【作者】 刘金兵；

【导师】 侯喜林；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2008， 博士

【摘要】 辣椒（Capsicum annuum L.）原产中南美洲,属茄科辣椒属一年生或多年生植物。它现在已经成为我国第二大蔬菜作物,年种植面积大约在135万公顷。辣椒具有明显的杂种优势,生产上以蕾期人工去雄授粉生产一代杂交种为主,而利用雄性不育尤其核质互作雄性不育（CMS）生产杂交种子是迄今为止简化制种工序、降低制种成本、保证杂交种子纯度、预防亲本流失的最有效途径。利用甜辣椒CMS系配制杂交品种是当今世界甜辣椒育种的热点。本文通过研究甜椒CMS的遗传规律、恢复系的筛选、创制与利用、CMS花粉败育机理及与不育基因相关的分子标记,对于探讨甜椒CMS雄性不育机理和分子标记辅助育种以及杂种优势利用均具有重要的理论指导意义。主要研究结果如下:1.选育了甜椒CMST6A和CMSMCA以辣椒CMS21A为母本,高灯笼形甜椒T6和长灯笼形甜椒MC为轮回父本,采用“饱和回交”法,经过连续多代回交和轮回父本自交,选育新的甜椒胞质不育系CMST6A和CMSMCA及其相对应的保持系T6B和MCB。CMST6A的不育性受一对隐性基因控制。I₂-KI染色后镜检表明,CMST6A不育花粉率占98.4%;CMSMCA不育花粉率占97.8%。2.甜椒CMST6A育性恢复基因分布测交鉴定育性结果表明,被测甜椒和辣椒品种可以划分为三个类型:保持型、恢复型、育性分离类型。尖辣椒品种中容易找到甜椒CMS育性恢复基因;而甜椒品种中容易找到保持CMS育性的不育基因。84-1、匈奥804、湘紫13、伏_M、线椒、NT2M、牛-1、5-2R₁和5-2R₃这9个恢复系的恢复基因处于同一位点。3.甜椒CMS恢复系的创造及其杂种优势利用利用花丝和花柱为白色的南京苜蓿园早辣椒、花丝和花柱为紫色的北方扁灯笼形甜椒,花丝和花柱为白色的高灯笼形甜椒5-2和甜椒CMS8A、CMS17A、CMS延A、CMS金A及辣椒CMS21A、CMS阴早A为材料,采用杂交、回交、自交和测交手段,育成与父本5-2性状近似的甜椒恢复系5-2R₁、5-2R₂和5-2R₃以及“三系配套”品种江蔬5号甜椒（CMS8A×5-2R₁）、牛角椒组合“CMS21A×5-2R₁”、“CMS阴早A×5-2R₁”,“CMST6A×匈奥804”、“CMSMCA×匈奥804”。4.甜（辣）椒自交系的RAPD分析采用RAPD技术分析了来自国内外42份甜辣椒自交系的DNA多态性。筛选出的23个特异多态性好引物共扩增出133条带,其中111条为多态带。UPGMA树状图显示,遗传距离为0.124时可将这些甜辣椒划分为3组。育性恢复辣椒在Ⅰ,Ⅱ、Ⅲ组内均有分布,说明在分子水平上恢复基因在供试辣椒自交系中的分布是随机的。5.甜椒CMS不育基因的RAPD标记和ISSR标记运用RAPD和ISSR技术分析了甜椒CMST6A和保持系T6B的基因组DNA差异。获得了一个与甜椒CMS不育性相关的特异片断RAPDAO19₁₄₀₀。序列测定结果表明,该片断序列全长为1378bp。根据序列测定结果,设计的一对特异引物将RAPDAO19₁₄₀₀标记转化为更稳定的SCAR标记SCAO19₄₉₈。ISSR分析研究得到一个与甜椒CMS不育相关的特异片断ISSR-8₈₄₀,该片段编码的氨基酸序列同源性检索发现与茄属番茄第4染色体上克隆的LE_HBa-20F17基因的序列有52%的同源性和67%的相似性。6.甜椒CMS系及其保持系的生化特性不育系中游离脯氨酸和游离赖氨酸含量均明显低于相应的保持系;不育花药中严重缺乏游离脯氨酸;不育花药中游离苏氨酸和精氨酸含量均明显高于相应的保持系;不育系中游离氨基酸总量则明显低于保持系。无论是在叶片中还是在花药中,不育系可溶性糖含量、可溶性蛋白质含量、CAT活性均低于其保持系;而SOD和POD活性则高于其保持系。说明上述物质含量和酶活性变化可能与甜椒CMS系的雄不育有关。更多还原

