用扩增片段的长度多态性分子标记探讨盾叶薯蓣的遗传多样性

用扩增片段的长度多态性(amplified fragment length polymorphism,AFLP)标记分析研究了中国5个盾叶薯蓣居群30个个体的遗传多样性。筛选出9对AFLP引物,从中检测到14698条清晰可见的条带,其中多态性带12628条,多态性比率85.92%。Shannon信息指数(I)为0.3656±0.1721,物种水平的Nei基因多样性(H)为0.2322±0.2200。遗传变异分析表明,物种水平的遗传分化系数Gst为0.4827,说明其群体间存在一定的遗传分化,居群间的基因流Nm为0.5358,居群间遗传交换较小。聚类分析结果显示5个居群盾叶薯蓣有较为丰富的遗传变异,且与地理分布有相关性。     

AFLP分子标记技术在昆虫学研究中的应用

AFLP分子标记技术是一种建立在PCR技术和RFLP标记基础上的新的DNA指纹分析技术 ,具有多态性丰富、结果稳定可靠、重复性好、所需DNA量少、可以在不知道基因组序列的情况下进行研究等特点 ,现已广泛用于构建遗传图谱、遗传多样性研究、系统进化及分类学、遗传育种和品质鉴定以及基因定位等方面。该文介绍了AFLP标记技术的原理以及在昆虫学研究中的应用。     

Establishment of the Amplified Fragment Length Polymorphism (AFLP) technique for genotyping of pollen beetle (Meligethes aeneus) - a noxious insect pest on oilseed rape (Brassica napus)

In order to conduct studies concerning genetic variability of pollen beetles (Meligethes aeneus), a genotyping protocol was established. No genome information is available for pollen beetles so the amplified fragment length polymorphism (AFLP) technique was chosen since it does not depend on any prior sequence information of the samples and also is a sensitive and robust technique. However, several modifications were needed in order to adapt the method for analysis of pollen beetles. Basic modifications included (i) alterations of DNA purification, (ii) use of two six-cutter restriction enzymes, (iii) and modified PCR conditions. This protocol resulted in a favourable number of fragments of an appropriate size range for standard gel analysis by a DNA sequencer applicable to a single insect and even body parts enabling different assays to be conducted on a single specimen. Pollen beetles from different areas of Sweden were analysed to verify the reproducibility and efficacy of the protocol as well as for phenetic analysis. The high reproducibility of the modified AFLP protocol allows it to be used as a reliable tool for genotype analysis of pollen beetles.     

Optimization of AFLP fingerprinting of organisms with a large-sized genome: a study on Alstroemeria spp.

 The recently introduced PCR-based DNA fingerprinting technique AFLP (amplified fragment length polymorphism) allows the selective amplification of subsets of genomic restriction fragments. AFLP has been used for multiple purposes such as the construction of linkage maps, marker saturation at specific genomic regions, analysis of genetic diversity and molecular phylogeny and cultivar identification. AFLP can be tailored by varying the number of selective nucleotides added to core primers and can allow accurate amplification, even in complex template mixtures generated from plant species with very large genomes. In this study Alstroemeria, a plant species with a very large genome, was tested for adapting the AFLP protocol. The results indicated that the estimated number of amplification products was close to the observed number when eight selective nucleotides were used but that seven selective nucleotides did not increase the number of amplification products fourfold. However, we found reproducibility in both +7 and +8 fingerprints. Various distributions of selective nucleotides over the various rounds of preamplifications were tested. Preamplification with four selective nucleotides followed by final amplification with eight selective nucleotides produced clear and reproducible AFLP patterns. The effects of GC content of primers and multiple preamplification steps were also discussed. Received: 16 March 1998 / Accepted: 14 July 1998     

AFLP分子标记技术及其在动物学研究中的应用

扩增片段长度多态性技术(AFLP)基于选择性扩增完全酶切消化后的基因组DNA片段,包括酶切与连接、选择性扩增、检测分析等3个步骤。该技术的运用不需要预知基因组的序列特征,具有较高的多态分辨力,产生的标记是显性标记,可适用于任何来源和各种复杂度的DNA。自AFLP技术问世以来,在酶切、扩增体系、检测和分析方法等方面不断得到改进。本文将以线虫、昆虫、鱼类、鸟类、家畜、鼠、人等为例,介绍近年来AHLP技术在动物或人的遗传图谱构建和QTL(quantitative trait loci)定位、生物多样性、性别决定和繁殖行为研究、疾病及疾病诊断研究等上的应用。     

