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Additional file 11: Figure S8. Log scale QQ-plots corresponding to association analysis performed independently on each trait and repetition. Three different models (i.e. EMMA, LFMM, MLM) taking into account relatedness and/or structure were used for association and ANOVA was used as a benchmark. For a given trait, the transformed data were used if at least one of the replicates failed to reach normality, otherwise non-transformed data were used.

Additional file 8: Figure S5. Correlations between traits and structure. For each ancestry group (A1 to A4), we made a Spearman’s rank correlation test and plotted it as a correlogram. Colors sign the intensity of the correlation and white stars were added when the correlation was significant given the threshold retained, i.e. either a p-value cutoff of 0.01 (a) or a False Discovery Rate of 5% (b).

Additional file 2:Figure S1. Positions of accessions with resistance alleles of RYMV1, RYMV2 and NLRRYMV3 genes on the genetic diversity tree. Susceptible accessions are colored in dark grey and accessions not evaluated for resistance in light grey. Adapted from the genetic tree of Orjuela et al. [20]. Figure S2. Characteristics of primers and amplified fragments for markers or Sanger sequencing. Genes are represented as grey boxes for exons and grey lines for introns. Primers are represented as triangles and the numbers below the triangle refer to the corresponding sequences. (a, b, c) Blue traits represent fragments that were amplified and then sequenced with the primers colored in red. (c) Amplification fragments corresponding to the CAPS or dCAPS markers designed on the CPR5–1 gene are represented as green traits. Additional information on these markers is provided in Additional file 2: Table S7. (d) Position of T-DNA insertions in the CPR5–1 gene in lines 3A-06612 and 3D-01842 are indicated. The T-DNA-specific and gene-specific primers used for sequencing the T-DNA flanking site and genotyping for the presence/absence of insertions are indicated in brown and blue, respectively. Table S7. Characteristics of CAPS and dCAPS markers. Marker names indicate whether there are CAPS or dCAPS markers and which alleles of the CPR5–1 gene they target. The bracketed number before the primer sequences refer to the reference of primers in Additional file 2: Figure S2. The size of the fragments expected in plants with the reference haplotype of CG14 (WT) or the alternate haplotypes (R) are indicated, except fragments below 30 bp that are uneasily detected by agarose electrophoresis. The CAPS-CPR5–1-R1 marker had already been described in Orjuela et al. [13].

Additional file 5: Figure S2. Genome wide Linkage disequilibrium (LD) decay.

Additional file 9: Figure S6. Linear scale QQ-plots corresponding to association analysis performed independently on each trait and repetition. Three different methods (EMMA, LFMM, MLM) taking into account relatedness and/or structure were used for association and ANOVA was used as a benchmark. For a given trait, the transformed data were used if at least one of the replicates failed to reach normality, otherwise non-transformed data were used.

Le riz est la principale céréale nourrissant l'humanité. L'augmentation de la populaiton mondiale, qui devrait atteindre 9 à 10 milliards d'individus d'ici à 2050, est principalement attendue dans les pays du Sud, où le riz constitue gébéralement l'aliment de base. Pour survivre cette évolution, la production devra augmenter de 50ù dans les 30 prochaines années, sur des surfaces arables qui sont, elles, en réduction.

The GLASS project (GLAberrima ASSembly), jointly between IRD and AfricaRice, aims to sequence de novo the two African NERICA parents, CG14 and TOG5681, representing the two opposite of the glaberrima variability. For that purpose we choose an hybrid approach combining the Illumina high-quality short reads and the PacificBioscience Long Reads technology. Tests were performed using Illumina data on TOG 5307 and TOG5681, using AbySS short-reads assembler, with 19x and 25x total data respectively. For TOG5307, we obtained 153'774 contigs (minimum size of 200b), mean size of 1.5 kb (N50 = 2.4kb, N90 = 637b). On TOG5681, we obtained 97'196 contigs (minimum size 200b), with a mean size of 2.8kb (N50 = 5kb, N90 = 1.3 kb). The overall assembly for each genome is of 0.56x for TOG5307 and of 0.69x for TOG5681. Just adding +6x of Illumina data almost doubled the assembly efficiency. PacBio data test on C2 and XL kits were also perfomed for CG14 data, providing respectively 143'456 and 99'488 contigs (mean size of 3140b and 3184b), with a 1.12x and 0.79x coverage. The C2 10x sequencing in PacBio for CG14 and TOG5681, and the 60x sequencing in Illumina for each is on the way. The annotation will be transfered from the Nipponbare MSU7.0 reference genome using tools such as QOD (Mancheron et al, 2010) and BLAST/BLAT based scripts. These sequences will be immediatly available for the whole community without restriction. The IRIGIN project from France Genomique (International RIce Genomic INitiative, http://irigin.org) is headed by IRD and CIRAD, and aimed to provide valuable genetic stocks genomic data in the frame of GriSP. African rice (300 O. glaberrima and 100 O. barthii) will be deeply resequenced (25x) and aligned on their reference (from the GLASS project). In the same time , NAMs population (indica x tropical japonica) from AfricaRice and CIAT will be genotyped (using low-coverage sequencing), to provide to the rice community the best genetic map ever created in plants, with the highest level of coverage ever seen. Thousand of terabytes of raw data and billions of polymorphic data will be gathered within this project, and will be distributed to the whole rice community as soon as possible.

