RAPD条件优化及天麻基因组DNA多态性分析

建立了RAPD扩增条件快速优化程序与方法．并应用于天麻基因组DNA扩增条件的优化及多态性的测定：获得了天麻基因组DNA的RAPD扩增优化条件和DNA指纹图谱;分析了模板DNA、引物、dNTP、Taq DNA聚合酶等的浓度和退火温度对RAPD扩增的影响．结果表明：天麻基因组DNA用引物S1扩增的片段具有更明显的多态性,这种指纹图谱更适合于天麻遗传分化研究;而用引物S12扩增的DNA指纹图谱具有更大的相似性,这种指纹图谱更适合于天麻真伪鉴别．该方法使RAPD扩增条件优化过程实现了程序化和数量化,是获得RAPD优化条件的简便快速、经济实用方法．应用该方法进行RAPD扩增,可获得图谱清晰、稳定可靠的实验结果．     

沙梨8个栽培品种的DNA指纹鉴定

利用RAPD标记技术,对8个沙梨(Pyrus pyrifolia)主栽品种进行RAPD分析。结果显示,从33个10碱基随机引物中筛选出了2个多态性高的引物S2075和S1296,扩增位点分别为11个和10个,以此为基础建立了8个品种的DNA指纹图谱;根据这2个引物中任何一个的扩增图谱均可以将这8个品种区分。研究结果为沙梨品种的区别与鉴定提供了一种有效方法。     

8份广西产绞股蓝种质的RAPD引物筛选及其遗传多样性分析

本研究的目的在于筛选合适的RAPD随机引物,应用RAPD技术对药用植物绞股蓝进行遗传多样性分析,并构建DNA指纹图谱。研究结果表明,我们利用生物信息学方法挑选出的20条引物中有19条引物的扩增条带清晰且多态性好;在清晰稳定出现的354条带中,294条具有多态性;其中有3条引物的扩增条带可清楚区分绞股蓝与混淆品种乌蔹莓,可建立其DNA指纹图谱。按UPGMA法进行聚类分析,计算其遗传相似系数,结果显示,8份绞股蓝供试材料聚为两类,聚类结果与其地理区域远近和生长环境一致。本研究中筛选出的19条引物适用于绞股蓝遗传多样性分析,且获得的DNA指纹图谱可用于鉴别绞股蓝。     

甘蓝品种'争春'和'寒光2号'的DNA指纹图谱构建

用SDS法提取甘蓝（Brassfca oleraceavat．capitata）品种‘争春’、‘寒光2号’及其各自亲本的基因组DNA,通过SRAP、RAPD两种分子标记方法,构建其DNA指纹图谱,用于种子纯度鉴定。利用30对SRAP引物组合和200个RAPD随机引物,以各品种及其亲本的基因组DNA为模板组进行筛选,结果显示：多数SRAP引物组合对模板组的扩增带型一致,少数组合扩增出差异,但未能找到具有互补差异的引物组合;通过RAPD标记方法筛选出能鉴定2个品种纯度的引物分别为S42、S103、S193和S42、S89、S151,其中引物S42对2组材料均能扩增出特异的RAPD指纹图谱,并将RAPD指纹图谱转变为相应的数字指纹。     

中国鸭茅主栽品种DNA指纹图谱构建

利用SSR标记和SCoT标记构建了我国主栽的21个鸭茅品种的DNA指纹图谱。从180对SSR引物和80个SCoT引物中,筛选出多态性高、谱带清晰的SSR引物和SCoT引物各24个。24对SSR引物在供试材料中共检测到186个条带,其中多态性条带为175个,品种特异条带6个,平均多态性比率94.03%,多态性信息量均值0.845,Shannon指数变幅0.4479~0.6549,基因多样性指数变幅0.2946~0.4633,可鉴别的品种数2~21个;利用24个SCoT引物在供试材料中共检测到321个条带,其中多态性条带为249个,品种特异条带6个,平均多态性比率76.33%,多态性信息量均值0.907,Shannon指数变幅0.2588~0.6329,基因多样性指数变幅0.1695~0.4451,可鉴别的品种数1~21个;5对SSR引物和5个SCoT引物在10个品种上具有唯一特征谱带,最终综合各项指标筛选出5个引物(A01E14、A01K14、B03E14、D02K13和SCoT23)上的37个条带用于鸭茅品种DNA指纹图谱构建,数据库中每个品种均具有唯一DNA指纹编码,构建的DNA指纹数据可用于鸭茅品种真伪鉴定,为品种权保护提供了科学依据。     

金银花五个品系的RAPD分析及DNA指纹图谱的建立

运用RAPD技术,对5个金银花品系进行遗传多样性研究并构建这5个金银花品系的DNA指纹图谱。从80个引物中筛选出25个带纹清晰,多态性好的引物用于实验。其中,引物SBSD06的扩增条带可以清楚明确区分5个品系,建立其DNA指纹图谱。在清晰、稳定出现的170条带中,153条带具有多态性。按UPGMA法进行聚类分析,计算其遗传相似系数,结果显示,金银花5个品系聚为两类,与其形态学分类结果相符。     

用随机扩增多态性DNA产物做探针产生鸡的DNA指纹图

我们用12个随机扩增多态性DNA(RAPD)引物对来自不同品系的4只鸡进行了RAPD分析,在扩增出的共99条带中,表现多态性的带为38条,占总带数的38％.回收了4个表现个体特异性的RAPD产物,当用鸡的基因组总DNA探针与它们杂交时,其中3个表现阳性,说明RAPD方法扩增出的高变异产物含有重复序列.用含重复序列的个体特异性RAPD产物作探针,与无关个体鸡基因组DNA的HaeⅢ酶切产物进行DNA印迹,获得了变异性较高的DNA指纹图谱.因此,高变异的RAPD产物可以有效地用作DNA指纹探针.     

