Pierre Larmande

Highly efficient production and characterization of T-DNA plants for rice ( Oryza sativa L.) functional genomics

AbstractnWe investigated the potential of an improved Agrobacterium tumefaciens-mediated transformation procedure of japonica rice (Oryza sativa L.) for generating large numbers of T-DNA plants that are required for functional analysis of this model genome. Using a T-DNA construct bearing the hygromycin resistance (hpt), green fluorescent protein (gfp) and β-glucuronidase (gusA) genes, each individually driven by a CaMV 35S promoter, we established a highly efficient seed-embryo callus transformation procedure that results both in a high frequency (75–95%) of co-cultured calli yielding resistant cell lines and the generation of multiple (10 to more than 20) resistant cell lines per co-cultured callus. Efficiencies ranged from four to ten independent transformants per co-cultivated callus in various japonica cultivars. We further analysed the T-DNA integration patterns within a population of more than 200 transgenic plants. In the three cultivars studied, 30–40% of the T0 plants were found to have integrated a single T-DNA copy. Analyses of segregation for hygromycin resistance in T1 progenies showed that 30–50% of the lines harbouring multiple T-DNA insertions exhibited hpt gene silencing, whereas only 10% of lines harbouring a single T-DNA insertion was prone to silencing. Most of the lines silenced for hpt also exhibited apparent silencing of the gus and gfp genes borne by the T-DNA. The genomic regions flanking the left border of T-DNA insertion points were recovered in 477 plants and sequenced. Adapter-ligation Polymerase chain reaction analysis proved to be an efficient and reliable method to identify these sequences. By homology search, 77 T-DNA insertion sites were localized on BAC/PAC rice Nipponbare sequences. The influence of the organization of T-DNA integration on subsequent identification of T-DNA insertion sites and gene expression detection systems is discussed.

A new cacao linkage map based on codominant markers: development and integration of 201 new microsatellite markers

A linkage map of cacao based on codominant markers has been constructed by integrating 201 new simple sequence repeats (SSR) developed in this study with a number of isoenzymes, restriction fragment length polymorphisms (RFLP), microsatellite markers and resistance and defence gene analogs (Rgenes-RFLP) previously mapped in cacao. A genomic library enriched for (GA)n and (CA)n was constructed, and 201 new microsatellite loci were mapped on 135 individuals from the same mapping population used to establish the first reference maps. This progeny resulted from a cross between two heterozygous cacao clones: an Upper-Amazon Forastero (UPAxa0402) and a Trinitario (UFxa0676). The new map contains 465 markers (268 SSRs, 176 RFLPs, five isoenzymes and 16 Rgenes-RFLP) arranged in ten linkage groups corresponding to the haploid chromosome number of cacao. Its length is 782.8xa0cM, with an average interval distance between markers of 1.7xa0cM. The new microsatellite markers were distributed throughout all linkage groups of the map, but their distribution was not random. The length of the map established with only SSRs was 769.6xa0cM, representing 94.8% of the total map. The current level of genome coverage is approximately one microsatellite everyxa03xa0cM. This new reference map provides a set of useful markers that is transferable across different mapping populations and will allow the identification and comparison of the most important regions involved in the variation of the traits of interest and the development of marker-assisted selection strategies.

OryGenesDB: a database for rice reverse genetics

Insertional mutant databases containing Flanking Sequence Tags (FSTs) are becoming key resources for plant functional genomics. We have developed OryGenesDB (), 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.

Oryza Tag Line, a phenotypic mutant database for the Génoplante rice insertion line library

To organize data resulting from the phenotypic characterization of a library of 30 000 T-DNA enhancer trap (ET) insertion lines of rice (Oryza sativa L cv. Nipponbare), we developed the Oryza Tag Line (OTL) database (http://urgi.versailles.inra.fr/OryzaTagLine/). OTL structure facilitates forward genetic search for specific phenotypes, putatively resulting from gene disruption, and/or for GUSA or GFP reporter gene expression patterns, reflecting ET-mediated endogenous gene detection. In the latest version, OTL gathers the detailed morpho-physiological alterations observed during field evaluation and specific screens in a first set of 13 928 lines. Detection of GUS or GFP activity in specific organ/tissues in a subset of the library is also provided. Search in OTL can be achieved through trait ontology category, organ and/or developmental stage, keywords, expression of reporter gene in specific organ/tissue as well as line identification number. OTL now contains the description of 9721 mutant phenotypic traits observed in 2636 lines and 1234 GUS or GFP expression patterns. Each insertion line is documented through a generic passport data including production records, seed stocks and FST information. 8004 and 6101 of the 13 928 lines are characterized by at least one T-DNA and one Tos17 FST, respectively that OTL links to the rice genome browser OryGenesDB.

OryGenesDB 2008 update: database interoperability for functional genomics of rice

OryGenesDB (http://orygenesdb.cirad.fr/index.html) is a database developed for rice reverse genetics. OryGenesDB contains FSTs (flanking sequence tags) of various mutagens and functional genomics data, collected from both international insertion collections and the literature. The current release of OryGenesDB contains 171 000 FSTs, and annotations divided among 10 specific categories, totaling 78 annotation layers. Several additional tools have been added to the main interface; these tools enable the user to retrieve FSTs and design probes to analyze insertion lines. The major innovation of OryGenesDB 2008, besides updating the data and tools, is a new tool, Orylink, which was developed to speed up rice functional genomics by taking advantage of the resources developed in two related databases, Oryza Tag Line and GreenPhylDB. Orylink was designed to field complex queries across these three databases and store both the queries and their results in an intuitive manner. Orylink offers a simple and powerful virtual workbench for functional genomics. Alternatively, the Web services developed for Orylink can be used independently of its Web interface, increasing the interoperability between these different bioinformatics applications.

