A modified AFLP with fluorescence-labelled primers and automated DNA sequencer detection for efficient fingerprinting analysis in plants

A modified AFLP (amplified fragment length polymorphism) technique is described. Fluorescence-labelled primers were used in the selective amplifications. The amplified fragments were detected on denaturing polyacrylamide gels using an automated ALF DNA sequencer with the fragment option. The modified AFLP technique avoids the use of isotopes or silver staining, but gives a much higher resolution than other AFLP detection systems.     

Evidence for range stasis during the latter Pleistocene for the Atlantic Coastal Plain endemic genus,Pyxidanthera Michaux

The general phylogeographical paradigm for eastern North America (ENA) is that many plant and animal species retreated into southern refugia during the last glacial period, then expanded northward after the last glacial maximum (LGM). However, some taxa of the Gulf and Atlantic Coastal Plain (GACP) demonstrate complex yet recurrent distributional patterns that cannot be explained by this model. For example, eight co‐occurring endemic plant taxa with ranges from New York to South Carolina exhibit a large disjunction separating northern and southern populations by >300 km. Pyxidanthera (Diapensiaceae), a plant genus that exhibits this pattern, consists of two taxa recognized as either species or varieties. We investigated the taxonomy and phylogeography of Pyxidanthera using morphological data, cpDNA sequences, and amplified fragment length polymorphism markers. Morphological characters thought to be important in distinguishing Pyxidanthera barbulata and P. brevifolia demonstrate substantial overlap with no clear discontinuities. Genetic differentiation is minimal and diversity estimates for northern and southern populations of Pxyidanthera are similar, with no decrease in rare alleles in northern populations. In addition, the northern populations harbour several unique cpDNA haplotypes. Pyxidanthera appears to consist of one morphologically variable species that persisted in or near its present range at least through the latter Pleistocene, while the vicariance of the northern and southern populations may be comparatively recent. This work demonstrates that the refugial paradigm is not always appropriate and GACP endemic plants, in particular, may exhibit phylogeographical patterns qualitatively different from those of other ENA plant species.     

橡胶树AFLP银染体系的建立和优化

目的:扩增片段长度多态性(AFLP)为遗传图谱的构建及育种的辅助选择提供了有力的工具。建立一套适合橡胶树的AFLP技术优化体系。方法:以197个GT1×IAN873橡胶树杂交群体为材料,通过对影响AFLP的多种关键因素如模板DNA质量、酶切连接体系、酶切连接反应时间、预扩增体系、选择性体系的分析进行研究。结果与结论:找出一套适于热带植物基因组DNA提取及橡胶树AFLP技术。用改进的CTAB法,经多次抽提纯化,用细玻璃棒挑出DNA得到高质量的模板DNA;酶切连接时DNA模板为250ng,反应体系采用各3U的EcoRⅠ/MseⅡ/T4连接酶,反应时间为9h;预扩增模板为稀释1/2的酶切连接产物,用量为1μL;选择性扩增要用稀释至1/20的预扩增产物。     

Genetic structure of a hybrid zone between two violets,Viola rossii Hemsl. and V. bissetii Maxim.: dominance of F1 individuals in a narrow contact range

The genetic composition of a hybrid zone can provide insight into the evolution of diversification in plants. We carried out morphological and amplified fragment length polymorphism analyses to investigate the genetic composition of a hybrid zone between two violets, Viola bissetii Hemsl. and Viola rossii Maxim. Our aim was to clarify the formation and maintenance of hybrids between these Viola species. We found that most hybrid individuals (V. bissetii × V. rossii) were of the F₁ generation, with a few of the F₂ generation. We found no backcrosses. The scarcity of post‐F₁ hybrids indicates that a species barrier is established between the parental species. The F₁‐dominated hybrid zone occupied only a narrow, intermediate ecotone between the parental habitats, suggesting that selection by environmental factors against hybrids may help to maintain the current conditions in this hybrid zone.     

