A Plant-Transformation-Competent BIBAC-Based Integrated Physical Map Of Japonica Rice(Oryza Sativa L.ssp.japonica Cv. Nipponbare) And Two BIBAC/TAC Libraries Of Wild African Rice(Oryza Barthii)

Following the release of the genome sequence of rice, the experimental determination of the function(s) of every gene of the genome has been targeted as a goal for the coming decades. To facilitate large-scale functional analysis of the rice genome sequence experimentally, I have developed a genome-wide, plant-transformation-competent BAC/BIBAC-based, integrated physical and genetic map of Oryza sativa L. ssp. japonica cv. Nipponbare. This map was developed from two BAC libraries and one plant transformation-competent BIBAC library of Nipponbare using the DNA sequencing gel-based restriction fingerprinting method. The map was constructed from 20,835 clones, which represents 7.2 haploid rice genome equivalents. The clones were fingerprinted using Hind III and Hae III on DNA sequencing gels and assembled into overlapping clone contigs. The map consists of 579 contigs, spanning 525 Mb of the 430-Mb rice genome. The reliability of the map was verified using different strategies including DNA marker screening and clone end-sequence alignment. Six hundred fifty two rice BIBAC/BAC end sequences (rBESs) of 637 BIBAC/BAC clones were generated for the vast majority of the map contigs to integrate with the rice genome sequence and facilitate the utility of the map in subsequent research. As a part of physical map project, a fine integrated BIBAC/BAC-based physical map of rice chromosome 8 has also been produced by landing 59 genetic markers and 36 end-sequenced clones to the contigs. This chromosomal map consists of 11 contigs with a total length 38.9 Mb. There are some distinguishing characteristics of our physical map and would be more powerful tool for rice genome research compared to other physical maps published so far. First, the BIBACs of this new map were especially designated to be transferred into plants by the Agrobacterium-mediated transformation method, therefore, the integrated map has provided a readily used platform for large-scale functional analysis of the genome sequence, including the gene and non-gene sequences, through Agrobacterium-mediated transformation of the BIBAC clone inserts into plants. Secondly, the BIBACs were constructed with an AT-rich enzyme (Hind III) and a vectorsystem, differing from the other two BAC libraries with Bam HI and Eco RI, thus, the BIBACs are complementary to the other BACs in this map and desirable for closing the gaps in the sequence map. Third, tagged with 652 rBESs, this map may be used to fill the gaps of the existing rice sequence maps by alignment of the both sequences. Fourth, an integrated genetic/physical map for chromosome 8 is detailed in this map, which will benefit many aspects of genome research on the chromosome. Additionally, the successful development of the BAC/BIBAC-based physical map of the rice Nipponbare genome has further demonstrated that the DNA sequencing gel-based restriction fingerprinting method is reliable and powerful for genome-wide physical mapping with large-insert BACs and /or BIBACs.To enhance the functional analysis of genome sequence and positional cloning of genes of the wild species of rice, two large-insert plant transformation-competent BIBAC and TAC libraries were constructed for Oryza barthii, a wild ancestor of African cultivated rice. These two libraries were constructed from Bam HI partial digested genomic DNA of O. barthii and cloned in the binary vector pCLD04541 and TAC vector pYLTAC7, respectively. The BIBAC and TAC library have been arrayed in 40 384-well micro-plates respectively and characterized. The BIBAC library has an average insert size of 130 kb, 2% empty clones and estimated to covers 3.2 haploid genome equivalents; while the TAC library has an average insert size of 116 kb, less than 8% of empty clones and estimated to covers 2.7 haploid genome equivalents. In combination, these two libraries contain 30,720 clones with an average insert size of 123 kb, representing approximately 5.9 haploid genome equivalents and providing a greater than 99% probability of isolating a single copy DNA sequence from the lib...