Véronique Jorge

Bud set in poplar – genetic dissection of a complex trait in natural and hybrid populations

• The seasonal timing of growth events is crucial to tree distribution and conservation. The seasonal growth cycle is strongly adapted to the local climate that is changing because of global warming. We studied bud set as one cornerstone of the seasonal growth cycle in an integrative approach. • Bud set was dissected at the phenotypic level into several components, and phenotypic components with most genetic variation were identified. While phenotypic variation resided in the timing of growth cessation, and even so more in the duration from growth cessation to bud set, the timing of growth cessation had a stronger genetic component in both natural and hybrid populations. • Quantitative trait loci (QTL) were identified for the most discriminative phenotypic bud-set components across four poplar pedigrees. The QTL from different pedigrees were recurrently detected in six regions of the poplar genome. • These regions of 1.83-4.25 Mbp in size, containing between 202 and 394 genes, form the basis for further molecular-genetic dissection of bud set.

Genetic linkage maps of Populus alba L. and comparative mapping analysis of sex determination across Populus species.

White poplar (Populus alba L.) is native to Eurasia and is unexploited for its growth potential and stress-adaptive mechanisms. A better knowledge of its genome will allow for more effective protection and use of critical genetic resources. The main objective of this study was the construction of highly informative P. alba genetic maps. Two genotypes were selected from contrasting natural Italian populations and crossed to generate an F1 mapping pedigree. Amplified fragment length polymorphism and simple sequence repeat markers were used to genotype 141 F1 individuals. The pseudo-testcross strategy was applied for linkage analysis. The generated maps showed good overall colinearity to each other and allowed for a complete alignment with the 19 haploid chromosomes of the Populus genome sequence. The locus that determines sex as a morphological trait was positioned on a non-terminal position of LG XIX of the female parent map. Comparison among Populus species revealed differences in the location of the sex locus on LG XIX as well as inconsistencies in the heterogametic sex. The genetic analysis of the sex locus in P. alba provides insights into sex determination in the genus and is useful for the identification of sex-linked markers and the early assessment of plant gender. Furthermore, these genetic maps will greatly facilitate the study of the genomics of Populus and how it can be exploited in applied breeding programs.

Integrating genome annotation and QTL position to identify candidate genes for productivity, architecture and water-use efficiency in Populus spp

BackgroundHybrid poplars species are candidates for biomass production but breeding efforts are needed to combine productivity and water use efficiency in improved cultivars. The understanding of the genetic architecture of growth in poplar by a Quantitative Trait Loci (QTL) approach can help us to elucidate the molecular basis of such integrative traits but identifying candidate genes underlying these QTLs remains difficult. Nevertheless, the increase of genomic information together with the accessibility to a reference genome sequence (Populus trichocarpa Nisqually-1) allow to bridge QTL information on genetic maps and physical location of candidate genes on the genome. The objective of the study is to identify QTLs controlling productivity, architecture and leaf traits in a P. deltoides x P. trichocarpa F1 progeny and to identify candidate genes underlying QTLs based on the anchoring of genetic maps on the genome and the gene ontology information linked to genome annotation. The strategy to explore genome annotation was to use Gene Ontology enrichment tools to test if some functional categories are statistically over-represented in QTL regions.ResultsFour leaf traits and 7 growth traits were measured on 330 F1 P. deltoides x P. trichocarpa progeny. A total of 77 QTLs controlling 11 traits were identified explaining from 1.8 to 17.2% of the variation of traits. For 58 QTLs, confidence intervals could be projected on the genome. An extended functional annotation was built based on data retrieved from the plant genome database Phytozome and from an inference of function using homology between Populus and the model plant Arabidopsis. Genes located within QTL confidence intervals were retrieved and enrichments in gene ontology (GO) terms were determined using different methods. Significant enrichments were found for all traits. Particularly relevant biological processes GO terms were identified for QTLs controlling number of sylleptic branches: intervals were enriched in GO terms of biological process like ‘ripening’ and ‘adventitious roots development’.ConclusionBeyond the simple identification of QTLs, this study is the first to use a global approach of GO terms enrichment analysis to fully explore gene function under QTLs confidence intervals in plants. This global approach may lead to identification of new candidate genes for traits of interest.

