Collection And Sequence Analysis Of 10,081 Full-length CDNA From Indica Guangluai 4 And Construction Of Single Chromosome DNA Library Using A Laser Microbeam Trap

The indica subspecies of cultivated rice occupies the largest area of rice production in the world. In this study, we collected and completely sequenced 10,081 full-length cDNAs from Oryza sativa L. ssp. indica cv. Guangluai 4 (NCGR-cDNAs) using an advanced full-length cDNA capture technique.Transcriptome comparison between indica and japonica (KOME full-length cDNAs) showed 2,693 NCGR unique cDNAs and 7,388 NCGR-KOME cDNA homologue pairs. Of the 2,693 NCGR-cDNAs that have no homologies in KOME cDNAs, 1,751 matched to japonica genomic sequences, and were expressed in both varietyes, while their expression varies between two subspecies. Sixty-four cDNAs that had no matches in japonica genomic sequences, matched indica 93-11 genomic sequences; the majority of these cDNAs were specifically expressed in Guangluai 4. PCR screening, in 3 indica varieties and 5 japonica varieties, further confirmed their unique presence to indica varieties. Of the remaining 878 cDNAs that had no matches in either Nipponbare or 93-11 genomic DNA sequences, part were confirmed for their rice origin by Southern blot analysis.Among 7,388 NCGR-KOME cDNA homologues, 7,346 were predicted to have open reading frames (ORFs). Of these ORFs, 2,487(33.8%) were identical at protein levels between indica and japonica subspecies, and 1, 273 (17.3%) were with single nucleotide polymorphisms (SNPs). The remaining 3,586 (48.8%) cDNAs showed substantial differences at protein level because of SNPs, insertions and deletions (Indels), and non-homologue sequences (NHS). The percentage of identical full-length cDNAs between indica and japonica including 5'and 3'UTRs, was only 7.5%. These cDNA variations between indica and japonica might distinguish the phenotypic changes of the two cultivated rice subspecies. We aligned the NCGR-cDNA clones to the corresponding rice genomic sequences and identifyied 7,573 transcription units (TU). We identified 1,382 indica alternative splicing transcripts corresponding to 540 TUs. Among them, 93 AS were conserved in indica and japonica. We also found 32 pairs of anti-sense transcripts in the NCGR-cDNAs. Our NCGR-cDNA resource for indica rice provides a platform for genome-wide comparison of two subspecies both in gene structure and further verification in biological function. Analysis of these cDNAs extends known rice genes and identifies new ones in indica variety.For the construction of rice chromosome specific DNA library, we developed a laser micro-manipulation system and applied it to the isolation of individual rice chromosomes directly from a metaphase cell. The cell wall was cut by an optical scissors, and the released single chromosomes were captured by an optical trap. Then, the single chromosomes were collected by a 2μm micropipette and chromosome-specific library was constructed by linker adaptor PCR. The rice chromosome 4-specific library was confirmed by PCR amplification with rice chromosome 4-specific primers. The developed method can also be applied to the preparation of other subcellular structures and to the cloning of single macromolecule through a laser microbeam trap.