A collection of accessions of Burkina Faso rice germplasm was evaluated for resistance using four Rice yellow mottle virus (RYMV) isolates: Ng122, Ng144, B27 and BF1. B27, an isolate from Benin was used first, followed by Ng122 and Ng144 (isolates from Niger), and BF1 an aggressive isolate from Burkina Faso was used last to assess the accessions status against RYMV. Fourteen-day-old plantlets were inoculated and symptoms scored fortnightly from 14 to 56 days post inoculation (dpi). Plant height of all accessions was recorded at 49 dpi with isolates Ng122 and Ng144. The Oryza sativa accessions of the collection were highly susceptible except one (BM24), which combined partial resistance and tolerance. Twenty one O. glaberrima accessions out of 48 were found resistant to Ng122 and Ng144. When these 21 accessions were subsequently screened with the aggressive RYMV strain BF1, eight of them displayed a delay in the appearance of RYMV symptoms while two showed resistance. The new sources of resistance identified in this study, could be exploited in breeding to control the spread of RYMV in Africa.

The GLASS project (GLAberrima ASSembly), joint project between Institut de recherche pour le développement (IRD) and AfricaRice, aims to sequence de novo the two African NERICA parents, CG14 and TOG5681, representing the two extremes of Oryza glaberrima variability. For that purpose, we chose a hybrid approach combining the illumina high-quality short reads and the PacificBioscience Long Reads technology. Tests were performed using Illumina data on TOG5307 and TOG5681, using AbySS short-reads assembler, with 19x and 25x total data, respectively. For TOG5307, we obtained 153,774 contigs (minimum size of 200b), mean size of 1.5 kb (N50 = 2.4kb, N90 = 637b). On TOG5681, we obtained 97,196 contigs (minimum size 200b), with a mean size of 2.8kb (N50 = 5kb, N90 = 1.3 kb). The overall assembly for each genome is 0.56x for TOG5307 and 0.69x for TOG5681. Just adding +6x of Illumina data almost doubled the assembly efficiency. PacBio data test on C2 and XL kits were also performed for CG14 data, providing, respectively, 143,456 and 99,488 contigs (mean size of 3140b and 3184b), with a 1.12x and 0.79x coverage. The C2 10x sequencing in PacBio for CG14 and TOG5681, and the 60x sequencing in Illumina for each is underway. The annotation will be transferred from the Nipponbare MSU7.0 reference genome using tools such as QOD and BLAST/BLAT based scripts. These sequences will be immediately available for the whole community without restriction. The IRIGIN project from France Genomique (International RIce Genomic INitiative) is headed by IRD and Centre de coopération internationale en recherche agronomique pour le développement (CIRAD), and aims to provide valuable genetic stocks genomic data in the frame of the Global Rice Science Partnership (GRiSP). African rice (300 O. glaberrima and 100 O. barthii) will be deeply resequenced (25x) and aligned on their reference (from the GLASS project). At the same time, NAMs population (indica x tropical japonica) from AfricaRice and the International Center for Tropical Agriculture (CIAT) will be genotyped (using low-coverage sequencing), to provide the best genetic map ever created in plants, with the highest level of coverage ever seen. Thousand of terabytes of raw data and billions of polymorphic data will be gathered within this project, and will be distributed to the whole rice community as soon as possible.

A collection of 312 accessions of Burkina Faso rice germplasm was evaluated in Cotonou, Benin and Montpellier, France for resistance using four Rice yellow mottle virus (RYMV) isolates. In Cotonou, the accessions were screened using one isolate from Benin (B27) and two isolates from Niger (Ng122 and Ng144). The experiment was conducted in a screenhouse. Fourteen-day-old plantlets were inoculated and symptoms scored fortnightly from 14 to 56 days after inoculation (dai). Plant height was recorded at 49 dai after all accessions were inoculated with isolates Ng122 and Ng144. Double Antibody Sandwich-Enzyme-Linked Immunosorbent Assay (DAS-ELISA) was used to assess RYMV into rice leaves. In Montpellier, aggressive isolate from Burkina Faso BF1 was used to assess the accessions against RYMV. The experiment was conducted in the greenhouse under controlled conditions (28-32°C, 12 h of light per 24 h and 80-90% relative humidity). Inoculation process and symptoms scoring were performed on rice leaves as in Benin and reverse-transcription polymerase chain reaction (RT-PCR) was used to check the presence of the allele rymvl -3 in resistant accessions. The results of both experiments showed that the Oryza sativa accessions of the collection were highly susceptible, except for BM24, which combined partial resistance and tolerance. Twenty-one O. glaberrima accessions out of 48 were resistant to Ng122 and Ng144. When these 21 accessions were subsequently screened with the aggressive RYMV strain BF1, eight of them displayed a delay in the appearance of RYMV symptoms, while two showed resistance. The molecular characterization of the resistant accessions showed that they do not have the allele rymvl-3. These accessions could have new resistance alleles. Therefore, the new sources of resistance identified in this study could be exploited in breeding to tackle the spread of RYMV in Africa.

