【作者】 叶要妹；

【导师】 包满珠；

【作者基本信息】 华中农业大学 ， 植物学， 2008， 博士

【摘要】 百日草是园林中广泛应用的一年生草花,而目前国内市场的百日草栽培品种都是国外进口的F₁代商业品种,每年因为购买种子要用去大量的外汇,所以培育具有自主知识产权的百日草新品种有十分重要的意义。本文利用定位观测、显微观察、系谱单株选择、测交或回交、分子标记等方法对百同草的开花授粉生物学特性、自交系的选育和遗传多样性预测、雄性不育系的获得等进行了系统的研究。主要研究结果如下:1.百日草的开花授粉生物学。百日草在8:30～16:00开花,舌状花柱头保持膨胀,花柱挺立可达7～10d;联苯胺—过氧化氢测定柱头可授性的结果显示,花后1～3d可授率为79.9%～83.1%,4～6d为56.1%～63.0%,7～10d为37.0%～43.3%;丫状时期、丫状尖端稍弯曲时期、柱状时期田间授粉的结实率分别为76.3%、48.5%和36.8%。百日草花粉属于三核型的花粉,离体萌发的最佳培养基组合为ME₃+16%PEG₄₀₀₀+12%蔗糖,且上午10:00比下午3:00采集的花粉萌发率高。花粉寿命短,在常温、干燥条件下贮藏,花粉的寿命只有18h,在4℃湿润条件下贮藏也不超过1d。从制种角度来看,武汉市百日草一年中可进行两季种子生产,种子生产的最适播种期,春播为2月下旬～3月上旬;夏播为6月下旬～7月中旬。授粉后25d采收,种子的活力最高。2.自交系选育。多数百日草自交结实率低,花期用3%NaCl处理的比蕾期授粉更能提高自交结实率。经单株多代选择获得了23个花色丰富、瓣型、株型不同的百日草和1个小百日草自交系,为杂交育种提供了基础材料。3.百日草雄性不育系的获得。通过两种途径获得育性1:1分离的雄性不育系:1)F₁代商业品种（利用不育系制种）自交F₂代获得不育株,与F₁代回交;2)多代自交出现的不育株与同一自交系可育株测交。然后在不育系内进行兄妹交、可育株自交,结合与多个自交系（恢复系）测交进行不育类型鉴定。获得了百日草深红色单隐性雄性不育两用系AH209AB、桔红色单隐性雄性不育两用系J16601AB等6个不育系。这些不育系的雄性不育株花瓣退化或丝状,雄蕊退化成丝状,内无花粉,外观呈绒毛状,具有败育彻底,不育性稳定,不受环境因素的影响,在育种上具有重要的利用价值。4.表型性状与RAPD、ISSR分子标记评价20个百日草自交系的遗传多样性。为了正确地选择百日草杂交育种的优良亲本,利用表型性状、RAPD和ISSR标记对百R草自交系间进行了遗传多样性的评估。通过对34个表型性状的分析,结果表明花色不是百日草表型特征分类的重要指标,而花径、株型和瓣型能很好地鉴别百日草自交系聚类群。从147条RAPD引物和44条ISSR引物筛选到具有多态性的RAPD引物12条,ISSR引物9条。RAPD引物共扩增出147条带,多态性位点数为100,多态性位点比率为68.03%,平均多样性指数为0.3559,有效等位基因数为1.4169;ISSR引物共扩增出128条带,多态性位点数为97,多态性位点比率为76.38%,平均多样性指数为0.4013和有效等位基因数为1.4728。试验表明,两种分子标记结果较为相似,呈极显著的正相关（r=0.733）。聚类分析将20个自交系分为2类:一类群来源于虹越种子集团公司:另一类群来源于甘肃酒泉种子集团公司,聚类群体与原产地相关。形态特征与分子标记（RAPD和ISSR）也具有显著的正相关（r=0.381,0.377）,但分析亲缘关系特别是系谱关系时,分子标记优于形态标记。ISSR是一种多态性和重复性优于RAPD的分子标记,育种过程中,适宜的分子标记结合表型性状分析,能更准确地评价百日草自交系间的遗传多样性。更多还原

