Construction Of Rice Bac-Based Physical Maps And Localization Of Related Database, Analysis And Query Systems

Rice, a staple food crop, is also a model plant for monocotyledon research. Genomic libraries with large inserts and physical maps are a valuable resource for genome sequence assembly, comparative genomic analysis, gene cloning and functional analysis, and so on. In this dissertation, we describe the construction of a BAC-based physical maps for rice Zhonghua11and93-11, and then, used them to do comparative genomics study and reassembly of93-11WGS sequence.Zhonghua11, as an important host rice variety for its high transformation efficiency through Agrobacterium, has been used to produce a large number of T-DNA insertion mutants to facilitate the identification, isolation and analysis of many functional genes. Here, we constructed a BAC library consisting of38432clones, with average insert size of124kb, arrayed them in96well384plates, fingerprinted and end sequenced half of clones in library. End sequencing produced35919reads with at least100high-Quality base (Phred Q>16) that containing5.93%organic sequence,31.93%repeat sequences. Besides31.93%previously identified repeate sequences,18additional Zhonghua11specific high-frequent fragments were identified. Four fragments show specific repetitiveness in O. rufipogon, another four fragments show specific repetitiveness in O. australiensis. Fingerprinting was successful for17527clones,16042of which were assembled into648contigs. BESs were used to anchor contigs to Nipponbare genome sequences. Through comparison, a global view of structural variations, substitutions, InDels and differences of repetitive sequences within the japonica subspecies was presented for the first time. The two genomes are generally highly syntenic and conserved but substancial variations were discovered. Contractions/expansions as large as140kb were found at contig level. ZH11-specific Repeat sequences were detected. In the tri-alignment regions using the indica variety93-11sequence as an outgroup, we found that:1) the substitutional rates of the two japonica-indica inter-subspecies comparison combinations are very close but almost a magnitude higher than that between ZH11and Nipponbare within the japonica subspecies;2) the substitution rate occurred in ZH11is lower than that in Nipponbare (52.6%);3) Of the indels found between ZH11and Nipponbare, the indels occurred in ZH11are15.8times of those in Nipponbare. Of the indels occurred in ZH11,75.67% are insertions and24.33%deletions. Of the indels occurred in Nipponbare,48.23%are insertions and51.77%deletions. The divergent times between ZH11and Nipponbare, ZH11and93-11, and Nipponbare and93-11were estimated to be about0.13,0.39and0.50MY respectively. The ZH11comparative map covered four Nipponbare physical gaps, detected assembly errors of the Nipponbare sequence and was integrated with the FSTs of a large ZH11T-DNA insertion mutant library.The genetic information of other rice varieties, such as BES of Kaslath, genome sequence of Nipponbare, were used to guide the assembly of93-11WGS sequence. Considering the divergence between varieties may misguide the assembly, we contructed the physical map of93-11in the hope of employing it to improve the quality of93-11assembly. The BAC library used here consists of36864clones, all of which were fingerprinted and end sequenced. Analysis of32205useful fingperprints assembly32205clones into285contigs. End sequencing produced65119BESs with total length of39864033bp. Through aligning BES to reference genome sequence,268clone contigs were anchored to Nipponbare genome sequence, while272clone contigs were anchored to93-11genome sequence. There were371paired ends hit to Nipponbare genome sequence in the same orientation, which may be caused by the assembly error in assembly of physical map or Nipponbare genome sequence, or the innate difference between the genome of93-11and Nipponbare. There were406paired ends hit to93-11genome sequence in the same orientation, which may be caused the assembly error in assembly of physical map or93-11genome sequence. FISH results of clones from7contigs showing different location in Nipponbare and93-11genome sequence verified the correctness of physical assembly and indicated that adjust WGS sequence based on physical map can improve the quality of93-11genome assembly.