International audience; We report here the molecular and phenotypic features of a library of 31,562 insertion lines generated in the model japonica cultivar Nipponbare of rice (Oryza sativa L.), called Oryza Tag Line (OTL). Sixteen thousand eight hundred and fourteen T-DNA and 12,410 Tos17 discrete insertion sites have been characterized in these lines. We estimate that 8686 predicted gene intervals--i.e. one-fourth to one-fifth of the estimated rice nontransposable element gene complement--are interrupted by sequence-indexed T-DNA (6563 genes) and/or Tos17 (2755 genes) inserts. Six hundred and forty-three genes are interrupted by both T-DNA and Tos17 inserts. High quality of the sequence indexation of the T2 seed samples was ascertained by several approaches. Field evaluation under agronomic conditions of 27,832 OTL has revealed that 18.2% exhibit at least one morphophysiological alteration in the T1 progeny plants. Screening 10,000 lines for altered response to inoculation by the fungal pathogen Magnaporthe oryzae allowed to observe 71 lines (0.7%) developing spontaneous lesions simulating disease mutants and 43 lines (0.4%) exhibiting an enhanced disease resistance or susceptibility. We show here that at least 3.5% (four of 114) of these alterations are tagged by the mutagens. The presence of allelic series of sequence-indexed mutations in a gene among OTL that exhibit a convergent phenotype clearly increases the chance of establishing a linkage between alterations and inserts. This convergence approach is illustrated by the identification of the rice ortholog of AtPHO2, the disruption of which causes a lesion-mimic phenotype owing to an over-accumulation of phosphate, in nine lines bearing allelic insertions.

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial blight in rice, one of the most devastating diseases of rice worldwide. African X. oryzae pv. oryzae strains belong to a clear genetic group distinct from those of Asia. Three new races of the pathogen were characterized among strains from West Africa. We evaluated 107 Oryza glaberrima accessions for resistance to bacterial blight under greenhouse conditions. Six-week-old seedlings were inoculated with five different African X. oryzae pv. oryzae strains originating from the West African nations of Burkina and Mali and representing different races (A1, A2, and A3). Philippine X. oryzae pv. oryzae strain PXO86 (race 2) was also used. Most (48%) of the accessions of O. glaberrima were highly susceptible to X. oryzae pv. oryzae strains from Burkina, while 20 and 36 were resistant to X. oryzae pv. oryzae strains from Mali and the Philippines, respectively. CAPS markers and dot blot assays were used for detection of resistance genes xa5 and Xa21 from a selected set of O. glaberrima accessions. Our results suggest that the O. glaberrima germplasm contains a narrow genetic base for resistance to X. oryzae pv. oryzae. Sources of resistance identified among O. glaberrima are recommended for rice breeding programs to develop bacterial blight-resistant cultivars for West Africa.

REFUGE is an international hosting platform for the elucidation of gene function using rice as a model species. It offers international scientists, notably nonrice specialists, an access to know-how, bioinformatic, biological, and molecular resources, allowing the use of rice as a model system to investigate gene function through functional genomics strategies. PhD and postdoctoral researchers, notably from the South, are the first target beneficiaries of this initiative. Visiting scientists have access to REFUGE's know-how, including bioinformatic searches, preparation of T-DNA vectors, high-throughput production of rice transformants, molecular characterization of transformants, genotyping of insertion lines from local and international collections; growth, crossing, and phenotyping in containment greenhouse; and access to cell imaging and genotyping platforms. Hosted scientists have the opportunity to carry out one to several visits from 1 to 3 months, the REFUGE staff taking care of the materials produced between two visits. The REFUGE platform provides supervision of the hosted scientists or students and covers bench fees. Visitors have to find their own travel and subsistence funds. However, REFUGE can provide assistance to scientists notably from South countries, to find an appropriate funding source for travel and subsistence. Applications should be submitted at http://www.refuge-platform.org and will be readily examined by the REFUGE scientific committee. Funded by Agropolis Fondation, http://www.agropolis-fondation.fr/, REFUGE is a collaborative venture between the joint research unit DAP -INRA, CIRAD and Montpellier University and Agronomy School- and the 'Plant genome and development' laboratory at IRD and Perpignan University. (Texte intégral)

