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Oaks are an important part of our natural and cultural heritage. Not only are they ubiquitous in our most common landscapes1 but they have also supplied human societies with invaluable services, including food and shelter, since prehistoric times2. With 450 species spread throughout Asia, Europe and America3, oaks constitute a critical global renewable resource. The longevity of oaks (several hundred years) probably underlies their emblematic cultural and historical importance. Such long-lived sessile organisms must persist in the face of a wide range of abiotic and biotic threats over their lifespans. We investigated the genomic features associated with such a long lifespan by sequencing, assembling and annotating the oak genome. We then used the growing number of whole-genome sequences for plants (including tree and herbaceous species) to investigate the parallel evolution of genomic characteristics potentially underpinning tree longevity. A further consequence of the long lifespan of trees is their accumulation of somatic mutations during mitotic divisions of stem cells present in the shoot apical meristems. Empirical4 and modelling5 approaches have shown that intra-organismal genetic heterogeneity can be selected for6 and provides direct fitness benefits in the arms race with short-lived pests and pathogens through a patchwork of intra-organismal phenotypes7. However, there is no clear proof that large-statured trees consist of a genetic mosaic of clonally distinct cell lineages within and between branches. Through this case study of oak, we demonstrate the accumulation and transmission of somatic mutations and the expansion of disease-resistance gene families in trees.

Oaks are an important part of our natural and cultural heritage. Not only are they ubiquitous in our most common landscapes' but they have also supplied human societies with invaluable services, including food and shelter, since prehistoric times(2). With 450 species spread throughout Asia, Europe and America(3), oaks constitute a critical global renewable resource. The longevity of oaks (several hundred years) probably underlies their emblematic cultural and historical importance. Such long-lived sessile organisms must persist in the face of a wide range of abiotic and biotic threats over their lifespans. We investigated the genomic features associated with such a long lifespan by sequencing, assembling and annotating the oak genome. We then used the growing number of whole-genome sequences for plants (including tree and herbaceous species) to investigate the parallel evolution of genomic characteristics potentially underpinning tree longevity. A further consequence of the long lifespan of trees is their accumulation of somatic mutations during mitotic divisions of stem cells present in the shoot apical meristems. Empirical(4) and modelling(5) approaches have shown that intra-organismal genetic heterogeneity can be selected for(6) and provides direct fitness benefits in the arms race with short-lived pests and pathogens through a patchwork of intra-organismal phenotypes(7). However, there is no clear proof that large-statured trees consist of a genetic mosaic of clonally distinct cell lineages within and between branches. Through this case study of oak, we demonstrate the accumulation and transmission of somatic mutations and the expansion of disease-resistance gene families in trees.

Neighbor joining tree of the indica panel. The colors corresponds to the subpopulations defined by Structure. The accessions in black are admixed (PPTX 163Â kb)

AGAP : équipe ID / GE2pop; Leucine-Rich Repeats Receptor-Like Kinase (LRR-RLK) genes represent a large and complex gene family in plants, mainly involved in development and stress responses. These receptors are composed of an LRR-containing extracellular domain (ECD), a transmembrane domain(TM) and an intracellular kinase domain (KD). To provide new perspectives on functional analyses of these genes in model and non-model plant species, we performed a phylogeneticanalys is on 8,360 LRR-RLK receptors in 31 angiosperm genomes (8 monocots and 23dicots). We identified 101 orthologous groups (OGs) of genes being conserved among almost all monocot and dicot species analyzed. We observed that more than 10% of these OGs are absent in the Brassicaceae species studied. We show that the ECD structural features are not always conserved among orthologs, suggesting that functions may have diverged in some OG sets. Moreover, we looked at targets of positive selection footprints in 12 pairs of OGs and noticed that depending on the subgroups, positive selection occurred more frequently either in the ECDs or in the KDs.

