11种兰属植物DNA的提取及RAPD-PCR实验体系的建立与优化

采用改进的CTAB-DNA提取方法，从11种兰属植物的嫩叶中提取总DNA。所得的DNA样品的A260／A280值在1．7～1．9之间，琼脂糖凝胶上主带清晰，较少降解，样品纯度高，DNA量大。另外，对影响RAPD-PCR的Mg^2＋、dNTPs、Taq酶、引物浓度等因素进行了优化。确定优化的反应体系为：75ng模板DNA，1×Buffer，2．5mmol／LMg^2＋，0．15mmol／LdNTPs，0．75UTaq酶，引物浓度0．4μmol／L，反应总体积为25山。该体系在20个供试兰属实验材料中获得较好的扩增结果。     

Molecular systematics in Chrysanthemum × grandiflorum (Ramat.) Kitamura

An attempt was made to understand the molecular systematics and genetic differences between 10 original chrysanthemum cultivars and 11 mutants. The similarity among the cultivars and mutants varied from 0.17 to 0.90 using RAPD analyses, a simple but efficient method to distinguish cultivars and to assess parentage. Two distinct groups were found. Two cultivars were present as a separate group showing differences from all other cultivars. Mutants with different flower colour could be identified at the molecular level using RAPD technique holding promise to identify unique genes as SCAR markers. A high genetic distance among the different chrysanthemums showed that there exists a possibility of introgressing new and novel genes from the chrysanthemum gene pool.     

Use of random amplified polymorphic DNA (RAPD) to identify races 1, 2, 4 and 8 of Fusarium oxysporum f. sp. dianthi in Italy

The RAPD fingerprinting procedure was used in combination with pathogenicity assays on differential cultivars to characterize a representative collection of 72 Fusarium spp. isolates of different geographic origin collected from diseased carnation. In F. oxysporum f. sp. dianthi, isolates were grouped according to the physiologic race: group 1 included isolates of race 4; group 2 was formed by isolates of race 2 and single representatives of races 5 and 6; group 3 included isolates of races 1 and 8. No correlation was found between RAPD data and geographic origin of the isolates tested: representatives of race 2 isolated in Italy, Israel and Japan had the same amplification profile. Three isolates which showed a low level of pathogenicity on all carnation cultivars tested shared an identical amplification pattern and are probably saprophytic F. oxysporum. Finally, two F. redolens isolates from Japan and seven non-pathogenic isolates of F. proliferatum collected from diseased carnation in Italy, Israel and The Netherlands were clearly distinguishable according to their RAPD fingerprint. The results are discussed in relation to previous studies on the genetic diversity of F. oxysporum f. sp. dianthi and to the development of forma specialis- and pathotype-specific diagnostic tools.     

Genetic diversity within commercial populations of watercress (Rorippa nasturtium-aquaticum), and between allied Brassicaceae inferred from RAPD-PCR

Commercial productivity of watercress (Rorippa nasturtium-aquaticum) can be adversely affected by the pathogenic crook-root fungus, Spongospora subterranea f.sp. nasturti, and watercress viruses. As there are no effective control measures for these diseases, attempts have been made to breed varieties resistant to the crook-root pathogen. This work has been hindered by a lack of knowledge of the genetic base of commercial watercress, and the genetic distance between watercress and allied Brassicaceae which have been identified as candidates for hybridisation programmes. We measured the diversity within these two groups using the RAPD-PCR fingerprinting technique and analysed the data by both distance methods and principal co-ordinate analysis. Little genetic diversity was found within commercial watercress populations. However, watercress formed a unique cluster genetically distinct from other Rorippa species, but equidistant to Cardamine species. It was placed closer to Barbarea verna. This revised version was published online in August 2006 with corrections to the Cover Date.     

利用RAPD-PCR与ISSR-PCR标记技术分析长江口刀鲚的群体遗传结构

利用RAPD-PCR及ISSR-PCR两种分子标记技术对长江出海口两年3个群体的52条刀鲚（Goiliaectenes）进行群体遗传结构的分析。在3个群体中，利用RAPD-PCR标记技术，15个10bp随机引物共检测到110条带，多态性为0．490～0．657，Shannon多样性指数为18．63～22．38，Nei平均遗传距离为0．1195～0．1454；而利用11个ISSR引物所获得的相应结果分别为67、0．576～0．682、11．56～13．66以及0．117～0．147。相关性分析表明这两种技术所获得的上述数据呈正相关（r=0．95，P〈0．05）。AMOVA结果显示，3个群体按采集时间划分成的两年之间的遗传变异不超过总变异的1．1％，同一年内群体间的遗传变异也分别只占总变异的6．99％（RAPD）和2．75％（ISSR-PCR），群体内各个体之间的遗传变异占总变异的96％（P〈0．0001），说明两年内所采集的3个群体之间基本上没有产生遗传分化。基于样品之间的Nei遗传距离所构建的Neighborjohing聚类图也清楚地显示出没有按采样的时间产生群体的遗传分化。对各样品之间的Nei遗传距离及Neighbor-joining聚类图分别经Mantel检测及支序分析，发现ISSR-PCR技术所获得的结果与RAPD-PCR技术的结果不存在明显的正相关，但ISSR-PCR标记技术具有在待测样品中能检测到高多态性等优点。     

