Establishment Of 1B Chromosome Specific DNA Library Of Wheat And Identification

The microdissection and microcloning of single chromosome of wheat (Triticum aestivum L.) was carried out. The IB chromosome specific DNA library of wheat was established and analyzed. The research will be useful for intensive molecular linkage map construction of chromosome IB and the isolation of important genes located on chromosome IB.1. Establishment of the method for single chromosome microdissection using microglassneedles, as well as amplification with the dissected chromosomeFreshly made mitosis metaphase spreads of wheat (Triticum aestivum L.) of cultivar "Jing 411" was used for microdissection. The chromosome IB with a satellite was isolated and transferred into an eppendorf tube with a glass neddle. After deproteinization with proteinase K and restriction, the DNA of chromosome IB was amplified by two rounds of linker adaptor PCR. The size of the amplified DNA varied from 300 to 2500 bp (with dominance in 1000 bp). The PCR products originated from the genome of wheat, which were verified by Southern hybridization. Compared with previous reports, there are some advantages in our method: the performance is easier, the dissection is more precise and the cost is low. This is the first report about the microdissection and amplification of chromosome IB.2 Construction of specific DNA library from chromosome IB of wheat and analysis of the clonesThe chromosome 1B DNA library was established by cloning the second round PCR product into pUCm-T vectors. The clone number was about 2.48X 105 and 100 clones from the library were analyzed by dot hybridization and plasmid restriction. The size of clone inserts varied from 500 to 2000 bp (with an average of 1000 bp approximately). It consisted of 39.6% single/low-copy-sequence clones, 50.4% medium/high-copy-sequence clones and 10% empty clones. Compared with previous reports, the length of inserts reported here was obviously longer. The library will provide plenty of chromosome-specific probes for genetic mapping.Two microsatellite primers located on chromosome 1B were used for verification of the PCR product origination. It was confirmed that the amplification DNAs came from IB chromosome. On the other hand, it showed that the microclone library contained abundant single sequence repeats and have potential usage for exploring chromosome specific microsatellite markers.3 PCR amplification of DNAs of chromosome IB and initial analysis of the resultTwo primers were designed according to published HMW-GS gene sequences of wheat and used to amplify DNA using the second round LA-PCR products as the template. A specific sequence which has no homology with the sequence registered in Genbank was obtained. Further study of this sequence needs to be done to verify if it is specific DNA sequence of chromosome IB.