不同寄主植物上烟蚜DNA多态性的RAPD-PCR分析

用RAPD-PCR技术研究了桃树、油菜和烟草上烟蚜的DNA多态性。结果表明,用相似性指数(SI)和Nei的遗传距离(D)及聚类分析对本研究筛选的三个引物OPX-04、OPX-06和OPX-19的扩增结果进行比较,除OPX-06的D值聚类可将各寄主植物上的烟蚜分开外,其余均可将样本分为两类,即来自桃树的烟蚜和来自油菜、烟草上的烟蚜。这说明与桃树的烟蚜相比,在DNA水平上,油菜上的烟蚜与烟草上的烟蚜更为接近。结果还表明,在烟蚜的体色方面,与黄绿色相比,红色与褐色更为接近。     

EMS对丽格海棠离体诱变的生物学效应

将不同浓度EMS处理的丽格海棠叶片作为外植体进行离体再生,以期获得性状优异的变异材料。结果表明:随EMS浓度的增加,其诱导率和增殖倍数降低,而变异率增加,0.4%(20℃,8h)EMS为丽格海棠叶片外植体再生的半抑制剂量,该浓度为诱变处理的适合浓度。对21株表型变异植株进行RAPD-PCR分析表明,有12株的电泳条带表现出多态性,进一步证明这些变异是由于基因组DNA的变异而引起的。     

松材线虫RAPD-PCR反应体系的优化与分子鉴定标记的筛选

应用随机引物S344、S356和S360比较分析了PCR热循环复性温度、Mg2+浓度和模板浓度对松材线虫基因组DNA的RAPD扩增结果的影响,旨在建立松材线虫的最佳反应体系,并以此进行松材线虫的分子鉴定标记筛选。结果表明:37℃的复性温度、3.0 mmol.L-1Mg2+、10μL反应体系中松材线虫基因组DNA模板1~25 ng是研究松材线虫RAPD特征的最佳反应体系。利用此反应体系,通过对100个随机引物的分析,获得了3条松材线虫区别于拟松材线虫的分子鉴定标记。     

平菇RAPD—PCR反应体系优化研究

以平菇黑丰268为试材,对RAPD—PCR反应体系的各项影响因素进行了优化,建立了平菇RAPD—PCR反应体系。结果表明,平菇RAPD—PCR最优体系为：20μl PCR反应体系中,Mg^2＋浓度为1．4mmol／L,模板DNA为50ng,引物浓度为0．6—0．8μmol／L,dNTPs浓度为250μmol／L,Taq酶为1．2U,10×Buffer2．0μl。     

干用辣椒RAPD-PCR反应体系及扩增程序的优化

以干用辣椒叶片为材料,研究了干用辣椒RAPD分析过程中的影响因素,包括Taq酶、Mg2+、dNTPs、引物、模板DTA浓度、退火温度、退火时间及循环次数等,建立适合干用辣椒RAPD反应的PCR体系,即25μL反应体系中含有Taq酶1.5 U、Mg2+2.5 mmol/L,dNTPs 0.6mmol/L、引物0.8μmol/L、模板DNA 60 ng.扩增程序为:94℃预变性4 min;94℃变性1 min,37℃退火1 min,72℃延伸1.5 min,40个循环;最后72℃延伸5 min.该优化体系在干用辣椒RAPD分析中获得较理想的扩增结果,为应用RAPD技术对干用辣椒遗传多样性奠定基础.     

洋葱(Allium cepa L.)RAPD-PCR反应体系及扩增程序的优化

为建立多态性高、稳定性好的洋葱RAPD-PCR反应体系,采用正交设计,研究了Taq酶、Mg2 、引物和dNTP 4种RAPD-PCR反应组分浓度变化对扩增结果的影响,在此基础上对模板DNA用量、扩增程序中退火温度和反应循环次数进行了筛选。试验结果表明,洋葱20μl RAPD-PCR优化反应体系为1×Buffer、2.0 mmo1/L Mg2 、1.0 UTaqDNA聚合酶、200μmo1/L dNTP、0.6μmo1/L引物、2%甘油和15 ng DNA模板;PCR扩增程序为94℃预变性4 m in;94℃变性30 s,35℃退火40 s,72℃延长1.5 m in,45个循环;72℃保温延伸7 m in。     