The obscure events contributing to the evolution of an incipient sex chromosome in Populus: a retrospective working hypothesis

Genetic determination of gender is a fundamental developmental and evolutionary process in plants. Although it appears that dioecy in Populus is genetically controlled, the precise gender-determining systems remain unclear. The recently released second draft assembly and annotated gene set of the Populus genome provided an opportunity to revisit this topic. We hypothesized that over evolutionary time, selective pressure has reformed the genome structure and gene composition in the peritelomeric region of the chromosome XIX, which has resulted in a distinctive genome structure and cluster of genes contributing to gender determination in Populus trichocarpa. Multiple lines of evidence support this working hypothesis. First, the peritelomeric region of the chromosome XIX contains significantly fewer single nucleotide polymorphisms than the rest of Populus genome and has a distinct evolutionary history. Second, the peritelomeric end of chromosome XIX contains the largest cluster of the nucleotide-binding site–leucine-rich repeat (NBS–LRR) class of disease resistance genes in the entire Populus genome. Third, there is a high occurrence of small microRNAs on chromosome XIX, which is coincident to the region containing the putative gender-determining locus and the major cluster of NBS–LRR genes. Further, by analyzing the metabolomic profiles of floral bud in male and female Populus trees using a gas chromatography-mass spectrometry, we found that there are gender-specific accumulations of phenolic glycosides. Taken together, these findings led to the hypothesis that resistance to and regulation of a floral pathogen and gender determination coevolved, and that these events triggered the emergence of a nascent sex chromosome. Further studies of chromosome XIX will provide new insights into the genetic control of gender determination in Populus.

Identification of quantitative trait loci affecting ectomycorrhizal symbiosis in an interspecific F1 poplar cross and differential expression of genes in ectomycorrhizas of the two parents: Populus deltoides and Populus trichocarpa

A Populus deltoides × Populus trichocarpa F1 pedigree was analyzed for quantitative trait loci (QTLs) affecting ectomycorrhizal development and for microarray characterization of gene networks involved in this symbiosis. A 300 genotype progeny set was evaluated for its ability to form ectomycorrhiza with the basidiomycete Laccaria bicolor. The percentage of mycorrhizal root tips was determined on the root systems of all 300 progeny and their two parents. QTL analysis identified four significant QTLs, one on the P. deltoides and three on the P. trichocarpa genetic maps. These QTLs were aligned to the P. trichocarpa genome and each contained several megabases and encompass numerous genes. NimbleGen whole-genome microarray, using cDNA from RNA extracts of ectomycorrhizal root tips from the parental genotypes P. trichocarpa and P. deltoides, was used to narrow the candidate gene list. Among the 1,543 differentially expressed genes (p value ≤ 0.05; ≥5.0-fold change in transcript level) having different transcript levels in mycorrhiza of the two parents, 41 transcripts were located in the QTL intervals: 20 in Myc_d1, 14 in Myc_t1, and seven in Myc_t2, while no significant differences among transcripts were found in Myc_t3. Among these 41 transcripts, 25 were overrepresented in P. deltoides relative to P. trichocarpa; 16 were overrepresented in P. trichocarpa. The transcript showing the highest overrepresentation in P. trichocarpa mycorrhiza libraries compared to P. deltoides mycorrhiza codes for an ethylene-sensitive EREBP-4 protein which may repress defense mechanisms in P. trichocarpa while the highest overrepresented transcripts in P. deltoides code for proteins/genes typically associated with pathogen resistance.

Characterization of the Poplar Pan-Genome by Genome-Wide Identification of Structural Variation

Many recent studies have emphasized the important role of structural variation (SV) in determining human genetic and phenotypic variation. In plants, studies aimed at elucidating the extent of SV are still in their infancy. Evidence has indicated a high presence and an active role of SV in driving plant genome evolution in different plant species. With the aim of characterizing the size and the composition of the poplar pan-genome, we performed a genome-wide analysis of structural variation in three intercrossable poplar species: Populus nigra, Populus deltoides, and Populus trichocarpa. We detected a total of 7,889 deletions and 10,586 insertions relative to the P. trichocarpa reference genome, covering respectively 33.2 Mb and 62.9 Mb of genomic sequence, and 3,230 genes affected by copy number variation (CNV). The majority of the detected variants are inter-specific in agreement with a recent origin following separation of species. Insertions and deletions (INDELs) were preferentially located in low-gene density regions of the poplar genome and were, for the majority, associated with the activity of transposable elements. Genes affected by SV showed lower-than-average expression levels and higher levels of dN/dS, suggesting that they are subject to relaxed selective pressure or correspond to pseudogenes. Functional annotation of genes affected by INDELs showed over-representation of categories associated with transposable elements activity, while genes affected by genic CNVs showed enrichment in categories related to resistance to stress and pathogens. This study provides a genome-wide catalogue of SV and the first insight on functional and structural properties of the poplar pan-genome.

