Construction Of The Bacterial Artificial Chromosome Library For Gossypium Herbaceum Var. Africanum

G. herbaceum var. africanum is the only wild type of two diploid cultivated cotton, and is the donorof A sub-genome in the tetraploid cotton. It has both the fiber production of cultivated cotton and theexcellent characters of insect and disease resistance of wildlife. Its genome information is important todevelop the biological or non-biological stress gene. But there is seldom report on it for rare material.On the base of the construction of bacterial artificial chromosome （BAC） Library for tetraploidcotton, we improved some steps of constructing processes, so this study first constructed the BAClibrary of G. herbaceum var. africanum which is the the donor of A sub-genome. Then some SSRmarkers which located on linkage map and closely linked with resistant gene were used to screen theBAC library, the results were as follow.1. In this study, the materials were seedling cotyledon of Gossypium herbaceum var. africanumwith darkness culture and leaves of Gossypium tomentosum with darkness culture. According to themethod of Ma, we compared the high molecular weight （HMW） nuclear DNA preparation effect ofdifferent treatment of materials. the results were that, the plugs which were prepared with seedlingcotyledon were transparent, it showed that HMW nuclear DNA extracted from seedling cotyledon waspure and nearly has no foreign substance pollution such as gossypol acetate and so on. However, theplugs which were prepared with true leaves were brown, it showed that HMW nuclear DNA extractedfrom true leaves was impure and has oxidation of gossypol acetate. But these two kinds of plugs weredetected by pulsed field gel electrophoresis（PFGE） showed that, the main bands of two kinds of HMWnuclear DNA was obviouse and had the same size, the following study showed that two kinds of HMWnuclear DNA were digested by restriction enzyme and had high transformation efficiency. thetransformation products were detected that both of the insert fragment were satisfied the need of thestudy. the results explained that existence of gossypol acetate had no influence to costructe the BAClibrary.2. In the study, the first bacterial artificial chromosome library of G. herbaceum var. africanum withhigh quality and coverage percentage was constructed based on pIndigoBAC-5（Hind III-CloningReady）. The library included75000clones and represented an equivalent of about five fold size ofdiploid genome based on A-genome size of1667Mb. In the BAC library, most of the insert sizeconcentrated on100-150kb,100-120kb clones accounted for40%,120-150kb clones occupied26.2%,70%clones were more than or equal to100kb, the average insert size was about115kb and emptyclones were less than4%. The BAC library provided99.3%probability for isolating any interestedgenes or sequences in the library, the screening probability was more than international general standard,so it meaned that the construction of BAC library could meet the gene screened. The deep coverage andthe large insert size of the library provided an important genomic resource for the gene localization,map-based cloning, comparative genomics of A genome and other genome, classification andevolvement of cotton species.3. In the processes of constructing BAC library, the initial time50s of first selection to large DNA fragment was changed to10s. the results was that,100-200kb DNA fragment we wanted wererecoveried but it were not recoveried in previous study. At the same time,100-200kb DNA fragmentwere concentrated and had high transformation efficiency.4. In order to validate the genome coverage and the value of the BAC library,40super pools wereconstructed on the base of sigle clone of every384plate mixed together. nine SSR markers wereselected to screen40super pools, which were located on five different chromosomes A_1-a05, A_1-a07,A_1-a08, A_1-a09, A_1-a11and linked with the Verticillium wilt resistance. The results showed that therewere7pairs of SSR primers with which the positive clones were screened, and the hits were between1-14. The positive clones correlated with the Verticillium wilt resistance provided an important genomicresource to develop molecular markers, isolate disease resistant gene and controlling elements.