR primers. After testing 33 primer pairs in combination with nine restriction enzymes, we detected one mitochondrial marker, f13 which was amplified in Japanese larch and absent in European larch, and one chloroplast marker, ll- TaqI which showed different restriction patterns depending on the species. A restriction fragment of 601 bp was obtained in Japanese larch while two fragments of 120 bp and 481 bp were observed in European larch. These patterns were found in all 197 individuals tested from the two pure species. These markers were then used for the evaluation of the hybrid proportion in a seed lot produced from seed orchards; this was assessed as between 43% and 53% depending on the parental species. The male and female parental species could be determined for each progeny.

A Single Gene Cluster Controls Incompatibility and Partial Resistance to Various Melampsora larici-populina Races in Hybrid Poplars.

ABSTRACT Complete cosegregation for race-specific incompatibility with three Melampsora larici-populina rust races was observed in five F(1) hybrid progenies of Populus, with different patterns among the various progenies. A single gene cluster could explain these segregations: one locus with multiple alleles or two tightly linked loci controlling complete resistance to E1 and E3, and two tightly linked loci for E2. The random amplified polymorphic DNA marker OPM03/04_480 was linked to that cluster in all families (<1 cM). This marker accounted for more than 70% of the genetic variation for field resistance in each family (heritability approximately 0.40). The same marker accounted for up to 64% of the clonal variation for growth in the nursery under natural inoculum pressure; the weak tolerance to rust of F(1) interspecific hybrids was attributed to a genetic background effect. Partial resistance was split into epidemiological components (heritability ranged from 0.35 to 0.87). Genotypic correlations among resistance traits for the different races were high (0.73 to 0.90). However, correlations among different resistance components for a single race were not all significant. A major quantitative trait locus for all components of partial resistance to E2 was associated to the cluster controlling incompatibility to E1 and E3 and marked by OPM03/04_480 (R(2)from 48 to 68%).

Detection of a variable number of ribosomal DNA loci by fluorescent in situ hybridization in Populus species.

Fluorescent in situ hybridization (FISH) was applied to related Populus species (2n = 19) in order to detect rDNA loci. An interspecific variability in the number of hybridization sites was revealed using as probe an homologous 25S clone from Populus deltoides. The application of image analysis methods to measure fluorescence intensity of the hybridization signals has enabled us to characterize major and minor loci in the 18S-5.8S-25S rDNA. We identified one pair of such rDNA clusters in Populus alba; two pairs, one major and one minor, in both Populus nigra and P. deltoides; and three pairs in Populus balsamifera, (two major and one minor) and Populus euroamericana (one major and two minor). FISH results are in agreement with those based on RFLP analysis. The pBG13 probe containing 5S sequence from flax detected two separate clusters corresponding to the two size classes of units that coexist within 5S rDNA of most Populus species. Key words : Populus spp., fluorescent in situ hybridization, FISH, rDNA variability, image analysis.

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 Jaccard's coefficient between parents. Thirteen fragments differentiated the larch species. The genetic distance between individuals of the same species (D J =0.39 in the Japanese larch and 0.45 in the European larch) was lower than the genetic distance between species (D J =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.

Rapid control of purity for the cytoplasm of male-sterile seed stocks by means of a dot hybridization assay.

To produce hybrids, one member of the parental line is genetically made male-sterile. This male-sterile trait is encoded by mitochondria so that it is maternally inherited. Consequently, the progeny of a male-sterile plant is fully sterile. Nevertheless, during the handling of cytoplasmic male-sterile seed stocks, some mixture with seeds of the maintainer lines can occur. Up to the present time, the only way to check the homogeneity of the cytoplasmic male-sterile seed stock was to grow the plants until flowering time. We have developed a method which can be used immediately after the harvest, allowing us to check samples from both sunflower and sugar beet. We used the mitochondrial plasmid, present only in the maintainer lines, as a probe for the total nucleic acids prepared from the cytoplasmic male-sterile seed stocks which might be contaminated. The signals compared to those of samples artificially contaminated allow us to measure as few as one male-fertile seed in 1000 seeds in a rapid and accurate manner.