Background Plant root systems play a major role in anchoring and in water and nutrient uptake from the soil. The root cone angle is an important parameter of the root system architecture because, combined with root depth, it helps to determine the volume of soil explored by the plant. Two genes, DRO1 and SOR1, and several QTLs for root cone angle have been discovered in the last 5 years. Results To find other QTLs linked to root cone angle, a genome-wide association mapping study was conducted on two panels of 162 indica and 169 japonica rice accessions genotyped with two sets of SNP markers (genotyping-by-sequencing set with approximately 16,000 markers and high-density-rice-array set with approximately 300,000 markers). The root cone angle of all accessions was measured using a screen protractor on images taken after 1 month of plant growth in the Rhizoscope phenotyping system. The distribution of the root cone angle in the indica panel was Gaussian, but several accessions of the japonica panel (all the bulus from Indonesia and three temperate japonicas from Nepal or India) appeared as outliers with a very wide root cone angle. The data were submitted to association mapping using a mixed model with control of structure and kinship. A total of 15 QTLs for the indica panel and 40 QTLs for the japonica panel were detected. Genes underlying these QTLs (+/−50 kb from the significant markers) were analyzed. We focused our analysis on auxin-related genes, kinases, and genes involved in root developmental processes and identified 8 particularly interesting genes. Conclusions The present study identifies new sources of wide root cone angle in rice, proposes ways to bypass some drawbacks of association mapping to further understand the genetics of the trait and identifies candidate genes deserving further investigation. Electronic supplementary material The online version of this article (10.1186/s12284-017-0184-z) contains supplementary material, which is available to authorized users.

Neighbor joining tree of the japonica panel. The colors corresponds to the subpopulations defined by Structure. The accessions in black are admixed (PPTX 158Â kb)

Rice WAKs regulated by blast fungus infection. (PDF 35 kb)

Fungal growth in OE- WAK91 lines. WAK91 over-expresser lines were inoculated with M. oryzae (GY11 moderately virulent isolate) and at the indicated time points after inoculation, the development stage of the fungus was observed under the microscope; four categories of growth stages were counted (100 interaction sites/condition). This experiment was repeated 3 times and significant differences (t-test; Pâ

Disease symptoms in T0 transgenic plants over-expressing OsWAK91 or OsWAK112d. Unique rice T0 plants over-expressing OsWAK91 (A, B, C; 5 lines starting with “OE”) or OsWAK112d (D, E, F; 6 lines starting with “OX”) were produced. Plants transformed with the empty vector are also shown (A; 4 lines starting with “EV”). The transgene expression level was normalized using Actin and is expressed as Arbitrary Unit (A, C). The values presented are unique values since each represents a unique T0 plant. Given the extremely low expression levels and thus variability in empty vectors, the very high values in over-expressor lines was considered as significant. Plants were inoculated with the virulent isolate FR13 of Magnaporthe oryzae and lesion number was quantified 7 dpi (B, E). Examples of symptoms are also shown (C, F). The experiments in panels A/B and C/D have not been conducted at the same time but in two separate experiments. The difference between empty vectors can thus be attributed to differences in the inoculum pressure (higher in the case of A/B). (PDF 2113 kb)

Wak insertion mutants. A. For each OsWAK gene, the different splicing forms are shown as well as the position of the T-DNA insertion site. The small arrows designate the primers (Additional file 6) that were used to genotype the plants and the primers used for measuring gene expression by quantitative RT-PCR. B. Transcript levels for the corresponding gene as measured by quantitative RT-PCR in mutant plants (MUT) compared to the corresponding null-segregant (WT) plants. Gene expression was normalized using actin. The values represent the percent of gene expression as compared to WT (100Â %); the values are the mean and standard deviation calculated from three biological replicates. (PDF 97 kb)

Primers used for this study. (PDF 66 kb)

