Isolation And Amplification Of Single Chromosomes And A Preliminary Trial Of High-throughput Screening Of Chromosome-specific CDNA Probes In Jute

The technology of chromosome microdissection and microcloning is useful for genetic research in plants. However, as most plants have small chromosomes that are difficult to be distinguished, the technology has not been widely applied to plants. In the present study, with jute as a model, which has typical small chromosomes, the isolation and amplification of single chromosomes and the high-throughput screening of chromosome-specific cDNA probes using uncloned DNA libraries of single chromosomes and cDNA macroarray were investigated. Major results are as follows:1) Single chromosomes of Corchorus olitorius L Minyin NO.l and Corchorus capsularis L. Gaoxiongqingpi were successfully isolated by micromanipulation with glass needles, 80 chromosomes were obtained for each species.2) The isolated jute chromosomes were amplified by LA-PCR or DOP-PCR. After two rounds of PCR amplification, the DNA fragments ranged from 250bp to 2000bp were obtained by LA-PCR and 100bp to 600bp were obtained by DOP-PCR. 45 chromosomes were amplified by LA-PCR and 5 chromosomes were amplified by DOP-PCR for Minyin NO.l and Gaoxiongqingpi. Therefore, 50 uncloned single-chromosome DNA libraries were established for both Minyin NO.l and Gaoxiongqingpi. Southern blot verified that the PCR products were from the jute genomic DNA.3) cDNA libraries of both Minyin NO.l and Gaoxiongqingpi. Both libraries had a liter of 1.8-1.9 X106 pfu/ml.4) High-throughput screening of probes through hybridization between cDNA macroarrays and isotope-labled PCR products of single chromosomes wastried. Result suggested the feasibility of the approach for obtainingchromosome-specific probes.