利用扩增片段长度多态性分析鉴别炭疽和蜡样芽孢杆菌

目的:利用扩增片段长度多态性(AFLP)分析建立鉴别炭疽芽孢杆菌和蜡样芽孢杆菌的分子生物学方法。方法:3株炭疽芽孢杆菌和3株蜡样芽孢杆菌基因组经限制性内切酶EcoRⅠ和MseⅠ酶切后与对应接头连接,通过预扩增和选择性扩增获得特异性DNA片段,将片段进行毛细管电泳,并利用GeneScan和BioNumerics软件对电泳数据进行分析。结果:选择性扩增最佳引物组合为EcoRⅠ-G/MseⅠ-A,其扩增片段在100~500 bp范围内的有效数量为40~50条;比较炭疽芽孢杆菌和蜡样芽孢杆菌的AFLP特征峰值图和DNA指纹图谱,确定了5个有明显差异的片段区。结论:利用AFLP分析可对芽孢杆菌属中相近的炭疽芽孢杆菌和蜡样芽孢杆菌进行鉴别,该方法可作为炭疽芽孢杆菌传统鉴定方法的补充。     

广东地区嗜肺军团菌的扩增片段长度多态性分析

【目的】了解广东部分地区2002～2007年环境水中嗜肺军团菌的基因型及遗传学关系。【方法】参考欧洲军团菌感染工作组(the European Working Group for Legionella Infections,EWGLI)制定的分型草案(1.2版),采用PstⅠ酶对我室保存的2株标准菌株及41株不同时间、不同地点的嗜肺军团菌环境分离株进行扩增片段长度多态性(amplified fragment length polymorphism,AFLP)分析,电泳图谱与EWGLI进行比对。【结果】AFLP分型结果稳定,重复性好;43株嗜肺军团菌,其不同来源菌株呈现多态性分布,经AFLP分析得到33个基因型,辨别力指数为99.79%,其中Kingmed AFLP 011为优势基因型。按Dice系数≥0.8,可分为18个群,Kingmed AFLPD群为优势群,占总菌株数的34.88%。由电泳图谱的相似性,初步推断存在EWGLI 001 Lugano型、002 Lugano、012 Rome、013 London、014 London型、015 Dresden型、021 Lyon等7个基因型,以及多个未报道的新基因型。【结论】广东地区嗜肺军团菌的基因型十分丰富,AFLP技术是其分子流行病学研究的有效手段。     

放线菌的单酶切AFLP基因分型研究

使用MseI限制性内切酶对放线菌链霉菌属中的12株菌基因组DNA进行酶切,与接头连接后,引物使用一个选择性碱基对模板进行PCR扩增,PCR产物在琼脂糖凝胶上进行电泳检测。结果表明,对于MseI内切酶产生的模板,选择性碱基采用A、T、C和G都能够获得清晰丰富的条带。MseI内切酶产生的图谱上有72个位点,多态性位点71个,达98．6％。因此,使用MseI内切酶适合构建放线菌的单酶切AFLP指纹图谱。     

华北地区小丛红景天种群的AFLP遗传多样性

利用扩增片段长度多态性(AFLP)标记, 对分布于华北地区5个山脉的25个小丛红景天(Rhodiola dumulosa)自然种群的776个样品进行了遗传多样性和遗传结构的研究。结果表明: 华北地区小丛红景天种群具有较高的遗传多样性, 4对选择扩增引物共扩增出398条清晰的条带, 其中多态带312条, 种群的平均多态位点百分率为78.46%, 种群总的Nei’s基因多样性为0.364 9, 总Shannon多态性信息指数为0.542 2。华北地区小丛红景天种群间的遗传分化系数G_st = 0.150 7, 基因流Nm = 2.817 9, 表明种群间遗传分化较低, 有一定的基因交流。AMOVA分析结果也表明: 华北地区小丛红景天的遗传变异主要存在于种群内, 地理单元间有一定的遗传分化, 而种群间的遗传分化较低。STRUCTURE的分析和UPGMA聚类分析结果一致, 结果显示地理分布距离相近的种群优先聚在一起。Mantel检验也进一步证实, 华北地区小丛红景天种群的遗传距离与地理距离间呈显著的正相关关系(r = 0.512 9, p < 0.001)。种群的遗传多样性与海拔呈显著的负相关关系(p < 0.05), 而与坡向没有显著相关性。用Dfdist软件分析海拔对遗传多样性的影响, 结果表明没有显著的受选择位点。     

Clarification of the genetic component of hybrids between Phyllodoce caerulea and Phyllodoce aleutica (Ericaceae) in Hokkaido,northern Japan

Natural hybridization provides great opportunities to understand the interaction of genetics and ecology in determining species boundaries. We examined the genetic relationships of Phyllodoce taxa and revealed that most hybrids were fertile F₁s and an extremely small number of backcross and no F₂ plants were established in natural conditions. Because this trend was irrespective of regions, we conclude that negative endogenous selection may act after the germination of F₁ seeds and prevent the establishment of later‐generation hybrids. Based on these results, we discuss the ecological and evolutionary significance of natural hybridization in Phyllodoce taxa.