International audience

Equipe 4; National audience; Depuis quinze ans, la population de cartographie de variété indica-japonica dite «IR64-Azucena» jouit d'une popularité inégalée auprès des phytogénéticiens puisqu'elle permet de localiser plus facilement les gènes intervenant dans la résistance aux insectes ravageurs ainsi qu'aux maladies d'origine fongique, bactérienne ou virale, la tolérance abiotique au stress, l'architecture de la plante et la qualité du grain. Un projet en cours s'appuie sur une nouvelle lignée de populations consanguines recombinantes issues du croisement IR64-Azucena pour analyser la plasticité phénotypique du riz en phase précoce de développement de la plante, en comparant le pourcentage de répartition de la biomasse entre racines et pousses, l'architecture de la plante et le rythme de développement selon trois scénarios de croissance: en carence de phosphore, sous stress osmotique et dans des conditions de culture hydroponique sous-optimales.

The new resources developed are already used in many fine mapping projects. This integrative genetic map provides a common representation framework and is a potential tool for gene positional cloning, marker-assisted selection and in silico QTL/genes co-localisation. It can also be used for comparative genomic studies by using anchor probes as bridge points to test local synteny and candidate genes of other cereals. The next step will be the integration of markers from other major rice genetic maps (Nipponbare x Kasalath) to enlarge the compilation to other genes and QTLs.

We investigated the diversity of the Tos17 retrotransposon in the Oryza genus using a combination of Southern hybridization with a reverse transcriptase (RT) probe and an adapter-PCR mediated amplification which allowed the sequencing of the genomic regions flanking Tos17 insertions. This analysis was carried out in a collection of 96 Oryza accessions covering the diversity of the genus and 246 accessions of a core collection of O. sativa representative of the diversity of the species. Our Southern hybridization results show that Tos17 is present in all the accessions of the A-genome species, except in the South American species O. glumaepatula and in the African species O. glaberrima and O. breviligulata. Surprisingly, a hybridization signal was observed in O. officinalis, O. minuta and O. latifolia. In O. sativa, the number of putative copies of Tos17 per accession ranged from 1-11. Moreover, multivariate analysis based on presence/absence of putative copies yielded varietal clusters consistent with the isozyme classification of rice. Adapter PCR amplification and sequencing of flanking regions of Tos17 insertions in Oryza species yielded 124 good sequences which were anchored on the rice genome sequence, overall revealing 71 insertions of Tos17 in most the Oryza species, which corresponded to 36 orthologous sites. In O. sativa the same approach yielded 461 good sequences, revealing 62 positions in the 6 varietal groups, representing 28 orthologous insertion sites. Comparison of these insertions among Oryza species in one hand and between cultivated and wild Oryza species on the other hand will be presented and discussed.

The objective of this project, funded by the Genomics initiative Génoplante, is to generate through Agrobacterium-mediated transformation a library of 50,000 insertion lines of rice cv. Nipponbare. In a 2 year time-frame, we produced more than 39,400 primary transformants, with an average efficiency of 4.2 independent transformation events per cocultured callus. Each line was found to harbour an average of 2.2 and 3.2 copies of the T-DNA and of new inserts of the endogenous ty1-copia retrotransposon Tos17 respectively. 89% of the 14,893 walk-PCR products of amplification of the regions flanking the left border of T-DNA inserts yielded a readable sequence. A first survey of 7,480 genomic sequences against the rice BAC/PAC sequences detected 5,791 hits (77%), consistent with the percentage of the rice genome sequenced at time of analysis (76%). 4,303 (56%) T-DNA insertion sites were assigned to at least one position on the rice genome. The T-DNA density averaged 17.9 insertions per Mb sequenced and ranged from 14 to 20 insertions per Mb across the 12 chromosomes. A more detailed examination of the distribution of T-DNA inserts along the Chromosome 1 showed that 668 (92.8%) of 720 T-DNA insertions are assigned to a unique location. The results also demonstrated that i. the centromeric region exhibits a lower insertion density whereas higher insertion density is observed in the subtelomeric regions, ii. T-DNA inserts are very rarely found in repetitive DNA, iii. T-DNA seems to preferentially integrate promoter and intron regions of the predicted rice genes. Frequencies of putative insertions in both chr1 genes predicted by TIGR annotation and transcription factors identified by the whole genome sequencing (Goff et al 2002) led to the same conclusion that about 7-10% of the rice gene complement is already covered by the 6,111 tagged lines characterized in the collection. (Texte intégral)