Phylogenetic tree of the Arabidopsis WAK and EGF-containing OsWAK genes from rice. The proteomes of Arabidopsis thaliana (TAIR release 9: 33,200 sequences) and Oryza sativa (TIGR Release 6.0: 67,393 sequences) were downloaded from the GreenPhyl database ( http://www.greenphyl.org/cgi-bin/index.cgi ) (Conte et al., 2008). We retrieved OsWAK genes proceeding into three steps. First, we ran the hmmsearch program (Eddy, 2009) to search for kinase Hidden Markov Model (HMM) profile (PF00069.16) (Sonnhammer et al., 1998) into Arabidopsis and Oryza sequences. We retrieved 3185 proteins containing a kinase motif. On this set of sequences, we again used the hmmsearch program seeking this time EGFs HMM profiles (PF00008.18, PF09120.1, PF07974.4, PF04863.4 and PF07645.6). From this second screen, we retrieved 248 proteins (33 from Arabidopsis thaliana and 215 from Oryza sativa). We extracted the kinase domain sequences of these proteins and aligned them with the E-INS-i program (default parameters) from the MAFFT website ( http://mafft.cbrc.jp/alignment/software/ ). Based on this alignment, we generated a phylogenetic tree by the maximum likelihood method with 100 bootstrap replicates. All the genes with a WAK signature, explicitly containing both EGF motif(s) and kinase domain, were grouped in the tree with a bootstrap value of 86. All other genes outside this clade have been considered as outgroup. All manipulations on phylogenetic trees have been performed with the treedyn program ( http://www.treedyn.org/ ). Empty circles: newly annotated OsWAK genes; black squares: OsWAK genes known to be differentially expressed upon infection; empty squares: WAK genes known to be involved in fungal resistance; asterics: OsWAK genes with an ACF kinase domain. (PDF 59 kb)

FSPM analysis of root systems requires structural data obtained on large data set. This paper describes a processing pipeline developed to extract automatically the architecture of root system from images databases.

The function of a small number of Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) genes has been studied, mostly in Arabidopsis thaliana. They are involved in several important biological processes, including development, resistance/defence and adaptation to biotic and abiotic stresses. We are interested in deciphering the function of these genes in rice. In this aim, we decided to take advantage of the availability of T-DNA and Tos17 insertion mutant libraries of rice cv. Nipponbare to start a systematic screen of knock-out mutants of these genes. The rice genome contains ~320 LRRRLK genes. Our search in international collections of insertion mutant lines which are catalogued by Flanking Sequence Tag (FST) information from most of the rice insertional mutagenesis projects around the world revealed that 238 insertional mutant lines are available for the 323 genes. Phenotypical evaluation of homozygous mutants is performed under various biotic and abiotic stresses and mutant plants are observed for phenotypes at several stages of growth, from seedlings in Petri dishes to flowering and grain development stages in the greenhouse. Thus, with this project, we aim first to decipher and explore new genes involved in defence and developmental responses to stresses. Second, our analysis will be of great benefit to provide potential targets for cereal improvement, notably in the breeding of drought-tolerant and pathogen-resistant crops. This project is supported by grant #ANR-08-GENM-021 from Agence Nationale de la recherche (ANR), France.

Deciphering the genetic and molecular control of the development of the root system and its adaptive response to the availability of nutrients and water is of primary importance for the sustainable establishment of the rice crop under adverse environments. Rice displays a complex root structure-comprising several root types and a large variation in root architecture that reflects adaptation to cropping systems. Our group is exploring various approaches to investigate the control of constitutive development of roots and its adaptation in response to abiotic constraints. These include i. the detection of QTLs involved in root system development though whole genome or targeted association mapping ii. the investigation of the molecular control of root meristem maintenance and function, tissue specification, and lateral and adventitious (nodal) root formation. Root typology, meristem organization and tissue specification have been investigated in comparison to Arabidopsis. Specific anatomical features include radial patterning, variation in number of radial cells, number of cells constituting the quiescent centre and tissues participating in lateral root formation (Rebouillat et al, in press). Recent progresses in two photon imaging now permit a non destructive in situ observation throughout the root tissue.in notably visualizing cell walls, nuclei and starch granules without specific staining. A 4D imaging of the rice root is under construction. This tool will allow to precisely analyze cell lineages using GFP-marked Enhancer Trap lines and will permit to study protein trafficking and interactions in specific cell types during root growth. A genome-wide comparison of A. thaliana root development genes through reverse genetics in rice has been undertaken. Using the phylogenomic database GreenPhylDB (http://www.greenphyldb.cirad.fr) we extracted rice orthologs for a total of 60 genes involved in root development in A. thaliana. Based on the reference rice root typology and root meristem fate we identified surprisingly several important genes missing in rice. Functional analyses of A. thaliana and O. sativa orthologs are underway using a reverse genetics (http://www.orygenesdb.cirad.fr) approach and will give new insight on divergence/conservation on root meristem patterning and maintenance in both species. (Texte intégral)