【Abstract】 Zinnia elegans,an important annual ornamental plant,are usually planted in the garden via F₁ seeds in China.However,most of Zinnia elegans cultivars depend on importation,the high cost spended in it necessitated us to develop our own breeding system of Zinnia elegans for expoilted novel cultivars adapting to native climate.In this study,the flowering and pollination biology of Zinnia elegans was investigated. Pedigree selection method and backcross and testcross were used for selecting inbred lines and male sterile lines.And molecular markers were developed to identify the genetic diversity of inbred lines.The results were presented as following:1.The flowering and pollination biology.Zinnia elegans flowers open at 8:30～16:00 in daytime.When it flowers,the stigmas of florets stand upright and keep high turgidity for 7～10 days.The stigmatic pollen receptivity for 1～3d,4～6d,7d and 10d was 79.9%～83.1%,56.1%～63.0%,37.0%～43.3%respectively according to the benzidine-H₂O₂ test.The seed set of the Y stigma phase,the tip of Y slightly curling stigma phase and columnar stigma phase was 76.3%,48.5%and 36.8%respectively by the test of artificial pollination.The seed set of no-treatment,detruncating one lob of stigma and style of detruncating stigma was 48.8%,30.9%,19.9%respectively.The optimum in vitro pollen germination medium of Zinnia elegans was ME₃ basal medium supplemented with 16%PEG₄₀₀₀ and 12%sucrose.The pollens collected at 10:00 am have much higher germination rate than those collected at 3:00 pm.Mature pollen grain was tricellular type,the viability of pollen lasted for 18h at room temperature under dry condition,and no longer than one day at 4℃and moist condition.The optimum twice sowing time for seed production in Wuhan is late February to early March for spring sowing and late June to mid-July for summer sowing.Seeds should be collected 25 days after pollinationand had a high rate of germination and long storage life.2.Selecting and breeding of inbred lines.Since selfing of Zinnia elegans get lower seed set rate,pollinate at flowering period with 3%NaCl treatment can get higher seed set rate than pollinate at bud phase.The 23 inbred lines of Zinnia elegans for diverse colors, petal type,plant shape and 1 inbred line of Zinnia angustifolia were obtained by repeated selfing and continuous selection.3.Production of male sterile lines of Zinnia elegans.The male sterile lines were obtained in two pathways:One is to back cross the F₁ generation with male sterile plants that segregated among selfed F₂ generation continuously,and another way is to testcross the fertile plants from self-F₅ generation with male sterile plants that segregated from the same inbred line.Through these ways,a half of male-sterile plants could be obtained.The male-sterile lines were analyzed and identified through sibmating,selfing of fertile plants, and testcrossing with a number of inbred lines（restorer lines）,and proved that it was a type of stable male sterile lines.Six recessive nucleus male sterile lines such as scarlet AH209AB,orange-red J16601AB were obtained.The petal of male sterile plant degraded as a thread-like structure,the stamens were villiform in appearance and no pollens were formed,stably sterility,were free from the impact of environmental factors.The male sterile lines are of great use value in F₁ seeds production.4.A comparative analysis of genetic diversity of Zinnia elegans inbred lines using morphological traits,RAPD and ISSR.In order to select the fight lines for crossing of Zinnia elegans,genetic diversity was assessed by morphological traits,RAPD and ISSR data.Analysis of thirty-four morphological Waits showed that flower color was not the important index for morphological classification and flower diameter,plant shape and petal pattern can identify 20 inbred lines of Zinnia elegans well.147 RAPD markers fragments amplified with 12 arbitrary primers,and 128 ISSR markers fragments generated from 9 primers were employed to discriminate polymorphism between 20 accessions.The number of polymorphic loci,the percentage of polymorphic loci, Shannon’s Information index（Ⅰ） and effective number of alleles（Ne） was 100,68.03%, 0.3559,1.4169 for RAPD markers,and 97,76.38%,0.4013 and 1.4728 for ISSR markers. The Mantel-test indicated that there was a significant correlation（R=0.733） between RAPD and ISSR markers.The clustering analysis showed that the grouping had a correlations with the geographical origin and flower color was not the most important character of the classification of inbred lines of Zinnia elegans.In contrast,the morphological matrix had a low correlation with RAPD,ISSR data matrices（R=0.381, 0.377）.When analyzed relationship,especially pedigree relations,molecular marker was superior to morphological marker for assessing genetic diversity among the different accessions.ISSR was better than RAPD in the aspect of polymorphism and repeatability. The suitable molecular marker,combined with morphological characteristics,can provide more accurate results in the breeding process when evaluated genetic diversity among inbred lines.更多还原