【Abstract】 Capsicum annuum L.,an annual or perennial plant in capsicum genera of solanaceae originated from middle-south America.Up to now,pepper has become the second largest vegetable crop,planted about 1.35 million hectare area under cultivation in China.Pepper’s heterosis is very obvious while the production of hybrid F₁ seeds is mainly propagated by man-emasculation in bud stage.But cytoplasmic male-sterility（CMS） of pepper is the most important way of utilization of heterosis.Using CMS pepper to produce hybrids can greatly decrease working procedures and costs,and guarantee hybrid seeds purity,and prevent parents to be stolen.So,using CMS pepper to select hybrids is one of the most intense popular in the present world pepper breeding.Studies mainly focus on the inheritance of male-sterile characters,creation and application of restorer lines,pollen abortive mechanism and molecular markers linked to CMS.Results were as follows:In the first part,it introduced the breeding of CMST6A and CMSMCA.Sweet pepper CMST6A and its maintainer line T6B,CMSMCA and its maintainer line MCB were successfully bred by hot pepper CMS21A crossed sweet pepper T6 or MC and backcrossed more than six generations.Heredity analyses showed that sterility of CMST6A was controlled by a pair of recessive genes.The observation with I₂-KI showed that sterile pollen percentage of CMST6A and CMSMCA were 98.4%and 97.8%respectively.In the second part,it studied the distribution of restorer genes of sweet pepper CMST6A.Identifying BC₁ fertility indicated that pepper cultivars can be divided into three groups:maintainers,restorers and segregations.CMS fertile restorer genes can be mainly found in long hot cultivars,while CMS maintainer genes easily found in sweet cultivars. Restorer genes of nine cultivars,84-1,Xiong ao 804,Xiangzi 13,Fu_M,Linear hot pepper, NT2M,horn.1,5-2R₁ and 5-2R₃,were located in the same gene site.In the third part,creation and heterosis application of CMS restorer lines in sweet pepper were studied.Sweet pepper restorer lines 5-2R₁,5-2R₂ and 5-2R₃ were developed by northem flat lantern sweet pepper,alfalfa garden early-maturing hot pepper in Nanjing and high lantern sweet pepper 5-2 by means of cross,backcross,self-pollination and testcross.CMS sweet pepper variety Jiangshu No.5 is bred by CMS8A crossed restorer line 5-2R₁,and other four new combinations were selected,Such as "CMS21A×5-2R₁", "CMSYinzaoA×5-2R₁","CMST6A×Xiong ao 804" and "CMSMCA×Xiong ao 804".In the fourth part,the RAPD analysis was conducted on 42 pepper inbred lines from China and overseas.Twenty-three primers selected from 340 random primers were used for 42 pepper samples.A total of 133 DNA bands were amplified,among which 111 were polymorphic.These 133 bands were analyzed using the UPGMA cluster software.The results showed that these inbred lines could be classified into 3 groups when the genetic distance was 0.124.Fertility restorer hot peppers were distributed in the groupⅠ,groupⅡand groupⅢ.It was observated that the restorer genes were random in these tested hot peppers in the molecular level.In the fifth part,RAPD and ISSR analysis were conducted on the genomic DNA of CMST6A and its maintainer T6B in sweet pepper.The specific fragment RAPDAO19₁₄₀₀ and ISSR-8₈₄₀ in CMS line were found.Sequencing analysis of AO19₁₄₀₀ indicated that the length of the sequence was 1378 bp.According to the DNA sequence,one pair of primers was designed and the RAPD markers were transformed into SCAR marker SCAO19₄₉₈. Sequencing analysis of ISSR-8 showed that the length of the sequence was 840bp,with 52%identities and 67%positives of its amino acid sequence with clone LE_HBa-20F17 on chromosome 4 from S.lycopersicum.In the last part,it studied biochemical characters in sweet pepper CMS Lines and its maintainer lines.Compared with maintainer lines,the contents of free proline and free lysine in sterile lines CMSZTA,CMS17A and CMS8A were obviously lower than maintainer lines ZTB,17B and 8B.Free threonine and arginine contents in sterile lines were obviously higher than maintainer lines.Total content of free amino acids in anther in sterile lines was significantly lower than maintainer lines.The content of soluble sugar and protein and the activity of CAT were low in both leaves and buds while the activities of SOD and POD were higher in CMS line than maintainer line.These results suggest that the variation of free amino acids,soluble sugar,protein content and enzyme activities were related to pollen abortion in CMS.更多还原