To contribute to the international effort of production of sequence-indexed insertion libraries in rice, we have generated a collection of 30,000 Nipponbare lines which are characterized by 25,000 and 15,000 flanking sequence tags of T-DNA and Tos17 inserts respectively. These lines are being field evaluated and seed increased in CIAT, Colombia and the phenotypes documented in the Oryza. Tag Line database at http://urgi.versailles.inra.fr/OryzaTagLine/. This work was conducted in the frame of the national genomics initiative Génoplante (http://genoplante.com) and also supported by the National Sequencing Centre, Génoscope. In France, several research groups are now using these resources to decipher the molecular control of grain filling processes, response to biotic and abiotic stresses and of plant architecture determinants. Our group has focused its research on root development and plasticity in response to osmotic stress. Aside forward genetic screens which have yielded tagged mutants currently under analysis, a medium scale reverse genetics screen was rondncted in the frame of projects supported by the Generation Challenge Program and the European Union. This screen included genes identified through expression profiling in osmotic- and salt stressed tissues (SSH microarrays) or orthologs of Arabidopsis developmental and stress-related genes, and also a long term, overall investigation of the role of the rice Leucine-rich repeat receptor like kinase (LRR-RLK) (320 genes), CLV3 like (30 genes) and WOX (15 genes) genes in development and biotic/abiotic stress response. Due to the recent establishment and characterization of large libraries of insertion lines in rice, 961 insertions (i.e. 3.8 per gene) have been identified for 255 (70%) of the LRR-RLK genes in the FST databases (http://OryGenesDB.cirad.fr). 102 lines from international collections have been tested by Southern using a gene specific probe and a rearranged fragment was observed in 81 lines. Once homozygous plants were identified, their progeny. was screened along with the WT sibling progeny for alteration in development and abiotic stress tolerance (PEG 20% and NaCl 150 mM). Putative homozygous-lethal lines have also been identified. So far 24 lines with homozygous progenies have been phenotyped and 4 exhibited a phenotype linked with the FST in the gene under investigation. A T -DNA insertion in a leucine-rich repeat receptor protein kinase ortholog to the EXS precursor, known as MSP1 in rice, conducts to panicle sterility. Another T-DNA insertion in a BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 precursor exhibited extra long rachis branches. A third T-DNA insertion, in a receptor-like protein kinase precursor, exhibited shorter culms and thinner panicle rachis branches. Last, a CL3 like putative ligand of a LRR-RLK, exhibited enhanced leaf and root growths. These phenotypes are being characterized in detail. We could anticipate that this satisfactory frequency of phenotypes (17%) could be increased in testing the behaviour of the lines under other stresses, such as pathogen infection. (Texte intégral)

To contribute to the international effort of production of sequence-indexed insertion libraries in rice, we have generated a collection of 30,000 Nipponbare lines which are characterized by 25,000 and 15,000 flanking sequence tags of T-DNA and Tos17 inserts respectively. These lines are being field evaluated and seed increased in CIAT, Colombia and the phenotypes documented in the Oryza Tag Line database at http://urgi.versailles.inra.fr/OryzaTagLine/. This work was conducted in the frame of the national genomics initiative Génoplante (http://genoplante.com) and also supported by the National Sequencing Centre, Génoscope. In France, several research groups are now using these resources to decipher the molecular control of grain filling processes, response to biotic and abiotic stresses and of plant architecture determinants. Our group has focused its research on root development and plasticity in response to osmotic stress. Aside forward genetic screens which have yielded tagged mutants currently under analysis, a medium scale reverse genetics screen was conducted in the frame of projects supported by the Generation Challenge Program and the European Union. This screen included genes identified through expression profiling in osmotic- and salt stressed tissues (SSH microarrays) or orthologs of Arabidopsis developmental and stress-related genes, and also a long term, overall investigation of the role of the rice Leucine-rich repeat receptor like kinase (LRR-RLK) (320 genes), CLV3 like (30 genes) and WOX (15 genes) genes in development and biotic/abiotic stress response. Due to the recent establishment and characterization of large libraries of insertion lines in rice, 961 insertions (i.e. 3.8 per gene) have been identified for 255 (70%) of the LRR-RLK genes in the FST databases (http://OryGenesDB.círad.fr). 102 lines from international collections have been tested by Southern using a gene specific probe and a rearranged fragment was observed in 81 lines. Once homozygous plants were identified, their progeny was screened along with the WT sibling progeny for alteration in development and abiotic stress tolerance (PEG 20% and NaCl 150 mM). Putative homozygous-lethal lines have also been identified. So far 24 lines with homozygous progenies have been phenotyped and 4 exhibited a phenotype linked with the FST in the gene under investigation. A T-DNA insertion in a leucine-rich repeat receptor protein kinase ortholog to the EXS precursor, known as MSP1 in rice, conducts to panicle sterility. Another T-DNA insertion in a BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 precursor exhibited extra long rachis branches. A third T -DNA insertion, in a receptor-like protein kinase precursor, exhibited shorter culms and thinner panicle rachis branches. Last, a CL3 like putative ligand of a LRR-RLK, exhibited enhanced leaf and root growths. These phenotypes are being characterized in detail. We could anticipate that this satisfactory frequency of phenotypes (17%) could be increased in testing the behaviour of the lines under other stresses, such as pathogen infection. (Texte intégral)

