| The wild germplasm is a valuable gene pool that can be used to broaden the genetic background of modern crops. Because of the massive international exchange of germplasm and repeated use of the limited number of productive parental materials in breeding programs, new varieties are usually derived from crosses among genetically related modern varieties. The genetic diversity in improved rice has narrowed, limiting the further increase in yield potential of newly developed varieties. There is thus an urgent need to explore and utilize the wild Oryza germplasm to meet various challenges affecting rice production.O. officinalis well ex watt, a species with a diploid CC genome, occurs wildly in the South and Southeast Asia. O. officinalis has been intensively studied.Some of these genes including resistance to brown planthopper (BPH), white backed planthooper (WBPH) and bacterial blight (BB) have been transferred from O. officinalis into several rice breeding lines by interspecific hybridization and backcrossing.The bacterial artificial chromosome (BAC) cloning system has provided a powerful tool for physical mapping, map-based cloning and genome sequencing in genome research. While BAC vectors are designed for cloning large fragment of DNA, they are not engineered for transformation of the cloned DNA back into plant genomes. A new generation of vector (termed binary-BAC or BIBAC) has been designed that is capable of replicating in both Escherichia coli and Agrobacterium tumefaciens and is able to deliver a large insert of DNA directly into the plant genome via Agrobacterium-mediated transformation. BIBAC libraries not only can be used as large-insert DNA libraries in genome research, but also facilitate gene discovery and functional studies by direct transformation of BIBACs carrying the genes, gene clusters (e.g., genes of disease resistance) or QTL of interest, into plant. While the BIBAC is proved as a useful system for transfer of large DNA fragments into the plant genome in dicotyledon, the construction of BIBAC library and the transfer of large insert DNA have not yet been reported in monocotyledonous plants.DNA transfer to plants has been accomplished by many methods, including Agrobacterium-mediated transformation, biolistic transformation (particle bombardment), and microinjection. Whereas plant transformation with DNA fragments up to circa 20 kb is routine, success in stable plant transformation with DNA fragments larger than 50 kb is limited. The technologies of cloning and transferring of large DNA fragments in plants are important for high-efficiency identification of new genes and study of gene functions. A reliable system for transforming large fragments of DNA into plant make it feasible to introduce a natural gene cluster or a series of previously unlinked foreign genes into a single locus. Thus, several diseases and/or pest resistance genes or genes encoding the enzymes of metabolic pathways can be simultaneously introduced in onlytransformation step. Large insert transformation would make it feasible to study the expression of plant genes or gene clusters in their native genomic context and might eliminate site-dependent gene expression, which can be serious problem in plant transformation experiments.To develop the resource for transferring and identifying valuable genes and carrying out genome-related research in O. officinalis, we constructed the first genomic library of wild rice using a new generation of vector BIBAC2. The library consists of 55,296 clones and stored in 144 384-well plates. A random sampling of 140 clones indicated an average insert size of 71 Kb at range of 15-235 Kb and 4.8% empty vectors. Four wheat chloroplast probes and four maize mitochondrial probes were hybridized separately to the library, showing that contamination with organellar DNAs is very low (0.61% and 0.04%, respectively). The BIBAC library provides 5.3 haploid genome equivalents, implying a 99.5% probability of recovering any specific sequence of interest. A stability test indicated that the... |
PDF Full Text Request