SSR Markers Location And Utilization Of Restorer Gene For Cytoplasmic Male Sterility In Gossypium Harknessii

The phenomenon of cytoplasmic male sterility （CMS） exists generally in the natural world, whichplays an important role in crop breeding and heterosis utilization. Gossypium harknessii is a vitualcytoplasmic male sterile material in cotton, whose prominent virtues are thorough pollen abortion,steady sterility and easy to be utilized in achieving three line combination. The utilization of CMS linesis much more effective and economical in producing commercial hybrids, because it can avoid handemasculation and has outstanding advantages of labour saving, cheap price, and high quality of hybridsand so on. At present, Gossypium harknessii has realized completely system of “three lines”, however,this system has not been widely used because of the lack of elite restoring lines. Therefore, study ongenetic and localization of restorer genes in Gossypium harknessii, will not only provide services toselect superior restorer lines with molecular marker assisted and test seeds genetic purity, but also layfoundation for studying the mechanism of nucleo-cytoplasmic interactions and cloning restorer gene.The experiment materials are CMS line ZBA, maintainer line ZB, restore line Zhonghui-46and F₂population of ZBA×Zhonghui-46and Near-isogenic lines （NILs） of fertility restorer genes of（ZBA×Zhonghui-46）×ZB8combination. By studying the segregations pattern of the fertilities ofF₂population and NILs, the inheritance of the fertility restorer gene was identified. SSR markers werechosen on fertility restorer gene of NILs by bulked segregation analysis （BSA） strategy,and subsequently Rf1gene was mapped and seeds purity were tested with these polymorphic markerslinked to fertility restorer gene and a SCAR marker closely linked to sterile gene. The main resultsobtained were as follows:1. Plants of the NILs were consisted of223fertile plants and186sterile plants, and plants of the F₂population were consisted of502fertile plants and193sterile plants. Based on the χ2test, the ratioof segregation was1:1in NILs and3:1in F₂population, respectively. This result confirmed thatfertility restoration was controlled by one dominant restorer gene.2. The fertile and sterile bulks were conducted by bulked segregant analysis to screen for markerslinked to the Rf1restorer gene, and13SSR molecular markers screening against the Rf1gene wereconducted from the8242pairs of SSR primers, which3marks of COT010, CGR5340andNAU3938are published for the first time. Combined with fertility survey results, the separation ofNILs of409individuals and F₂of695individuals were analyzed with the13polymorphic marks.The results showed that among495NILs plants, only one plants was represented fertile fragmentwhich was in accordance with its sterile fragment with NAU2650, while2markers ofCGR5340and BNL3535didn’t find exchange plant, one exchange plant was found with eachprimer.3. Rf1genetic linkage maps of NILs and F₂populations were made with JionMap3.0software,respectively, and the total genetic distance was all less than0.5cM. NAU2650was linked to Rf1with a genetic distance of0.245cM and12other markers showed co-segregated with Rf1in the NILs. A linkage map was constructed with the same13SSR markers based on an F₂population, too,with a total of0.35cM; and Rf1gene was located between the SSR markers BNL3535and CM003,which had a distance of0.042cM and0.119cM, respectively. By comparising genetic linkage mapsof cotton and analysising of the position of the13SSR markers closely linked to Rf1gene in cottonchromosome, we found that7out of13SSR markers were located on chromosome19（D5chromosome）. For SSR markers were more stable and could be used as anchor marker, Rf1genewas mapped in chromosome19.4. Seeds purity of Zhonghui-46was tested using the2co-dominant markers COT010and NAU3938,meanwhile, genotypes of the sterile line, maintainer line, restorer line and their F1seeds andconvertional hybrids were identified with the2markers CGR5340and NAU3938linked to fertilegene and a SCAR marker closely linked to sterile gene. PCR results showed that these markerscould produce qualified fragments, so these markers closely linked to Rf1gene, especiallyco-dominant markers, are very suitable for testing the “three system” and its hybrid seeds purity andselecting elite restoring lines by molecular marker-assisted selection.