Study On The Microdissection And Centromere In Cotton

Over the past nearly 20 years, the structure and function of centromere and filling the gap in centromere area of physical map have become one of the hotspots in molecular cytobiology and molecular cytogenetics, but there was no breakthrough in cotton centromere. Cotton is most important fiber crops in China even in the world, the study of cotton centromere, can not only resolve the long-term debate of theoretical issues, but also reference and help for cotton sequencing and genome assembly. In this study, we used microdissection and PCR technology, explore the cotton centromere. The main results were as follows:1. Establishment of chromosome or chromosome segment microdissection and LA-PCR technology in cotton. After pre-hypotonicity, enzymolysis, post- hypotonicity and squashed with cover slide, the mitosis metaphase chromosome slides suitable for cutting was obtained. Use LA-PCR technology, amplification the chromosome or fragment from microdissection, it can reduce the number of chromosome. Amplified product was verified by fluorescence in situ hybridization. We established a highly efficient, fast, easy-to use system of chromosome or fragment microdissection in cotton.2. When we got the centromere fragments from microdissection, they were amplified by LA-PCR, but there was no production. Maybe there be two reasons leading to this result: One reason is cotton chromosomes are very small, when we burn the end of chromosomes, we also destroy the centromere area.The other reason is that it's possible the centromere area dose'thave suitable Sau3A restriction enzyme cutting site. Which reason leads to the result, we still have to check in the future experiments.3. Microdissection 5th chromosome of G. arboretum Shixiyal and amplified by LA-PCR, then check the production by FSIH. It was revealed that all of the chromosomes have hybridization signal, and two chromosomes have strong signals. Double FISH was done ues 5th chromosome specific clone 87P01 and the LA-PCR production, it was found that the 87P01 signals were located on two chromosome which have stronger signals than others.4. Designed four pairs primers from A. thaliana centromere sequence (Cen1, Cen2) and retrotransposon sequence (CR1, CR2). But only one primer amplified production from G. arboretum genome DNA and G.raimondii genome DNA, then check the production by FISH, there was no signal neither on G. arboretum chromosome nor G.raimondii chromosome. It may be because that the centromere sequence and retrotransposon sequence between Gossypium and A. thaliana have very slow homology or no homology.5. Two primers were designed through G. barbadense PM-90 gypsp retrotransposon sequence, after amplified, the production were check by FISH, only primer K202 has signals on G. arboretum chromosome, and the signal located near telomere. When we check every segment of the production, it was found that one 818bp segment come into being the signal. Then we sequence the fragment and Blast in NCBI. found the sequence have 94% homology compared to gypsp retrotransposon and 98% homology compared to LIB5327-017-A1-N1-D12_P_S_Gh557 of microsatellite sequence in Ghirsutum.