Construction Of A Bacterial Artificial Chromosome (BAC) Library Of Pima90-53 And Identification Of BAC Clones Related With Verticillium Wilt Resistance Gene

Cotton is one of the main crops and source of the natural fibers. At present, Upland cotton and Sea Island cotton of the tetraploidy are main cultivatable species all over the world. They account for 90% and 8% of the cotton total yield in the world, respectively. The Sea Island cotton has good fiber quality and high Verticillium wilt resistance. So we constructed a library of the cultivar Pima90-53 which belonged to Sea Island cotton. It is important for us to construct Sea Island cotton BAC library with good fiber quality and high Verticillium wilt resistance. BAC clones which have important characters can be fished and analyzed utilizing molecular marker probes, and then find new molecular markers, isolate the important genes and launch functional and comparative genome research thoroughly. This libarary will play an important role in improving the level of breeding in cotton.The results are as follows:Using the resistant Sea Island cotton variety Pima90-53 as the material, in the foundation of our group of constructing BAC library, the research made some improvements, constructed BAC library of this variety complementarily and got 77,568 clones. Finally the clones of the library were 167,424.Insert sizes of 224 clones were checked. They varied between 50 and 260kb, with a combined average of 130kb. About 94.0% of the clones had inserts over 100kb and the empty clones accounted for less than 4.0%. Contamination of the genomic library with chloroplast clones was very low (0.2%). The library represents 7.2-fold genome equivalents. Randomly selected BAC clones were blotted and hybridized with a probe from the total genomic DNA of Pima90-53. Results of Southern blotting indicated that all of the BAC inserts from the BAC clones gave hybridization signals. To determine the stability of BAC clones in host E. coli strain, DH10B, 7 randomly selected BAC clones were analyzed at generation 0 to 100. No visible changes were observed. Two probes, including Fb38 (a putative G. barbadense fiber protein gene) and MYB transcription factor (involved in cotton fiber initiation), were chosen as probes for the library screening. Two positive BAC clones were isolated for Fb38 and forty for MYB from 43,776 BAC clones screened. The above results showed that this BAC library is of high quality and isimportant genome resource for map-based cloning of important genes, cotton physical mapping, genome sequencing and development of SSR primer.The fragment PR8 related with Verticillium wilt resistance was used as a probe to screen Bacterial Artificial Chromosome (BAC) library of a Sea Island cotton, Pima90-53 Four positive BAC clones, 127K13, 128D14, 169J3 and 178C5, were detected from 30,336 BAC clones. The BAC clone, 169J3, was digested with Sau3A I . After digestion, 2kb~4kb fragments were collected and ligated with pUC1185awH I -BAP to construct a subclone library. The library contained 4,224 clones, and inserts DNA ranged from lkb to 3kb, averaged 2kb. Twenty-four positive subclones were isolated and sequenced, we obtained a fragment related with Protein Transporter, this fragment has 2317 base pairs with five open reading frames. Blast analysis showed that this fragment has high homologous with genes induced by water, cold and phoma. This fragment may work in the signal conduction in cotton resistance response. It laied a foundation of obtaining the complete sequence of PR8 linked with the gene of Verticillium wilt resistance and developing SSR markers linked with the resistant gene.