Deciphering the genetic and molecular control of the develot of the root system and its adaptive response to the availability of nutrients and water is of primary importance for the sustainable establishment of the rice crop under adverse environments. Rice displays a complex root structure-comprising several root types and a large variation in root architecture that reflects adaptation to cropping systems. Our group is exploring various approaches to investigate the control of constitutive development of roots and its adaptation in response to abiotic stresses. Root typology, meristem organization and tissue specification have been investigated first in comparison to Arabidopsis. Specific anatomical features include radial patterning, variation in number of radial cells, number of cells constituting the quiescent centre and tissues participating in lateral root formation. Recent progresses in bi photon imaging now permit a non destructive in situ observation throughout the root tissue. This allows to precisely analyze cell lineages using GFP-marked Enhancer Trap lines and will permit to study protein trafficking and interactions in specific cell types. Cloning and characterization of architectural genes identified both through QTL analysis and screening of insertion mutants altered in root development is underway. A genome-wide comparison of A. thaliana root development genes through reverse genetics in rice is undertaken. We first developed a comparative database for genome-wide comparison of A. thaliana and O. sativa genic repertoire. GreenPhylDB (http://www.greenphyldb.cirad.fr) nowadays comprises the most complete list of plant gene families (6,421 families) which have been manually curated. GreenPhylDB also contains all the phylogenomic relations computed for 4,375 families. We extracted rice orthologs for a total of 60 genes involved in root development in A. thaliana. Based on the reference rice root typology and root meristem fate we identified surprisingly several important genes missing in rice. For instance, the PLT genes involved in stem-cell niche establishment in A.thaliana were all missing in rice suggeetinn Pither extensive redtinrlánry in A. thaliana or alternative molecular mechanism for root meristem maintenance in rice. Moreover, several genes involved in root hair pattering in A. thaliana were missing in rice and this feature can be tentatively linked to the absence of root hair radial pattering in rice. By using a thew phylogenomics tool integrated in GreenPhylDB (i-GOST for iterative Greenphyl Ortholog Search Tool) we also identify moss (Physcomitrella patens) orthologs for most of the A. thaliana root hairs genes suggesting that rhizoids in moss and root hairs in angiosperm are truly homologs structures. Functional analyses of A. thaliana and O. sativa orthologs are underway using a reverse genetics (http://www.orygenesdb.cirad.fr) approach and will give new insight on divergence/conservation on root meristem patterning and maintenance in both species. (Texte intégral)

Insertional mutant databases containing Flanking Sequence Tags (FSTs) are becoming key resources for plant functional genomics. We have developed OryGenesDB(http://orygenesdb.cirad.fr/), a database dedicated to rice reverse genetics. Insertion mutants of rice genes are catalogued by Flanking Sequence Tag (FST) information that can be readily accessed by this database. Our database presently contains 44166 FSTs generated by most of the rice insertional mutagenesis projects. The OryGenesDB genome browser is based on the powerful Generic Genome Browser (GGB) developed in the framework of the Generic Model Organism Project (GMOD). The main interface of our web site displays search and analysis interfaces to look for insertions in any candidate gene of interest. Several starting points can be used to exhaustively retrieve the insertions positions and associated genomic information using blast, keywords or gene name search. The toolbox integrated in our database also includes an 'anchoring' option that allows immediate mapping and visualization of up to 50 nucleic acid sequences in the rice Genome Browser of OryGenesDB. As a first step toward plant comparative genomics, we have linked the rice and Arabidopsis whole genome using all the predicted pairs of orthologs by best BLAST mutual hit (BBMH) connectors.

