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题名: 杜鹃花属迷人杜鹃的自然杂交起源研究
作者: 张敬丽
学位类别: 博士
答辩日期: 2007-06-01
授予单位: 中国科学院昆明植物研究所
授予地点: 昆明植物研究所
导师: 张长芹
关键词: 自然杂交 ; 物种形成 ; 杜鹃花 ; 地理分布 ; 形态学 ; 孢粉学 ; 传粉生物学 ; 分子生物学
学位专业: 植物学
中文摘要: 摘要:本文根据地理分布调查的结果,利用形态性状分析、孢粉学、传粉生物学和分子生物学的研究方法,对分布于云南省内的杜鹃花属(Rhododendron L.)常绿杜鹃亚属(Subgenus Hymanenthes (Bl.) K. Koch)的常绿杜鹃组(Sect. Ponticum G. Don)中三个不同亚组的推测亲本马缨花R. delavayi Franch.、大白花杜鹃R. decorum Franch. 和露珠杜鹃R. irroratum Franch. 的假设自然杂交种迷人杜鹃R. agastum Balf. f. et W. W. Smith(四种杜鹃花都是二倍体2n=26)进行了综合研究。结果证实了迷人杜鹃是马缨花和大白花杜鹃的自然杂交种,同时发现了露珠杜鹃也是马缨花和大白花的自然杂交种。具体研究结果如下: 1. 地理分布调查 本文对滇东地区的师宗县菌子山、沾益县马雄山和滇西地区大理苍山东坡、漾濞县内苍山西坡的大平地、大竹坪、金盏以及剑川县石宝山7个地点的杜鹃花地理资源分布情况进行了调查。结果表明,除大竹坪只有迷人杜鹃、马缨花与露珠杜鹃,没有发现大白花杜鹃;剑川县的石宝山只有马缨花、大白花杜鹃与迷人杜鹃,没有发现露珠杜鹃外,这四种杜鹃花在菌子山、马雄山、大平地和金盏脉地分布区域重叠。从调查的这几个地点水平分布看,马缨花是一个广布种,大白花杜鹃的分布较马缨花窄,露珠杜鹃和迷人杜鹃则与马缨花一样分布较广;从垂直分布看,大白花杜鹃基本分布在低海拔区域,马缨花则在不同海拔都可生长,而迷人杜鹃与露珠杜鹃基本分布在海拔较高的地方(不同地方略有差异)。 2. 形态性状分析 我们从七个自然地点(滇东部二个地点和滇西北五个地点)和昆明植物园于2005和2006年的不同月份分别采集到四种杜鹃花的标本,从质量性状与数量性状两个方面对这四种杜鹃花的各种形态,包括树形、叶、花、果实、种子和幼苗进行了统计比较和主成分分析,结果表明,马缨花和大白花杜鹃是区别明显的两个种,迷人杜鹃与露珠杜鹃的形态基本介于马缨花与大白花杜鹃之间。 3. 孢粉学 电镜扫描显示,迷人杜鹃、马缨花、大白花杜鹃和露珠杜鹃的花粉都是四合花粉,近球形,具三孔沟。马缨花的花粉粒最小37.00-51.60um,大白花杜鹃的花粉粒最大39.00-54.20um,而迷人杜鹃和露珠杜鹃的花粉粒大小介于二者之间分别为41.00-53.00um 和40.40-53.00um。花粉外壁纹饰为马缨花的纹饰较光滑,大白花杜鹃的最粗糙,迷人杜鹃和露珠杜鹃的纹饰则介于二者之间。 4.传粉生物学 本研究选取了师宗县菌子山作为自然居群,对迷人杜鹃、马缨花、大白花杜鹃和露珠杜鹃进行了开花物候、花粉活力、柱头可授性检测、传粉昆虫观察、人工授粉组合实验和种子萌发实验。结果表明,这四种杜鹃花的花期基本为3-5月,单花开花期可持续一周左右;花粉活力与柱头可授性在开花3-5天达到高峰。它们的主要传粉方式为虫媒传粉,共有的传粉媒介为中华蜜蜂;除露珠杜鹃外,其余三种杜鹃花都是自花不育,异花授粉可育,不同种之间可进行人工杂交,产生具有活力的种子,种子萌发率在65%以上。通过分析四种杜鹃花开花物候重叠、共有传粉昆虫及自花不育等现象,探讨了传粉昆虫和异花授粉的机制在自然杂交物种形成中的作用及适应能力。 5.细胞学 体细胞染色体观察结果表明,马缨花、迷人杜鹃、露珠杜鹃和大白花杜鹃也都为二倍体,染色体基数为x=13,同时也证实了迷人杜鹃应为二倍体杂交起源。但对减数分裂的观察,目前尚没有取得较好的结果。 6.分子生物学 本研究共采集到迷人杜鹃、马缨花、大白花杜鹃和露珠杜鹃的19个野外自然居群,共283个个体。从每个居群中随机选出1个到3个个体,对核基因ITS片段与叶绿体基因trnL-F片段进行测序,并根据ITS测序结果,对283个个体进行了PCR-RFLP分析。结果表明,马缨花与大白花杜鹃的核基因ITS序列和叶绿体基因trnL-F序列都能明显区分,迷人杜鹃与露珠杜鹃的核基因ITS序列在马缨花与大白花杜鹃相互区别的6个变异位点出现杂合带;经过对ITS区的克隆测序,迷人杜鹃和露珠杜鹃都有分别与马缨花和大白花杜鹃相同的两条ITS序列,同时又有第三种序列,即5个变异位点和马缨花相同,1个变异位点和大白花杜鹃相同;迷人杜鹃的叶绿体基因trnL-F序列全部和马缨花相同,露珠杜鹃的trnL-F序列部分和马缨花相同,部分和大白花杜鹃相同。ITS区的PCR-RFLP分析表明马缨花与大白花杜鹃存在广泛的基因交流,在漫长的杂交进化的过程中,杂交种可能存在与亲本多次回交的现象。
英文摘要: Abstract: According to the investigation of geographical distributions, the putative natural hybrid Rhododendron agastum Balf. f. et W. W. Smith and its assumed parents R. delavayi Franch., R. decorum Franch.and R. irroratum Franch are sympatrically distibuted in Yunnan, China. And the four species belongs to three subsection of the Section Ponticum under Subgenus Hymenanthes of Rhododendron. In this paper we study the morphological characters, the palynology, the pollination biology, sequences of the nuclear ribosomal DNA ITS region, the chloroplast DNA trnL-F intron-spacer and RFLP analysis of ITS sequences of them in order to confirm our assumed review of the relationship among them. We also compared these data