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Plant-transformation-ready, large-insert binary bacterial artificial chromosome (BIBAC) libraries are of significance for functional and network analysis of large genomic regions, gene clusters, large-spanning genes, and complex loci in the postgenome era. Here, we report the characterization of a plant-transformation-ready BIBAC library of the sequenced Arabidopsis genome for which such a library is not available to the public, the transformation of a large-insert BIBAC of the library into tobacco by biolistic bombardment, and the expression analysis of its containing genes in transgenic plants. The BIBAC library was constructed from nuclear DNA partially digested with BamHI in the BIBAC vector pCLD04541. It contains 6144 clones and has a mean insert size of 108 kb, representing 5.2x equivalents of the Arabidopsis genome or a probability of greater than 99% of obtaining at least one positive clone from the library using a single-copy sequence as a probe. The transformation of the large-insert BIBAC and analyses of the transgenic plants showed that not only did transgenic plants have intact BIBAC DNA, but also could the BIBAC be transmitted stably into progenies and its containing genes be expressed actively. These results suggest that the large-insert BIBAC library, combined with the biolistic bombardment transformation method, could provide a useful tool for large-scale functional analysis of the Arabidopsis genome sequence and applications in plant-molecular breeding. Key words: Arabidopsis thaliana, high-molecular-mass DNA transformation, binary bacterial artificial chromosome, functional genomics, molecular breeding. En cette ere post-genomique, les banques de clones avec des inserts de grande taille dans des chromosomes bacteriens artificiels competents en vue de la transformation vegetale (BIBAC) sont importantes en vue d'analyses fonctionnelles et de reseaux portant sur de grandes regions genomiques, des amas de genes, des genes de grande taille et des locus complexes. Les auteurs decrivent ici la caracterisation d'une banque de clones BIBAC pour le genome sequence d'Arabidopsis, une espece pour laquelle une telle ressource n'existait pas dans le domaine publique. Les auteurs decrivent la transformation du tabac par biolistique a l'aide d'un grand clone BIBAC et l'analyse subsequente de l'expression des genes introduits chez les plantes transgeniques. La banque BIBAC a ete produite a partir d'ADN nucleaire partiellement digere avec BamHI, lequel a ete introduit dans le vecteur BIBAC pCLD04541. La banque totalise 6144 clones dont la taille moyenne est de 108 kb, ce qui correspond a une couverture de 5,2x du genome d'Arabidopsis et qui confere une probabilite de 99% d'identifier au moins un clone positif en employant comme sonde une sequence presente en simple copie. La transformation avec un clone BIBAC et l'analyse des plantes transgeniques obtenues ont montre que non seulement ces plantes possedaient l'insert BIBAC intact, mais aussi que l'insert BIBAC etait transmis de maniere stable et que les genes continuaient de s'exprimer activement. Ces resultats suggerent que cette banque BIBAC a grands inserts, combinee avec la transformation biolistique, fournissent un outil precieux pour l'analyse fonctionnelle a grande echelle du genome d'Arabidopsis et ouvre la voie a des applications en amelioration genetique. Mots-cles: Arabidopsis thaliana, transformation avec de l'ADN de grande taille, chromosomes bacteriens artificiels et binaires, genomique fonctionnelle, amelioration genetique assistee d'outils moleculaires. [Traduit par la Redaction], Introduction As numerous genomes are being sequenced and decoded rapidly, significant challenges have arisen on how to functionally characterize and efficiently use the sequences in plant genetic improvement. Multiple approaches [...]

Abstract: The successful assignment of the existing genetic linkage groups (LGs) to individual chromosomes and the second-generation linkage map obtained by mapping a large number of bacterial artificial chromosome (BAC) [...]

Abstract: Sequencing of the rice genome has provided a platform for functional genomics research of rice and other cereal species. However, multiple approaches are needed to determine the functions of [...]

The patatin multicopy gene family encodes the major storage protein in potato tubers and is organized as a single cluster in the potato genome. We sequenced a 154-kb bacterial artificial chromosome (BAC) clone containing a portion of the patatin gene cluster. Two putatively functional patatin genes were found in this BAC. These two genes are embedded within arrays of patatin pseudogenes. Using a chromatin immunoprecipitation method we demonstrate that the dramatic increase of patatin gene expression during the transition from stolons to tubers coincides with an increase of histone H4 lysine acetylation. We used 3' rapid amplification of cDNA ends to profile expression of different patatin genes during tuber development. The profiling results revealed differential expression patterns of specific patatin gene groups throughout six different stages of tuber development. One group of patatin gene transcripts, designated patatin gene group A, was found to be the most abundant group during all stages of tuber development. Other patatin gene groups, with a 48-bp insertion in the 3'-untranslated region, are not expressed in stolons but display a gradual increase in expression level following the onset of tuberization. These results demonstrate that the patatin genes exhibit alterations in chromatin state and differential transcriptional regulation during the developmental transition from stolons into tubers, in which there is an increased demand for protein storage.

The genome of the model plant species Arabidopsis thaliana has recently been sequenced. To accelerate its current genome research, we developed a whole-genome, BAC/BIBAC-based, integrated physical, genetic, and sequence map of the A. thaliana ecotype Columbia. This new map was constructed from the clones of a new plant-transformation-competent BIBAC library and is integrated with the existing sequence map. The clones were restriction fingerprinted by DNA sequencing gel-based electrophoresis, assembled into contigs, and anchored to an existing genetic map. The map consists of 194 BAC/BIBAC contigs, spanning 126 Mb of the 130-Mb Arabidopsis genome. A total of 120 contigs, spanning 114 Mb, were anchored to the chromosomes of Arabidopsis. Accuracy of the integrated map was verified using the existing physical and sequence maps and numerous DNA markers. Integration of the new map with the sequence map has enabled gap closure of the sequence map and will facilitate functional analysis of the genome sequence. The method used here has been demonstrated to be sufficient for whole-genome physical mapping from large-insert random bacterial clones and thus is applicable to rapid development of whole-genome physical maps for other species.