| Cotton is one of the main natural fiber and major cash crops in the world. Among them Gossypium arboreum has a very economic high value in production because of its advantage about early maturity, stress resistance, insect resistance, high fiber strength. It is difficult to prepare good chromosome preparations of cotton, not only because of the cotton chromosomes are small and similar, but also there are much substance such as polysaccharide, tannins and gossypol in cotton cell. The study was aim at constructing the technique about chromosome microdissection, and a single chromosome library construction. Forthermore, cloning resistance gene analogs (RGAs) in a single chromosome pool from second Diolig-prime-polymerase chain reaction (DOP-PCR). We were looking forword to accumulating basic cotton genome research data in the end. The major findings are as follows:1. Complete the system of a single chromosome microdissection, protein removed, enzymolysis, PCR and verification of production in Gossypium arboreum. The high qulity mitosis metaphase chromosome membrane preparations of Gossypium arboreum suitable for laser cutting were obtained after dealed with the method of pre-hypotonicity, enzymolysis, post-hypotonicity and squashed with cover slide. Amplified production was obtained after the single chromosome was removed protein, enzymolysis and PCR. A verified system was constructed after integrating the method of Southern blotting, SSR primer amplification and fluorescence in situ hybridization. A highly efficient technique of cotton chromosome microdissection was completed.2. Studying on cotton chromosome painting. According to the study of chromosome painting, Gossypium arboreum has high homology with Gossypium herbaceum, it is speculated that the two species has close relationship. Compared with five tetraploid cotton, it was found that Gossypium arboreum had the lowest homology with Gossypium mustelinum, had lower homology with Gossypium tomentosum, Gossypium darwinii, but had higher homology with Gossypium hirsutum and Gossypium barbadense, and had highest homology with Gossypium hirsutum, which indicated that the degree or speed of evolution between the tetraploid cotton may different.3. Single chromosome library construction. A single chromosome library of the 7th chromosome of Gossypium arboreum Shixiya 1 was constructed. There were approximately 38,000 recombinant clones in the library. After analying the library it was found that the size of the inserted DNA sequences varied from 150bp to 1000bp and the average size was about 550bp, the coverage of library was about 1.9 times, the rate of no-load was 1%, the titer was 1.3×106pfu/mL, single and low copy sequences were over 59%. The construction of the 7th chromosome library of G. arboreum Shixiya 1 would facilitate the cloning and mapping of important genes, specific probe screening, genetic map saturation on this chromosome.4. RGA Cloning from a single cotton chromosome. About 30 DNA sequences were obtained after amplified with the second round DOP-PCR as plate and with disease-resistant homologous sequence as primer. Five classic nucleotide sequence L3, L11, L15, L20 and L27 were selected after cluster and sequencing among the 30 DNA sequences. Sequences alignment with other resistant gene analog homologous showed that the five sequences were the typical NBS-LRR-type resistant gene analog. Clustering analysis with several plant RGA sequences indicated that the sequences of L3, L11 and L15 in the same cluster and were highly homologous with one RGA sequence from Populus trichocarpa, NBS-LRR, mRNA (gi|224102274| ref|XM_ 002334160), L20 and L27 was in the other cluster and homologous with two RGA sequences, RGA17 (gi|53680927|gb|AY746413.1) and RGA12 (gi|53680917|gb|AY746408-.1) from Citrus reticulata Blanco. |
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