with an artificial hybrid between R. decorum (♀) × R. delavayi (♂). Based on the results, we conclude that R. agastum is a natural hybrid between a female R. delavayi and a male R. decorum, and R. irroratm is also a natural hybrid between R. decorum and R. delavayi. The main research results are summarized as follows: 1. Geographic distributions We totally investigated 7 sites of the geographic distributions of four Rhododendron species in Yunnan, China. Two sites including Junzishan in shizong county and Maxiongshan in zhanyi county of eastern Yunnan, and five sites of Dapingdi, Jinzan and Dazhuping in Yangbi county (western slope of cangshan), Shibaoshan in Jianchuan county and Dalicangshan (eastern slope) of the northwestern Yunnan. R. agastum, R. delavayi, R. decorum and R. irroratum are all sympatrically distributed in Junzishan, Maxiongshan, Dapingdi and Jinzhanmaidi. However, Dazhuping possesses R. delavayi, R. irroratum, and R. agastum, without R. decorum. And Shibaoshan possesses R. delavayi, R. decorum and R. agastum without R. irroratum. The results show that the four Rhododendron species are basically sympatrically distributed. R. decorum grows about the lower altitude, R. delavayi distributes more widely than the other three species in Yunnan Province. Except for horizontal distribution, the putative hybrids R. agastum and R. irroratum are occupant of the higher altitude. 2. Morphology We analyzed the morphological characters of the four species specimens which were collected in 2005 and 2006 from the above mentioned seven natural populations in the eastern and northwestern Yunnan and in Kunming Botanic Garden. We compared the qualitative characters and the quantitative characters of the four Rhododendron species, also used PAC analysis. The results show that morphology of R. delavayi and R. decorum are obviously distinct, and morphology of R. agastum and R. irroratum are intermediate between the two parents R. delavayi and R. decorum. 3. Palynology The pollen morphology of R. delavayi, R. decorum, R. agastum and R. irroratum are all pollen tetrad, approaching to sphericity, three apertures. The pollen size of R. delavayi and R. decorum are distinct, R. delavayi is the smallest (37.00-51.60um), but R. decorum is the largest (39.00-54.20um). The pollen size of R. agastum and R. irroratum are similar (41.00-53.00um and 40.40-53.00um), and are intermediate between R. delavayi and R. decorum. Extine of R. delavyi and R. decorum are distinct. R. delavayi is very smooth,but R. decorum have many coarse adorns. And extine of R. agastum and R. irroratum are intermediate between the two parents of R. delavayi and R. decorum. 