【作者】 李文强；

【导师】 赵建成；

【作者基本信息】 河北师范大学 ， 生态学， 2007， 硕士

【摘要】 真藓科Bryaceae植物的分子系统学研究是藓类植物研究中的新热点,RAPD是分子系统学研究中被广泛采用的一项重要技术。在真藓科Bryaceae中,丝瓜藓属Pohlia的位置、大叶藓属Rhodobryum属的地位和真藓科Bryaceae内几个属的系统排列关系一直存在争议。为了对上述问题进行探讨,本研究建立了真藓科Bryaceae植物RAPD的反应体系,构建了真藓科Bryaceae植物RAPD图谱,并利用RAPD图谱对真藓科Bryaceae植物进行了系统发育分析。通过比较DNA提取方法,本研究选择快速提取试剂盒成功提取所有标本基因组DNA,所提DNA完整性好,纯度高,能够满足RAPD的要求。通过单因素和正交试验对影响RAPD的因素进行考查,建立了优化的RAPD反应体系:dNTP为0.2mmol/L,随机引物0.4μmol/L,Taq DNA聚合酶1.25U,Mg2+为2mmol/L,模板DNA为50ng,扩增体系为25μl。扩增程序为:95℃预变性7min,然后94℃变性1min,36℃退火1min,72℃延伸2min,40个循环,72℃终延伸7min。以优化出来的PCR条件和PCR体系以标本Y1、Y2、Y3所提DNA为模板对250条引物进行筛选扩增,35条扩增稳定、富有多态性的引物被筛选出来。采用上述35条引物对40份标本所提DNA进行扩增,最终采用26条引物构建RAPD图谱,共扩增出198条稳定的条带,分子量在200bp~5,000bp之间,平均扩增条带7.61条,其中181条为多态性条带,占91.41%。表明该图谱能够反映出所研究类群的遗传多态性,可以作为系统发育分析的依据。采集RAPD多态性信息,运用系统进化分析软件PAUP4.0b8分别采用MP法、NJ法、UPGMA法对多态性信息进行分析,构建了MP、NJ、UPGMA系统发育树。所揭示的系统发育关系与传统划分并不一致,部分结果支持基于分子序列的研究结果。系统发育分析的结果显示:丝瓜藓属Pohlia更接近于提灯藓科Mniaceae而远离真藓科Bryaceae的类群,支持Cox等分子序列证据将丝瓜藓属Pohlia放在提灯藓科Mniaceae的安排;大叶藓属Rhodobryum与真藓属Bryum作为姊妹群存在,不支持Ochi将其作为真藓属Bryum内一个亚属的安排。真藓科Bryaceae和真藓属Bryum均为多系群,认为传统分类对一些类群的划分存在争议。可见RAPD可以为真藓科Bryaceae植物的系统发育研究提供分子证据,真藓科Bryaceae植物的系统发育关系有待进一步研究。更多还原

【Abstract】 The molecular systematics of Bryaceae is new focus in the study on moss. RAPD has been widely used for molecular systematics, and it’s an important technique in phylogeny analysis. The placement of Pohlia, the status of Rhodobryum genera and the phylogenetic relationship of several generas within Bryaceae are conflictive. In this study, the optimum random amplified polymorphic DNA (RAPD) reaction system for Bryaceae was built, then the RAPD profile of Bryaceae was construct. The phylogenetic relationships of the mosses in virtue of the RAPD profile in this study were explored.After the comparative method was performed, the genomic DNA extracted by rapid kit with good integrality and purity is enough to perform RAPD. The single factor level experiment and orthogonal test were used to analyze the factors influencing RAPD and the optimum reaction was established. The results showed that the suitable program was one cycle at 94℃for 7 min, 40 cycles at 94℃for 1min, 36℃for 1min, 72℃for 2 min and one cycle at 72℃for 7min. In the RAPD system of Bryaceae the optimal concentration of template DNA was 2ng/μl, primer 0.4μmol/L, dNTP 0.2mmol/L, Mg2+ 2mmol/L, Taq DNA polymerase 1.25U, total volum 25μl. Several DNA samples were selected to choose the compatible primers. The primers were selected by which stabilized products with great polymorphism were acquired. Thirty-five primers were selected from 250 primers by amplifing template DNA from sample Y1, Y2 and Y3 under the optimum reaction system.The selected primers were applied to amplify all DNA samples, and twenty-six primers were used to create the RAPD profile finally. Differential bands of RAPD appeared clearly among samples. 198 bands were amplified with these primers, in which 181 bands were polymorphic. The percentage of polymorphic bands was 91.41% and the mass weight is between 200bp~5000bp.The polymorphism information was acquired from gel imaging system. Phylogenetic analysis was carried out by using Paup4.0b8. Maximum parsimony analyses (MP), neighbour-joining analyses (NJ) and unweighted pair group method using arithmetic average analyses (UPGMA) from collected information. Phylogenetic trees were created and comparative analysis was performed. The phylogenetic relationships is different from traditional classification, supporting the results from molecular analyses.The result from phylogenetic trees created respectively by MP, NJ and UPGMA indicates that Bryaceae and Bryum are not monophyletic. The definition of some groups of Bryaceae and Bryum is doubtful in traditional classification based on morphological data. The analyses approves that Pohlia is more closely related to Mniaceae than Bryaceae, which supports placement of Pohlia in Mniaceae from Bryaceae based on DNA sequence data. Rhodobryum appears as sister of Bryum, which suggests that Rhodobryum ought to rank in Bryum genera. It may not be suitable to recognize Rhodobryum as subgeneras in Bryum.The polymorphic profile could reflect the polymorphism of the taxa and related taxa. RAPD can provide some molecular proof for Bryaceae’s phylogeny. There is no need to study deeply on Phylogenetic relationships of Bryaceae.更多还原