Potential for marker-assisted selection for forest tree breeding: lessons from 20 years of MAS in crops

For the most part, molecular markers and detection of quantitative trait loci have been developed for forest tree species in view to performing marker-assisted selection (MAS). However, MAS has not been applied to forest trees until now. In parallel, some success stories of MAS in crop breeding have been reported. Recently, genotyping techniques have undergone a tremendous increase in throughput, moving the trend from MAS to genomic selection. We analyzed 250 papers reporting the use of MAS in plant breeding and found that the most popular schemes used were gene pyramiding and marker-assisted backcross manipulating a single or very few genomic regions which have a major impact on crop value. We reviewed theoretical and simulation studies to identify the parametric space in which MAS is expected to bring about significant advantages over phenotypic selection. Then, we tried to explain why MAS has not been applied to forest trees and discuss the opportunities offered by recent advances in these species.

Qualitative and quantitative resistances to leaf rust finely mapped within two nucleotide-binding site leucine-rich repeat (NBS-LRR)-rich genomic regions of chromosome 19 in poplar.

• R(US) is a major dominant gene controlling quantitative resistance, inherited from Populus trichocarpa, whereas R(1) is a gene governing qualitative resistance, inherited from P. deltoides. • Here, we report a reiterative process of concomitant fine-scale genetic and physical mapping guided by the P. trichocarpa genome sequence. The high-resolution linkage maps were developed using a P. deltoides × P. trichocarpa progeny of 1415 individuals. R(US) and R(1) were mapped in a peritelomeric region of chromosome 19. Markers closely linked to R(US) were used to screen a bacterial artificial chromosome (BAC) library constructed from the P. trichocarpa parent, heterozygous at the locus R(US) . • Two local physical maps were developed, one encompassing the R(US) allele and the other spanning r(US) . The alignment of the two haplophysical maps showed structural differences between haplotypes. The genetic and physical maps were anchored to the genome sequence, revealing genome sequence misassembly. Finally, the R(US) locus was localized within a 0.8-cM interval, whereas R(1) was localized upstream of R(US) within a 1.1-cM interval. • The alignment of the genetic and physical maps with the local reorder of the chromosome 19 sequence indicated that R(US) and R(1) belonged to a genomic region rich in nucleotide-binding site leucine-rich repeat (NBS-LRR) and serine threonine kinase (STK) genes.

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.

Sequence Imputation from Low Density Single Nucleotide Polymorphism Panel in a Black Poplar Breeding population

Background Genomic selection accuracy increases with the use of high SNP (single nucleotide polymorphism) coverage. However, such gains in coverage come at high costs, preventing their operational implementation by breeders. Low density panels imputed to higher densities offer a cheaper alternative. Our study is one of the first to explore the imputation in a tree species: black poplar. About 1000 pure-breed Populus nigra trees corresponding to a subsample of the French breeding population were selected and genotyped with a 12K custom Infinium Bead-Chip. Forty-three of those individuals corresponding mostly to nodal trees in the pedigree were fully sequenced (reference), while the remaining majority (target) was imputed from 8K to 1.4 million SNPs using FImpute. Each SNP and individual was evaluated for imputation errors by leave-one-out cross validation in the training sample of 43 sequenced trees. Some summary statistics such as Hardy Weinberg Equilibrium exact test p-value, quality of sequencing, depth of sequencing per site and per individual, minor allele frequency, marker density ratio or SNP information redundancy were calculated. Principal component and Boruta analyses were used on all these parameters to rank the factors affecting the quality of imputation. Additionally, we characterize the impact of the relatedness between reference population and target population. Results During the imputation process, we used 7,540 SNPs from the chip to impute 1,438,827 SNPs from sequences along the 19 Chromosomes. At the individual level, imputation accuracy was very high with a proportion of SNPs correctly imputed between 0.84 and 0.99. The variation in accuracies was mostly due to differences in relatedness between individuals. At a SNP level, the imputation quality strongly depended on genotyped SNP density and to a lesser extent on the original minor allele frequency. The imputation did not appear to result in an increase of linkage disequilibrium. The genotype densification not only brought a better distribution of markers all along the genome, but also we did not detect any substantial bias in annotation categories. Conclusions This study shows that it is possible to impute low-density marker panels to whole genome sequence with good accuracy under certain conditions that could be common to many breeding populations.