4. Pollination Biology We investigated the flowering period, floral visitors and conducted a series of pollination experiments to examine breeding systems and hybrid compatibility in four Rhododendron species in Junzishan. We observed that R. delavayi was flowering from the beginning of March to the end of May, while R. agastum and R. irroratum were flowering from the beginning of March to the beginning of April. After their flowering periods were end, R. decorum began to flower until the end of May. The anthesis of the single flower in four Rhododendron species was about one week. Their pollen vigor and pistil reception are the highest within flowering 3-5 days. The floral visitors belong to Hymenoptera and Diptera Order. Apiscerana Fabricius is the mutual visitor of the four Rhododendron species, but Colletes sp is proprietary visitor for R. agastum and R. irroratum. We found that R. delavayi, R. agastum and R. decorum are self-incompatible except R. irroratum. No breeding barrier was detected in four Rhododendron species by artificial pollination, and the hybrid seeds were fertile. The hybrids between four species showed equivalent fitness as their parents. Based on the above resultd, we suggest that the role of potential interspecific pollination is essential to the hybrid speciation and evolution of Rhododendron species. 5. Cytology The observation of chromosomes revealed that Rhododendron delavayi, R. agastum, R. irroratum and R. decorum are all diploids, x=13, and confirmed that R. agastum is a diploid hybrid speciation. But the result of the meiotic observation is not very good in this study. 6. Molecular Biology We collected materials of Rhododendron delavayi, R. decorum, R. agastum and R. irroratum from 19 natural populations in different places, and randomly choose 1 to 3 individuals from every population to analysis their nuclear DNA ITS sequences and chloroplast DNA trnL-F sequences. According to the ITS sequences of the four Rhododendron species, three restriction enzyme Bsp68I, Bsh1285I and MvaI were used in the PCR-RFLP analysis. The nuclear DNA ITS sequences and chloroplast DNA trnL-F sequences revealed R. delavayi and R. decorum are clearly distinct, and the cloning ITS sequences of R. agastum and R. irroratum shows that the two hybrid have cloning ITS sequences from R. delavayi and R. decorum, and have another cloning ITS sequences. The trnL-F sequences of R. agastum are all from R. delavayi, but trnL-F sequences of R. irroratum are partly from R. delavayi, partly from R. decorum. PCR-RFLP analysis of ITS sequence reveals that R. agastum is a natural hybrid between R. delavayi and R. decorum and R. irroratum is also a hybrid between R. delavayi and R. decorum. During the course of hybrid evolution, backcrossing may be happened for many times.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.kib.ac.cn/handle/151853/176
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杜鹃花属迷人杜鹃的自然杂交起源研究.张敬丽[d].中国科学院昆明植物研究所,2007.20-25
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