【作者】 曹秀霞；

【导师】 邢朝柱；

【作者基本信息】 中国农业科学院 ， 作物遗传育种， 2012， 硕士

【摘要】 细胞质雄性不育（CMS）现象在自然界普遍存在，是作物杂交育种和杂种优势利用的重要途径之一。哈克尼西棉是棉花中重要的胞质不育材料，其突出优点是败育彻底、能稳定表现出雄性不育特性，并能实现“三系”配套。利用其“三系”生产杂交种可免除人工去雄，具有省工、生产成本低、杂交种子纯度高等优点，在规模化生产棉花杂交种中具有很高的应用价值。目前，哈克尼西棉已实现“三系”配套，但受恢复系资源匮乏的制约，长久以来难以在棉花杂交种生产上被广泛应用。因此，开展哈克尼西棉细胞质雄性不育育性恢复基因遗传和定位研究，将为分子标记辅助选育优良恢复系和分子检测鉴定“三系”及其杂交种纯度服务，也为进一步研究核质互作的作用机理、克隆恢复基因打下基础。本研究以哈克尼西棉细胞质雄性不育系、保持系和恢复系为亲本，经杂交、自交或连续多代回交培育的F₂群体与恢复基因近等基因系（NILs：near-isogenic lines）群体为研究材料，通过调查和分析两群体单株的育性，确认哈克尼西棉细胞质雄性不育系育性恢复基因的遗传模式；采用SSR分子标记技术结合群体分离分析法（Bulked segregant analysis, BSA），筛选与恢复基因连锁的分子标记，并对育性恢复基因进行遗传定位，并构建连锁图谱；同时，用筛到的与恢复基因紧密连锁的分子标记和课题已报道的与不育基因紧密连锁的SCAR标记来检测种子纯度。取得的主要实验结果如下：1.调查统计409株NILs群体单株和695株F₂群体单株的育性分离情况，在NILs群体中，有223个可育株，186个不育株；在F₂群体中，有502个可育株，193个不育株。经χ2检验，可育株与不育株在NILs群体中的分离比例符合1:1，在F₂群体中的分离比例符合3:1，表明哈克尼西棉细胞质雄性不育系的恢复基因受1对显性基因控制。2.采用SSR分子标记技术并结合BSA法，以NILs群体构建的不育DNA池和可育DNA池为标记筛选材料，对恢复基因进行分子标记筛选。在8242对SSR引物中，共筛选到13个与Rf1基因紧密连锁的分子标记，其中COT010、CGR5340和NAU3938这3个标记为首次发现。结合育性调查结果，利用这13个标记对NILs群体409株单株和F₂群体695株单株的分离情况进行了分析。结果发现，在13对多态性标记中仅引物NAU2650在409株NILs群体中出现1个交换单株，其余12对引物未发现交换单株；而在695株F₂群体中只有引物CGR5340和BNL3535未发现交换单株，其余11对引物则各发现1个交换单株。3.利用JionMap3.0软件分别构建了NILs群体和F₂群体的Rf1遗传连锁图谱，其总遗传距离都小于0.5cM。在用近等基因系群体构建的Rf1遗传连锁图谱中，标记NAU2650距Rf1基因为0.245cM，另12个标记则与Rf1基因共分离；在用F₂群体构建的Rf1遗传连锁图谱中，连锁图总的遗传距离为0.35cM，Rf1基因位于标记BNL3535和标记CM003之间，距2个标记分别为0.042cM和0.119cM。通过比较棉花遗传连锁图谱和分析这些标记在棉花染色体上的位置，发现在本研究获得的13对与Rf1基因紧密连锁的SSR标记中，7对标记位于第19染色体上（又名D5染色体），由于SSR标记常作为染色体定位的锚定标记，因此可以推测哈克尼西棉细胞质雄性不育育性恢复基因Rf1位于第19染色体上。4.利用本研究获得的与恢复基因紧密连锁的2对共显性标记COT010和NAU3938对中恢-46（恢复系）种子进行了纯度检测，同时借助新得到的与恢复基因连锁的标记CGR5340、NAU6466和课题已报道的与不育基因紧密连锁的SCAR标记检测了田间不育系、保持系、恢复系及其杂交F₁代及常规棉杂交种的基因型。PCR扩增结果表明，这些标记扩增条带清晰，扩增效果好，十分适用于棉花CMS“三系”及其杂交一代种子纯度检测和分子标记辅助选育优良恢复系。更多还原

【Abstract】 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.更多还原