P-TRAP: a Panicle Trait Phenotyping tool

BackgroundIn crops, inflorescence complexity and the shape and size of the seed are among the most important characters that influence yield. For example, rice panicles vary considerably in the number and order of branches, elongation of the axis, and the shape and size of the seed. Manual low-throughput phenotyping methods are time consuming, and the results are unreliable. However, high-throughput image analysis of the qualitative and quantitative traits of rice panicles is essential for understanding the diversity of the panicle as well as for breeding programs.ResultsThis paper presents P-TRAP software (Panicle TRAit Phenotyping), a free open source application for high-throughput measurements of panicle architecture and seed-related traits. The software is written in Java and can be used with different platforms (the user-friendly Graphical User Interface (GUI) uses Netbeans Platform 7.3). The application offers three main tools: a tool for the analysis of panicle structure, a spikelet/grain counting tool, and a tool for the analysis of seed shape. The three tools can be used independently or simultaneously for analysis of the same image. Results are then reported in the Extensible Markup Language (XML) and Comma Separated Values (CSV) file formats. Images of rice panicles were used to evaluate the efficiency and robustness of the software. Compared to data obtained by manual processing, P-TRAP produced reliable results in a much shorter time. In addition, manual processing is not repeatable because dry panicles are vulnerable to damage. The software is very useful, practical and collects much more data than human operators.ConclusionsP-TRAP is a new open source software that automatically recognizes the structure of a panicle and the seeds on the panicle in numeric images. The software processes and quantifies several traits related to panicle structure, detects and counts the grains, and measures their shape parameters. In short, P-TRAP offers both efficient results and a user-friendly environment for experiments. The experimental results showed very good accuracy compared to field operator, expert verification and well-known academic methods.

In-depth molecular and phenotypic characterization in a rice insertion line library facilitates gene identification through reverse and forward genetics approaches.

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.

The Rise and Fall of African Rice Cultivation Revealed by Analysis of 246 New Genomes

African rice (Oryza glaberrima) was domesticated independently from Asian rice. The geographical origin of its domestication remains elusive. Using 246 new whole-genome sequences, we inferred the cradle of its domestication to be in the Inner Niger Delta. Domestication was preceded by a sharp decline of most wild populations that started more than 10,000 years ago. The wild population collapse occurred during the drying of the Sahara.xa0This finding supports the hypothesis that depletion of wild resources in the Sahara triggered African rice domestication. African rice cultivation strongly expanded 2,000 years ago. During the last 5 centuries, a sharp decline of its cultivation coincided with the introduction of Asian rice in Africa. A gene, PROG1, associated with an erect plant architecture phenotype, showed convergent selection in two rice cultivated species, Oryza glaberrima from Africa and Oryza sativa from Asia. In contrast, a shattering gene, SH5, showed selection signature during African rice domestication, but not during Asian rice domestication. Overall, our genomic data revealed a complex history of African rice domestication influenced by important climatic changes in the Saharan area, by the expansion of African agricultural society, and by recent replacement by another domesticated species.

Gigwa-Genotype investigator for genome-wide analyses.

BackgroundExploring the structure of genomes and analyzing their evolution is essential to understanding the ecological adaptation of organisms. However, with the large amounts of data being produced by next-generation sequencing, computational challenges arise in terms of storage, search, sharing, analysis and visualization. This is particularly true with regards to studies of genomic variation, which are currently lacking scalable and user-friendly data exploration solutions.DescriptionHere we present Gigwa, a web-based tool that provides an easy and intuitive way to explore large amounts of genotyping data by filtering it not only on the basis of variant features, including functional annotations, but also on genotype patterns. The data storage relies on MongoDB, which offers good scalability properties. Gigwa can handle multiple databases and may be deployed in either single- or multi-user mode. In addition, it provides a wide range of popular export formats.ConclusionsThe Gigwa application is suitable for managing large amounts of genomic variation data. Its user-friendly web interface makes such processing widely accessible. It can either be simply deployed on a workstation or be used to provide a shared data portal for a given community of researchers.

Clever generation of rich SPARQL queries from annotated relational schema: application to Semantic Web Service creation for biological databases

BackgroundIn recent years, a large amount of “-omics” data have been produced. However, these data are stored in many different species-specific databases that are managed by different institutes and laboratories. Biologists often need to find and assemble data from disparate sources to perform certain analyses. Searching for these data and assembling them is a time-consuming task. The Semantic Web helps to facilitate interoperability across databases. A common approach involves the development of wrapper systems that map a relational database schema onto existing domain ontologies. However, few attempts have been made to automate the creation of such wrappers.ResultsWe developed a framework, named BioSemantic, for the creation of Semantic Web Services that are applicable to relational biological databases. This framework makes use of both Semantic Web and Web Services technologies and can be divided into two main parts: (i) the generation and semi-automatic annotation of an RDF view; and (ii) the automatic generation of SPARQL queries and their integration into Semantic Web Services backbones. We have used our framework to integrate genomic data from different plant databases.ConclusionsBioSemantic is a framework that was designed to speed integration of relational databases. We present how it can be used to speed the development of Semantic Web Services for existing relational biological databases. Currently, it creates and annotates RDF views that enable the automatic generation of SPARQL queries. Web Services are also created and deployed automatically, and the semantic annotations of our Web Services are added automatically using SAWSDL attributes. BioSemantic is downloadable at http://southgreen.cirad.fr/?q=content/Biosemantic.