滇山茶多倍体复合体起源和演化的分子细胞遗传学研究
其他题名Molecular cytogenetics studies on the origin and evolution of the Camellia reticulata polyploid complex
刘利勤
学位类型博士
导师顾志建
2009-05-25
学位授予单位中国科学院昆明植物研究所
学位授予地点昆明植物研究所
学位专业植物学
关键词滇山茶多倍体复合体 荧光原位杂交 基因组原位杂交 起源 演化
摘要滇山茶又称云南山茶花,为我国原产的十大名花之一,也是世界著名的观赏园艺植物。阐明滇山茶的起源特点和演化规律具有重要理论意义,而且对滇山茶的种质资源的合理开发利用有重要参考价值。本论文通过荧光原位杂交分析对滇山茶多倍体复合体以及相关的近缘种进行了研究,目的在于探讨该多倍体复合体的起源和演化。同时为进一步明确滇山茶多倍体复合体与近缘种的关系,利用扩增片段长度多态性分析和序列分析等分子系统学手段对滇山茶多倍体复合体及相关的近缘种进行了研究。研究结果如下: 1、通过核基因waxy片段和4个叶绿体基因片段(trnL-F、rpL16 intron、trnS-G和trnT-L)的最大简约性分析和联合分析。结果表明:滇山茶多倍体复合体与西南山茶、怒江山茶、石果毛蕊山茶、南山茶和多齿山茶关系较近。 2、利用AFLP分子标记对滇山茶多倍体复合体及近缘种进行聚类分析,实验结果支持上述结论。 3、在以滇山茶基因组DNA为探针,自身染色体DNA为靶DNA的荧光原位杂交中。所有的染色体均有非常强的信号分布。 4、以滇山茶四倍体染色体DNA为靶DNA,分别以滇山茶二倍体、西南山茶、怒江山茶、山茶、南山茶和多齿山茶的总基因组DNA为探针,进行荧光原位杂交。实验结果显示,当以怒江山茶、山茶、南山茶和多齿山茶为探针时,杂交信号主要出现在染色体的端部,其中以山茶的杂交信号最多。当分别以滇山茶二倍体和西南山茶为探针时,滇山茶四倍体一半的染色体具有杂交信号。因此推断:滇山茶四倍体由滇山茶二倍体和西南山茶杂交形成,滇山茶二倍体和西南山茶是滇山茶四倍体的主要供体种。 5、以滇山茶六倍体染色体DNA为靶DNA,分别以滇山茶二倍体、西南山茶、怒江山茶、山茶、南山茶和多齿山茶的总基因组DNA为探针,进行荧光原位杂交。实验结果显示,当以山茶、南山茶和多齿山茶为探针时,杂交信号主要出现在染色体的端部,其中以山茶的杂交信号最多。当分别以滇山茶二倍体、西南山茶和怒江山茶为探针时,滇山茶六倍体近三分之一的染色体具有杂交信号。因此推断滇山茶二倍体、西南山茶和怒江山茶是滇山茶六倍体的供体种。 6、另外分别以西南山茶、怒江山茶和山茶的总基因组为探针,对滇山茶二倍体进行荧光原位杂交分析。实验结果显示杂交信号主要出现在染色体的端部。 7、最终结果表明运用染色体基因组原位杂交(GISH)的方法和技术已探明了滇山茶六倍体三个主要亲本种基因组成分和其他近缘种渗入部分基因片段成分。可以说我们已获得了证明滇山茶六倍体起源的实验结果和理论。; Camellia reticulata, named Yunnan Camellia, is one of the ten well-known flower in China, is also famouse in the world as an important flowering ornamental species. Clarifying the orgin and evolution of C. reticulata will has some important theoretical significance, and has important value for the rational exploitation and utilization of C. reticulata germplasm resouces. In order to reveal the origin of the C. reticulata polyploid complex, FISH was used. Meanwhile, AFLP, rDNA waxy sequence and four cpDNA sequences were employed to clearify the relationships between C. reticulata polyploid complex and its related species. The main results are as follows: 1, Phylogeny of C. reticulata polyploid complex and its related species were studied by maximum parsimony analysis of nuclear DNA waxy sequence and 4 chloroplast DNA sequence (trnL-F, trnT-L, trnS-G and rpL16 intron), respectively and unitedly. The results showed that C. pitardii, C. saluenensis, C. semiserrata, C. polyodonta and C. mariei var. lapidea were close related species of C. reticulata polyploid complex. 2, Amplified fragment length polymorphisms (AFLP) was used to analyze the relationship between C. reticulata polyploid complex and its related species. The result was the same as that of gene sequence analysis. 3, Strong signals were observed on the whold chromosomes when the gDNA (genomic DNA) of C. reticulata was as its own probe. All of the signals were located on every chromosome. 4, Signals were observed at the end of chromosomes when tetraploid C. reticulata was the target, diploid C. reticulata, C. saluenensis, C. semiserata, C. polyodonta and C. japonica was the probe respectively. With diploid C. reticulata and C. pitardii as probe respectively, strong signals were observed at half of chromosomes in tetraploid C. reticulata. The results supported that tetraploid C. reticulata is an allotetraploid species, and diploid C. reticulata and C. pitardii are the diploid progenitors of tetraploid C. reticulata. 5, Signals were observed mainly at the end of chromosomes with hexaploid C. reticulata as the target, C. semiserata, C. polyodonta and C. japonica as probe respectively. Almost one third chromosomes of hexaploid C. reticulata have strong signals at the whole genome, with diploid C. reticulata, C. pitardii and C. saluenensis as probe respectively. The results supported that diploid C. reticulata, C. pitardii and C. saluenensis are the diploid progenitors of hexaploid C. reticulata. 6, There are signals at the end of chromosomes with the diploid C. reticulata as target, C. pitardii, C. saluenensis and C. japonica as probe respectively. 7, The GISH results proved that C. reticulata hexaploid contained one set of C. reticulata diploid, one set of C. pitardii and one set of C. saulensis and part set of C. japonica and some close realtive species.
页数122
语种中文
文献类型学位论文
条目标识符http://ir.kib.ac.cn/handle/151853/384
专题昆明植物所硕博研究生毕业学位论文
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刘利勤. 滇山茶多倍体复合体起源和演化的分子细胞遗传学研究[D]. 昆明植物研究所. 中国科学院昆明植物研究所,2009.
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