扩增片段长度多态性实验技术及在动物遗传多样性研究中的应用

扩增片段长度多态性(AFLP)是一种有效的分子遗传标记方法,具有经济、简便、模板需要量少、重复性高、结果可靠等优点。目前AFLP在动物方面的应用还不是很多,处于初级阶段,主要用于鉴定分类关系、种群遗传多样性分析、遗传连锁图谱构建等方面。     

AFLP标记的特点及其在昆虫学研究中的应用

扩增片段长度多态性（AFLP）是一种新兴的很有效的分子遗传标记方法, 它通过对基因组DNA限制性内切酶酶切片段进行选择性扩增而揭示多态性,具有快速、经济简便、不需要预先知道模板DNA的信息、模板需要量少、重复性高、结果可靠及具有很高的信息含量等优点。AFLP也具有缺点,主要是标记是显性的,同其他显性标记一样,不能区分杂合体和纯合体,因而不能更好地估算种群遗传的变异,对种群遗传结构的分析不能提供更多的统计信息;AFLP技术较复杂,而且经常使用放射性同位素,对模板DNA质量要求也较高。为了克服AFLP的这些缺点,人们又在其基础上发展了其他相关技术,例如AFRP、SAMPL、DALP和TE-AFLP等。目前AFLP在昆虫方面的应用还不是很多,处于初级阶段,主要应用在生态型鉴定、种群遗传分析、连锁图谱构建等方面,相信随着其技术的发展完善,必将会越来越多地应用于昆虫学的研究中。     

Evaluation of AFLP for genetic mapping in Pinus radiata D. Don

Efficient construction of reasonable density genetic linkage maps is an essential component of QTL detection programmes. The AFLP technique has been used to produce genetic linkage maps in a range of species. We have developed protocols to generate reproducible AFLP profiles in Pinus radiata and have evaluated the inheritance and informativeness of AFLP markers in this important timber species. The large genome size of P. radiata necessitated increased levels of selection at both the pre-amplification and selective amplification steps of the AFLP protocol to generate reproducible AFLP profiles. Once optimised ca. 41.3 scorable AFLP bands were resolvable through denaturing gels, of which 48.4% were polymorphic in a screen of eight unrelated trees. This level of polymorphism is ca. three times higher than with RAPD markers. The total number of bands and the number of polymorphismic bands per PCR were ca. halved when AFLPs were electrophoresed on non-denaturing gels and stained with ethidium bromide. Using the protocols developed, AFLP is an efficient method for generating the DNA markers required for genetic linkage map construction in P. radiata. This revised version was published online in June 2006 with corrections to the Cover Date.     

Suitability of genomic DNA synthesized by strand displacement amplification (SDA) for AFLP analysis: genotyping single spores of arbuscular mycorrhizal (AM) fungi

Limited biological samples of microbial origin often yield insufficient amounts of genomic DNA, making application of standard techniques of genetic analysis, like amplified fragment length polymorphism (AFLP), virtually impossible. The Phi29 DNA polymerase based whole genome amplification (WGA) method has the potential to alleviate this technical bottleneck. In the present work, we have sought to investigate the suitability of genomic DNA synthesized using Phi29 based WGA for AFLP analysis. We first used genomic DNA from Saccharomyces cerevisiae to optimize the protocol for the use of SDA-amplified DNA for AFLP analysis. Based on the optimized protocol we obtained AFLP fingerprints which were indistinguishable from the non-amplified genomic DNA. Finally, AFLP analysis was performed using SDA synthesized genomic DNA from single spores of various species of arbuscular mycorrhizal (AM) fungi. Unique and highly reproducible fingerprints for each species were obtained. The present study introduces the application of WGA-mediated AFLP to AM fungal biology; similarly, our protocol could be useful for other microbial genomes currently not amenable to genetic analysis owing to the paucity of starting template.     

Amplified fragment length polymorphism (AFLP): a review of the procedure and its applications

Amplified fragment length polymorphism (AFLP) is a novel molecular fingerprinting technique that can be applied to DNAs of any source or complexity. Total genomic DNA is digested using two restriction enzymes. Double-stranded nucleotide adapters are ligated to the DNA fragments to serve as primer binding sites for PCR amplification. Primers complementary to the adapter and restriction site sequence, with additional nucleotides at the 3′-end, are used as selective agents to amplify a subset of ligated fragments. Polymorphisms are identified by the presence or absence of DNA fragments following analysis on polyacrylamide gels. This technique has been extensively used with plant DNA for the development of high-resolution genetic maps and for the positional cloning of genes of interest. However, its application is rapidly expanding in bacteria and higher eukaryotes for determining genetic relationships and for epidemiological typing. This review describes the AFLP procedure, and recent, novel applications in the molecular fingerprinting of DNA from both eukaryotic and prokaryotic organisms. Received 19 December 1997/ Accepted in revised form 3 June 1998