Molecular characterization and PCR detection of the melon pathogen <Emphasis Type="Italic">Acremonium</Emphasis><Emphasis Type="Italic">cucurbitacearum</Emphasis>

Acremonium cucurbitacearum is a soil-borne pathogen that causes collapse of muskmelon and watermelon plants. Cluster analysis based on RAPD patterns, obtained from use of 25 primers, divided isolates of A. cucurbitacearum from Spain and USA into two major groups. Most isolates from the USA fell into group 1, however, genetic similarity was not highly correlated with geographical origins or with previously established VCG groups. Analysis of 5.8S-ITS sequences showed very little sequence variation among isolates of A. cucurbitacearum, most had identical 5.8S-ITS sequence. Nodulisporium melonis, previously reported to cause a similar disease in Japan, had a 5.8S-ITS sequence that was identical to that of isolate A-419 proposed as the type strain of A cremonium cucurbitacearum suggesting that the two fungal pathogens should be considered a single species. Phylogenetic analysis, based on the 5.8S-ITS region, indicated that A cremonium cucurbitacearum is a monophyletic taxon more closely related to Plectosphaerella cucumerina than to other species of the genus Acremonium. Based on the 5.8S-ITS nucleotide sequence, a polymerase chain reaction was designed and used for specific detection of A. cucurbitacearum in diseased plants.     

Molecular analysis of an interspecific hybrid ornamental eucalypt for parental identification

The parentage of ‘Urrbrae Gem’, an ornamental hybrid Eucalyptus, was investigated using RAPD-PCR. The female parent was known to be E. erythronema var. erythronema but previous opinion, based on adult morphological characters, placed the male parent as either E. stricklandii or E. gomphocephala. Samples of DNA, extracted from leaves of different individuals within each species, were amplified with six different 10-mer primers to produce RAPD fingerprints. These were used to generate a UPGMA dendrogram based on genetic similarities, and an ordination plot, derived by multi-dimensional scaling (MDS), and minimum spanning tree (MST) to show the relative dissimilarities between the individuals tested. The UPGMA dendrogram divided the samples into three clusters, with the hybrid slightly closer to E. stricklandii than E. gomphocephala. The MDS ordination and MST placed the hybrid between E. erythronema var. erythronema and E. stricklandii, supporting E. stricklandii as the male parent. This revised version was published online in August 2006 with corrections to the Cover Date.     

Optimizing the indirect extraction of prokaryotic DNA from soils

The objective of this work was to develop protocols to selectively extract prokaryotic DNA from soils, representative of the whole community, amenable to high-throughput whole genome shotgun sequencing. Prokaryotic cells were extracted from soils by blending, followed by gradient centrifugation. Detergent (sodium deoxycholate) was required for complete dispersion of soil aggregates and detachment of prokaryotic cells from a broad range of soil types. Repeated extractions of a given soil sample were critical to maximize cell yield. Furthermore, cells obtained through repeated extractions captured unique prokaryotic assemblages that would otherwise have been missed in single-pass extractions. DNA was isolated from extracted cells using one of the following treatments: i) lysozyme-SDS-proteinase K (enzymatic) digestion; ii) potassium ethyl xanthogenate digestion; or iii) enzymatic digestion of cells embedded in agarose plugs. In addition, these methods were compared to a commercial bead-beating extraction kit (MoBio UltraClean). Of the indirect DNA extraction methods, plug digestion generated the largest yields (up to 70% of yields obtained by direct DNA extraction) of high-molecular weight DNA (>400 kb). Thus, plug digestion is amenable to large-insert metagenomic library construction and analysis. Comparisons of banding patterns generated by RAPD-PCR and DGGE indicated that sequence composition and inferred community composition of a given extract varied greatly with DNA isolation method. While overall diversity did not change significantly with the cell lysis method, analysis of 16S rRNA gene clone libraries revealed that each extraction procedure produced unique distributions of prokaryotic phyla within the sample population.     

彩色甜椒基因组DNA提取方法的优化

以不同颜色的甜椒为试验材料,采用CTAB法、SDS法、ROSE法和氯化苄法提取彩色甜椒基因组DNA。结果表明,CTAB法所提取DNA的质量和纯度较高,以此DNA为模板进行RAPD-PCR分析,DNA扩增效果较好,带形清晰、整齐,说明CTAB法提取的DNA较为完整,能用作RAPD-PCR模板来开展彩色甜椒分子水平的研究。