Construction of a genomic library of wild rice and Agrobacterium-mediated transformation of large insert DNA linked to BPH resistance locus

Abstract

Here we report the first genomic library of wild rice constructed on a plant-transformation-competent binary vector (BIBAC2) and transformation of the large insert DNA into rice via Agrobacterium. We selected Oryza officinalis for genomic library construction. The library consists of 55,296 clones and stored in one hundred forty-four 384-well plates. Random sampling of 140 clones indicated an average insert size of 71 Kb at a 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 binary bacterial artificial chromosome (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 large DNA inserts were stable in this BIBAC vector both in host cells of Escherichia coli and Agrobacterium. Two restriction-fragment length polymorphism (RFLP) markers R288 and C820, which co-segregate with brown planthopper (BPH) resistance gene Qbp2, were used to screen the library, and identified seven and eight positive clones, respectively. The candidate clones of target gene isolated from the library are directly used to transform cultivated rice. After screening the Agrobacterium strains and helper plasmids, and using an improved procedure of transformation, a BIBAC clone with 120 Kb O. officinalis DNA insert was successfully transferred into the rice genome via Agrobacterium-mediated transformation. The system developed here should serve as source for gene discovery, gene cloning and genome-related research in wild rice.