Efficiency of reduced primer selectivity and bulked DNA analysis for the rapid detection of AFLP† polymorphisms in a range of crop species

Two approaches to optimise AFLP fingerprinting forthe rapid detection of genetic polymorphisms, i.e.reduced primer selectivity and bulked DNA analysiswere examined. The efficiency of reduced primerselectivity to increase the detection frequency ofgenetic polymorphisms and to obtain more informativefingerprinting profiles was tested in six differentcrops. The number of selective nucleotides was reducedto six in onion, to five in barley, potato, lettuceand cabbage, and to four in flax. This allowed therapid identification of several primer pairs that wereable to discriminate between closely relatedgermplasm. Reproducibility tests on replicate DNAsamples indicated no major negative effects on thereliability of the fingerprinting profiles due to theuse of less selective primers, although for onionpurified DNA was needed to avoid irreproducibleresults. In barley, flax and onion, a less thanfourfold increase in the number of fragments wasobserved when primer pairs were reduced by oneselective nucleotide. This result was attributed todifferent tolerance levels for amplificationmismatches between primer pairs of differentselectivity.The efficiency of bulked DNA analysis to detectgenetic polymorphisms was investigated in differentmixtures of two barley DNA samples. AFLP's of varyingintensity could still be recovered when the two DNA'swere mixed in a 1:1 ratio. However, the frequency ofrecovered bands quickly dropped when in the mixturesthe presence of the DNA carrying the fragments wasdecreased below 50%.The usefulness of the two approaches is discussed inrelation to various aspects of genetic resourcesmanagement.     

Evaluation of 'quick and dirty' DNA extraction methods for marker-assisted selection in rice (Oryza sativa L.)

Six simple methods for extracting DNA from rice seedlings were evaluated for marker‐assisted selection (MAS). The assessment of each method was based on PCR amplification of SSR markers, DNA yield and purity, time and cost. Based on these criteria, two methods were selected as being superior to other methods. The best two methods included the standard method developed at the International Rice Research Institute (IRRI), which utilizes a sodium dodecyl sulfate extraction buffer followed by chloroform/isoamyl alcohol extraction and a previously published method using sodium hydroxide and Tris. These two methods produced nearly identical PCR amplification results. The sodium hydroxide method is considerably simpler, quicker and cheaper than the standard IRRI method, and may be particularly useful for many applications of MAS or high‐resolution mapping. This method was also adapted into an effective high throughput method utilizing 96‐well plates emphasizing its versatility.     

Effect of Azospirillum brasilense inoculation on rhizobacterial communities analyzed by denaturing gradient gel electrophoresis and automated ribosomal intergenic spacer analysis

Nucleic acid-based techniques allow the exploration of microbial communities in the environments such as the rhizosphere. Azospirillumbrasilense, a plant growth promoting rhizobacterium (PGPR), causes morphological changes in the plant root system. These changes in root physiology may indirectly affect the microbial diversity of the rhizosphere. In this study, the changes in the rhizobacterial structure following A. brasilense inoculation of maize (Zea mays) plants was examined by PCR-denaturating gradient gel electrophoresis (DGGE) and automated ribosomal intergenic spacer analysis (ARISA), using two universal primers sets for the 16S rRNA gene, and an intergenic 16S-23S rDNA primer set, respectively. Similar results were obtained when using either ARISA or DGGE performed with these different primer sets, and analyzed by different statistical methods: no prominent effect of A. brasilense inoculation was observed on the bacterial communities of plant roots grown in two different soils and in different growth systems. In contrast, plant age caused significant shifts in the bacterial populations.     

长距离RT-PCR非特异性扩增的鉴定与排除

根据已经测定的乙脑病毒WHe株全基因组序列(GenBank EF107527),设计1对引物扩增基因组全长cDNA和2对特异性的鉴定引物。经RT-PCR技术扩增后,得到约9 kb的产物,经过反复实验,鉴定为外源性污染核酸扩增产物,并加以有效的排除,初步的研究表明该产物可能是由外源性污染核酸自身反向部分互补扩增所致。     

真空解冻装置的电气控制系统

本文介绍了真空解冻装置电气控制系统的设计思想与系统构成。该控制系统主要包括温度控制系统、真空度控制系统和定时控制系统。文中对系统主要电路的工作思路，性能和特点进行了分析，该系统经生产单位的试用，温度控制精度高，工作可靠。     

Gene amplification and insecticide resistance

Pesticide resistance in arthropods has been shown to evolve by two main mechanisms, the enhanced production of metabolic enzymes, which bind to and/or detoxify the pesticide, and mutation of the target protein, which makes it less sensitive to the pesticide. One route that leads to enhanced metabolism is the duplication or amplification of the structural gene(s) encoding the detoxifying enzyme, and this has now been described for the three main families (esterases, glutathione S-transferases and cytochrome P450 monooxygenases) implicated in resistance. More recently, a direct or indirect role for gene duplication or amplification has been described for target-site resistance in several arthropod species. This mini-review summarises the involvement of gene duplication/amplification in the insecticide/acaricide resistance of insect and mite pests and highlights recent developments in this area in relation to P450-mediated and target-site resistance.     

基于LAMP方法的苜蓿疫霉根腐病菌分子检测研究

苜蓿疫霉根腐病菌是我国检疫性有害生物之一.但是,目前国内尚无检测标准.笔者针对该病菌特异分子标记,设计了LAMP引物,建立了苜蓿疫霉根腐病菌恒温快速检测方法(巳申请发明专利).该方法快速、准确、无需PCR仪,很好地满足了口岸检疫部门的检测要求.     

ZL胶粉聚苯颗粒外保温施工技术--哈尔滨威德自动化焊接有限公司联合厂房施工实例

本文结合工程实践介绍了ZL胶粉聚苯颗粒外保温在高寒地区的施工方法和要点,并阐述了在高寒地区推广的可行性.     

适于贵州玉米种质DNA指纹库构建的SSR核心引物筛选

为了筛选出适用于贵州玉米种质DNA指纹库构建的引物,以21份贵州普通玉米自交系和36份糯玉米品种为材料,综合比较PIC值大小、扩增带型清晰度及引物的重复性高低,对87对SSR引物进行筛选,以确定贵州玉米种质DNA指纹库构建的核心SSR引物。结果表明:筛选出的23对和15对SSR引物可分别作为构建贵州普通玉米和贵州糯玉种质DNA指纹库的核心引物。