蜜蜂遗传多样性研究的RAPD-PCR反应体系的正交优化

利用正交设计L16（45）对蜜蜂基因组DNARAPD-PCR反应体系的5个因素（Tag酶,引物,Mg2＋,dNTP,模板）在4个水平上进行优化试验,筛选出各个反应因素的最佳水平,建立蜜蜂模板DNARAPD-PCR反应的最佳体系（25L）：10×PCRbuffer3.0L,Tag酶0.5U,引物浓度0.4mol/L,Mg2＋浓度3.0mmol/L,dNTP浓度0.5mmol/L,模板40ng。对蜜蜂DNARAPD-PCR最佳反应体系的退火温度进行了梯度试验,最佳退火温度为54℃。     

应用正交设计优化江南牡丹RAPD-PCR反应体系

以牡丹品种"云芳"基因组DNA为模板,采用正交实验设计L25(55)对影响牡丹RAPD-PCR反应的5因素(模板DNA、引物、Mg2+、dNTPs、Taq酶)在5个水平上进行优化试验。结果表明:最佳的RAPD-PCR的反应体系为:20μL体系中含有25 ng的模板DNA,0.2μmol/L的引物,Mg2+1.0 mmol/L,1×buffer反应缓冲液,DNTPs各为0.2 mmol/L,1.0 U的TaqDNA聚合酶。     

A duplex-PCR bioassay to detect a Trichoderma virens biocontrol isolate in non-sterile soil

This paper describes the identification and utilisation of a sequence-characterised amplified region (SCAR) marker specific for the Trichoderma virens biocontrol isolate GV4. The marker was developed from a RAPD-PCR amplification product unique to isolate GV4. When used as a hybridisation probe in Southern blot analysis, it hybridised to the DNA of the species T. virens alone and not to that of other Trichoderma species or closely related genera Gliocladium and Verticillium. The marker also produced a GV4-specific RFLP, distinguishing it from other T. virens isolates when probed to blots with HindII, BamHI or PstI genomic DNA digests. Primers designed from the sequence of the RAPD marker produced a diagnostic amplification product of 346 bp for GV4 alone, distinguishing it from all other test isolates. With the exception of one, test isolates did not produce an amplification product with the SCAR primers. The exception was a single Verticillium psalliotae isolate (ICMP5509) that produced a product of approx. 400 bp that was easily distinguished from the 346 bp product of GV4. The reliability of the SCAR-based diagnostic test was further improved with the introduction of a positive PCR reaction control to each test, achieved by converting the test to a duplex PCR system. Two universal primers flanking the two ITS and the 5.8S region of the ribosomal gene complex were introduced to each reaction to provide a test for PCR reaction inhibitors to eliminate false negatives in the diagnosis. Amplification of this multi-copy genomic region did not reduce diagnostic sensitivity of the single copy SCAR marker. To further increase the sensitivity of detecting GV4 propagules while maintaining a fast sample assessment assay, soil was amended with cornmeal, as a nutrient source, and a mix of antibiotics to favour Trichoderma growth. The soil mix was subsequently incubated for 5 d before total DNA was extracted. Under these conditions, the duplex soil PCR assay detected GV4 down to a concentration of 10 spores g⁻¹ soil in non-sterile agricultural field soil. This study is the first to report the use of a duplex-PCR diagnostic bioassay for a species within the Hypocrea/Trichoderma genus.     

蒌蒿DNA提取、RAPD优化及引物筛选初报

为了能从分子水平探讨蒌蒿资源的遗传多样性、蒿属的系统分类和种质资源的鉴定,在此,报道采用小量法(SDS法)和大量法(CTAB法)提取蒌蒿DNA,建立优化的RAPD-PCR体系,并进行引物初步筛选。结果显示,两种抽提方法都可有效抽提出高质量的DNA,大量法得率要明显高于小量法,但小量法抽提的DNA也足够RAPD-PCR反应几十至上百次,不同的实验室可根据实验的需要进行选择。对两种方法抽提的DNA,都进行PCR扩增体系梯度摸索,最优的RAPD-PCR反应条件为:25μl反应体系中,含DNA模板约20ng,10×TaqDNA聚合酶缓冲液2.5μl,2μldNTPs(2.5mM),Mg2 2μl(25mM),TaqDNA聚合酶0.15μl(5U/μl),引物0.5μl(25μM),其余以双蒸水补充。在剔除了重复性差,条带模糊和单态的引物后,有九个引物表现稳定,扩增出来的条带清晰、多态性高。