In the last 7 years, several independent national initiatives have been launched for generating rice insertion libraries, characterizing flanking sequences at insertion points and gathering phenotype and sequence information in web accessible databases (Hirochika et al 2004). This effort has lead to the generation of more than 460,000 T-DNA lines and the release of more than 67 000 Flanking Sequence Tags (FST) in public databases. It is presumed that the full characterization of all these lines with other concurrent initiatives using the maize Ac/Ds and En/Spm transposable elements and the tissue culture stimulated, and endogenous retrotransposon Tos17 will enable rice geneticists to find at least one insertion in any rice gene and several alleles in most of the genes. We will survey the progresses accomplished in generating and characterizing insertion libraries in rice and identifying or validating genes of interest. In France, an effort of generation of 30,000 insertion lines characterized by 25,000 and 18,000 flanking sequence tags of T-DNA and Tos17 inserts respectively has been supported by the national genomics initiative Génoplante and the National Sequencing Centre Génoscope. Several research groups are using these resources together with other tools and resources for deciphering the molecular control of the grain filling processes, the response to biotic and abiotic stresses and determinants of plant architecture. Our group has focused its research on root development and plasticity in response to osmotic stress using both forward and reverse genetics approaches. Recent advances will be presented. (Texte intégral)

Leucine-Rich Repeat Receptor-Like Kinases (LRR-RLKs) are plant-specific receptor kinases involved in development and defense. Taking advantage of the whole rice genome sequence, we performed an extensive sequence analysis of the 320 LRR-RLK sequences that we retrieved from the Oryza sativa sp. japonica genome database at The Institute for Genomic Research (TIGR). Our aim is to study LRR-RLKs involved in root rice development. A search in expressed genes databases shows that ~77% of these genes are potentially expressed in rice. This high percentage of expression might reflect the fact that we looked for sequences with putative functionally active domains, like the presence of cystein pairs, LRRs and kinase domains. Based on the alignment of kinase domains and phylogenetic analysis, these LRRRLKs sequences can be classified into 22 subgroups. Interestingly, the ~100 extra genes present in the rice genome compared to the Arabidopsis one belong to one single subgroup, LRR XII, whose known members are receptors involved in resistance/defense against bacteria. We find that these genes are distributed throughout the twelve rice chromosomes and that 45% of the genes are organized into 41 tandem clusters of 2 to 14 genes. Interestingly, 38.2% of LRR-RLKs belonging to subgroup LRR XII are localized on chromosome 11 with 89.7% of them organized in 5 clusters of 3 to 14 genes. Our results on the genomic organization of the LRR-RLKs also show that ~3.

Insertional mutant databases containing Flanking Sequence Tags (FSTs) are becoming key resources for plant functional genomics. We have developed OryGenesDB (http://orygenesdb.cirad.fr/), a database dedicated to rice reverse genetics. Insertion mutants of rice genes are catalogued by Flanking Sequence Tag (FST) information that can be readily accessed by this database. Our database presently contains 44166 FSTs generated by most of the rice insertional mutagenesis projects. The OryGenesDB genome browser is based on the powerful Generic Genome Browser (GGB) developed in the framework of the Generic Model Organism Project (GMOD). The main interface of our web site displays search and analysis interfaces to look for insertions in any candidate gene of interest. Several starting points can be used to exhaustively retrieve the insertions positions and associated genomic information using blast, keywords or gene name search. The toolbox integrated in our database also includes an "anchoring option" that allows immediate mapping and visualization of up to 50 nucleic acid sequences in the rice Genome Browser of OryGenesDB. As a first step toward plant comparative genomics, we have linked the rice and Arabidopsis whole genome using all the predicted pairs of orthologs by best BLAST mutual hit (BBMH) connectors.