GenomicsandComparativeGenomicsHybridization(CGH)StudiesofRiceChromosome4;DifferentiationofaMITESystemandIts Inference For A Diphyletic Origin Of Two Subspecies Of Asian Cultivated Rice And Molecular Mechanism Of Stress Response Of At Transposon Famil

We developed a Pool PCR (PCR with multiplex primer pairs and templates) method to select the specific clones that are located on rice chromosome 4 long arm region of 56.1-68 cM from rice genomic libraries. Compared with molecular hybridization techniques, Pool PCR method has much higher efficiency, sensitivity, specificity and reusability. We report a novel strategy in appropriately grouping the primers and a deliberately designed PCR condition for developing this approach. With this method, we identified 65 positive clones, and of which, 29 clones in 8 contigs were confirmed to extend a total length of 500 kb from the existing contigs. Clone end sequencing, restrictive enzyme fingerprinting and Southern blotting were used to further confirm the Pool PCR results. In analysis of rice chromosome 4 sequences, we adopted an integrated platform with several gene prediction software and manual check (based on EST and homology search). We focused on the chromosome 4 region of 7.9-111.0 cM, summing up to 24 BAC covering 3 Megabase(Mb). Totally we annotated 307 genes which were categorized into specific classes for further study. We used a rice chromosome 4 specific DNA chip including 15,146 subclones from Nipponbare variety to carry out indica-japonica Comparative Genomic Hybridization(CGH) study. We used genomic DNA of Nipponbare and Guanglu'ai 4 for this study. We found about 60 positive subclones in which there are copy number differences between Nipponbare and Guanglu'ai 4. Further analysis revealed that the positive subclones mostly represented the retroelements and some others were related to resistance genes. Real-time quantitative PCR was adopted to prove the copy number differences. We also found some of the differences existing between the 2 subspecies of rice. Furthermore, we observed the correlation between the copy number and gene expression activity. We exerted a survey on a Miniature Inverted Transposable Elements (MITE) system known as mPing in 102 varieties of Asian cultivated rice Oryza sativa. We found that mPing population could be generalized into two families of mPing-1 and mPing-2 according to their sequence structures. Further analysis showed that these two families of mPing had significant bias in their distribution pattern in two subspecies of rice Oryza sativa ssp. japonica and indica. Japonica has a higher proportion of mPing-1 as a general trait, whereas indica has that of mPing-2. We also examined the mPing system in a Double Haploid (DH) crossbreeding population of jingxi17 (japonica) and zhaiyeqing8 (indica) varieties, and observed that the mPing system was not tightly linked to major subspecies-determining genes. Furthermore, we checked mPing system in 28 accessions of Asian common wild rice Oryza rufipogon and found the existence of the mPing system in O.rufipogon. The distribution pattern of the mPing system in O.rufipogon indicated a diphyletic origin of Asian...