【作者】 朱明东；

【导师】 易自力； 蒋建雄；

【作者基本信息】 湖南农业大学 ， 遗传学， 2011， 硕士

【摘要】 自然杂交一直是分类学和进化生物学的热点。芒属植物是一类新型的能源植物,芒属植物的分类及进化研究一直有待完善,在野外收集野生种植资源时,常遇见表型特征介于芒与五节芒之间的植株,通过表型形态鉴定难以准确确定其分类地位。本研究拟通过分析芒与五节芒表型性状及Adh1基因序列,初步探讨芒与五节芒的自然杂交起源过程中基因及表型的渗透。具体结果如下：1、表型性状分析测定了芒与五节芒共13份材料17项表型性状,所有表型性状数据采用ZScores进行标准化转换,计算13个材料两两间的欧式距离,在MVSP3.2软件上计算做主成分分析并进行UPGMA聚类。形态性状聚类分析结果表明疑似杂交种AD431与五节芒类群(AA335和AD625)聚为一类,而疑似杂交种AA343、AD431、AD628、AD607、AD633和AD606则与芒类群(AD623、AD512、AD620、AD619和AD627)聚为一类。2、Adhl基因序列分析测定13份材料Adhl基因的全部序列,分析已测定序列的序列特征,并结合GeneBank上11份已知序列,采用最大简约法(Maximum parsimony, MP)和邻接法(Neighbor-joining, NJ)分别构建分子系统树。结果显示芒属植物的Adh1基因包括6个外显子和5个内含子,外显子片段大小为60-210bp,平均大小为114bp。在13分材料用来构树的19个序列中,序列大小范围为1413-1444bp,平均大小为1430bp,当用ClustalX对齐序列并排除空位和多重迭代后显示序列长度为1473bp。序列A,T,C,G碱基含量分别为24.7%,30.3%,20.5%,24.5%,序列有明显的T碱基偏好,AT碱基含量(55.0%)明显大于CG碱基含量(45%)。颠换值为16,转换值为27,转换(SV)明显大于颠换(SI),转换与颠换的比为1.7。采用MEGA4.0程序上分别构建最大简约树和邻接树,结果显示五节芒和芒的材料分别聚成两个明显分开的分支,而疑似杂交种中均检测到两种Adh1基因单倍型的存在,其中一种单倍型在系统树中与芒聚为一类,另一单倍型则与五节芒聚为一类。本研究结果表明：采用基于形态学性状的聚类分析并不能有效的将杂交种区分开来,而采用Adh1基因序列聚类分析则成功将杂交种鉴定出来。导致这两种聚类分析方法出现差异的本质原因在于：自然杂交所产生的杂交种,往往会与亲本再发生回交,即所谓的渐渗杂交现象,从而导致一个种群的基因逐渐渗入到了另一个种群的基因库中。种间的基因渗透,则降低了种群间的遗传差异性,使得种群间的遗传背景和表型性状趋于同化,从而导致这两个种群在进化上表现出趋近的亲缘关系。从本研究的测序结果可以发现,杂交种Adh1基因的内含子和外显子碱基序列均发生了改变,外显子碱基序列的变异更为丰富；而能够反映到形态上出现差别性状,仅仅只是那些由外显子上的显性基因所控制的少数性状,大量的中性突变在形态学上是没有反映的。因此基因序列聚类分析与形态性状聚类分析的结果出现差异是常见的,而基因序列聚类分析结果所反映出的物种变化更本质、更全面、更准确,用它所鉴定出的杂交种也更可靠。更多还原

【Abstract】 Penetration of natural hybrid has always been the hottest issue in the field of taxonomy and evolutionary biology, owing to be hindered by the traits of its wide range of origins, frequent occurrences, and difficulties in identifications. Miscanthus is a new type of energy plant, the study of whose classification and evolution need to be improved urgently. It’s hard to accurately distinguish a certain plant and identify its taxa by morphological phenotype whose characteristics is similar to and Miscanthus during the collection of wild plants resources. Though research and analysis to different phenotypes and Adh1 gene sequences of, a primary study into genetic and phenotypic penetration between sinensis and miscanthus in the process of the natural hybrid origin. The major results were as follows:1) morphological phenotype analyse.All of 13 materials’s 17morphological characters had been determined and analysed.All the morphological characters had been standardized by the ZScores and computed theirs distance. The date operating in the MVSP3.2 by the UPGMA method. The phylogenic analysis based on morphological characters indicated that one putative hybrid (AD431) clustered together with M. floridulus samples and other putative hybrids (AD623、AD512、AD620、AD619 and AD627) grouped with M. sinensis samples.2) Adhl gene analyseAll of 13 materials’s Adh1 gene sequence which building-up the phylogenetic trees using the MP method and the NJ method with the other 11 sequences registered in the GeneBank were mensurated and cluster analysed. The results showed that the Adhl gene of the Miscanthus Anderss include 6 exons and 5 introns, and the exon region is 60bp to 210bp,the average value is 114bp. The 19 sequences’s region is 1413bp to 1444bp and the average value is 1430bp. The length of sequence is 1473bp when analyse by the ClustalX. The A,T,C,G of the sequence take up 24.7%,30.3%, 20.5%,24.5%,and the sequence has the prominent T predilection, AT(55.0%) greater than CG(45%), and the ratio of between the SI(16) is 1.7two diverged types of Adhl gene sequences presented in all samples of putative hybrids, one of which is clustered with M. sinensis while the other is clustered with M. floridulus on the Adh1 phylogeny, suggesting that they are the true hybrids between M. sinensis and M. floridulus.The phylogenic analysis based on morphological characters can not verification the hybrids, and the Adhl gene sequence analyse can.The Introgressive hybridizationhas been always appears in the nature hybrids, this phenomenon bring about the divergence between two analyse. The heredity background and the morphological characters has been assimilated by the Introgressive of gene and the otherness of the species has been reduced. Acid base of those hybrids’s exons and introns has been changed and the exons are more plentifully. The disparate phylogenic controlled by the exons which in the dominant gene is the minority and the neutral mutation can’t been expressed in the phylogenic. The otherness of the gene sequence analyse bwtween the morphological characters analyse is familiar, and the sequence analyse is more essential, general, exacting and the results when identifying the hybrids is more exacting.更多还原