High Through-Put Genotyping For Rice Recombinant Inbred Lines (RILs) And Comparative Analysis Of Oryza AA CC Genomes

With the development of biotechnology, the next-generation sequencing technology makes up the shortage of conventional biological methods. The next-generation sequencing technology coupled with the growing number of genome sequences opens the opportunity to redesign genotyping strategies for more effective genetic mapping and genome analysis. We have firstly developed a bar-coding sequencing strategy and a high-throughput genotyping method for recombinant populations by using Illumina Genome Analyzer to generate low coverage of rice whole genome sequences. By detecting SNPs between the RILs and their parents, we designed a sliding window approach to collectively examine genome-wide single nucleotide polymorphisms (SNPs) for genotype calling and recombination breakpoint determination. Using this method, we constructed a genetic map for 150 rice recombinant inbred lines with an expected genotype calling accuracy of 99.94% and a resolution of recombination breakpoints within an average of 40 kb. In comparison to the genetic map constructed with 287 PCR-based markers for the rice population, the sequencing-based method was approximately 20 times faster in data collection and 35 times more precise in recombination breakpoint determination. Using the sequencing-based genetic map, we located a quantitative trait locus of large effect on plant height in a 100 kb region containing the rice'green revolution'gene. Through computer simulation, we demonstrate that the method is robust for different types of mapping populations derived from organisms with variable quality of genome sequences and feasible for organisms with large genome sizes and low polymorphisms. With continuous improvement of sequencing technologies, this genome-based method may replace the traditional marker-based genotyping approach to provide a powerful tool for large-scale gene discovery and for addressing a wide range of biological questions.Comparative analyses of genome structure and sequence of closely related species have yielded insights into the evolution and function of plant genomes. A total of 103,844 BAC end sequences delegated ~73.8 Mb of O. officinalis that belongs to the CC genome type of the rice genus Oryza were obtained and compared with the genome sequences of rice cultivar, O. sativa ssp. japonica cv. Nipponbare. We found that more than 45% of O. officinalis genome consists of repeat sequences, which is higher than that of Nipponbare cultivar. To further investigate the evolutionary divergence of AA and CC genomes, two BAC-contigs of O. officinalis were compared with the collinear genomic regions of Nipponbare. Of 57 genes predicted in the AA genome orthologous regions, 39 had orthologs in the regions of the CC genome. Alignment of the orthologous regions indicated that the CC genome has undergone expansion in both genic and intergenic regions through primarily retroelement insertion. Particularly, the density of RNA transposable elements was 17.95% and 1.78% in O. officinalis and O. sativa, respectively. This explains why the orthologous region is about 100 kb longer in the CC genome in comparison to the AA genome.