二十八份玉米自交系的RAPD亲缘关系分析

采用RAPD技术,对28份玉米自交系的亲缘关系进行分析。旨在DNA水平上揭示玉米自交系之间的亲缘关系,为进一步提高玉米杂种优势利用水平提供有益的信息从100个10bp随机引物中筛选出24个多态性较好的引物,对28份玉米自交系DNA进行扩增,扩增出24张DNA指纹图谱,其中多态性DNA谱带106条,占总扩增带数的64％。利用DNA扩增结果进行聚类分析,建立了28个玉米自交系的亲缘天系树状图,将供试材料划分为五个类群,RAPD分析结果与已知系谱的亲缘关系基本一致。     

32个柿主栽品种SSR图谱构建及遗传变异分析

以32份柿主栽品种为试验材料,利用SSR标记技术构建其指纹图谱并进行遗传多样性分析。从78对候选引物中筛选出20对多态性高、稳定性好的引物作为核心引物,构建柿主栽品种SSR指纹图谱。结果显示:(1)20对引物在32份材料中共扩增出183条多态性条带,每对引物扩增出3～20条不等,平均每对引物扩增出9.15条,多态性比率为81.3%。各个位点的杂合度在0.410 3～0.914 3之间,平均为0.702 7。(2)8对引物在12个品种上具有特征带型,采用5对引物进行组合鉴定即可将32个柿品种完全区分开。(3)依据SSR带型特征,对每对引物生成的不同带型直接编号,简化柿SSR带型记录方法,并利用每个品种的带型编号,建立其DNA指纹图谱,结果表明,核心引物组合法比特征谱带法更适用于构建中国柿主栽品种DNA指纹图谱。(4)根据系统聚类分析将32个柿主栽品种分为两大类,品种间的亲缘关系与地理来源具有一定的相关性。     

名贵茶花种质资源的RAPD分析

采用RAPD方法构建了国内外23个茶花品种的指纹图谱。从100个随机引物中筛选出的20个有效引物共产生136条DNA片段,遗传多态性带120条占总数88．2％。遗传相似性分析表明,各基因型间的Nei＇s相似系数分布在0．4386～0．8936之间,平均相似性系数为0．7668。通过非加权算术平均数聚类（UPGMA）法,绘制了它们的遗传关系树状图,23个品种可划分为3个类群。研究结果表明,RAPD技术可用于茶花品种的鉴别以及种质资源遗传多态性的研究。     

满天星试管苗与其玻璃化苗的RAPD指纹图谱分析

采用分离群体分组分析法(BSA),用100个随机引物对满天星的正常苗和玻璃化苗进行RAPD分析的结果表明,7个随机引物扩增出多态性差异条带。再用上述7个引物分别对试管苗及其玻璃化苗个体进行DNA的PCR扩增的结果显示,引物J20在2种苗中出现差异条带。     

应用DNA指纹图谱技术鉴定整肠生菌株BL63516的研究

目的建立应用DNA指纹图谱技术鉴定微生态制剂——整肠生菌株BL63516的方法,提高菌种鉴别水平。方法应用RAPD（随机扩增多态性）方法,采用50条随机引物对7株地衣芽胞杆菌进行基因组DNA指纹图谱分析,选择多态性好、重复性好、稳定性强的随机引物,对BL63516与其他地衣芽胞杆菌进行区分。结果发现选用引物$87或$88分别对7株地衣芽胞杆菌进行基因组DNA指纹图谱分析,BL63516菌株扩增的DNA片段的大小、数量均与其他地衣芽胞杆菌有明显差异。结论此方法具有可重复性,方便、快速和准确的优势,可用于微生态制剂整肠生菌株的鉴别。     

Rapid DNA extraction methods and new primers for randomly amplified polymorphic DNA analysis of Giardia duodenalis.

A randomly amplified polymorphic DNA (RAPD) procedure using simple genomic DNA preparation methods and newly designed primers was optimized for analyzing Giardia duodenalis strains. Genomic DNA was extracted from in vitro cultivated trophozoites by five freezing-thawing cycles or by sonic treatment. Compared to a conventional method involving proteinase K digestion and phenol extraction, both freezing-thawing and sonication were equally efficient, yet with the advantage of being much less time- and labor-intensive. Five of the 10 tested RAPD primers produced reproducible polymorphisms among five human origin G. duodenalis strains, and grouping of these strains based on RAPD profiles was in agreement among these primers. The consistent classification of two standard laboratory reference strains, Portland-1 and WB, in the same group confirmed previous results using other fingerprinting methods, indicating that the reported simple DNA extraction methods and the selected primers are useful in RAPD for molecular characterization of G. duodenalis strains.     

RAPD (arbitrary primer) PCR is more sensitive than multilocus enzyme electrophoresis for distinguishing related bacterial strains.

The RAPD (random amplified polymorphic DNA) fingerprinting method, which utilizes low stringency PCR amplification with single primers of arbitrary sequence to generate strain-specific arrays of anonymous DNA fragments, was calibrated relative to the widely used, protein-based multilocus enzyme electrophoretic (MLEE) typing method. RAPD fingerprinting was carried out on five isolates from each of 15 major groups of Escherichia coli strains that cause diarrheal disease worldwide (75 isolates in all). Each group consisted of isolates that were not distinguishable from one another by MLEE typing using 20 diagnostic enzyme markers. In our RAPD tests, three or more distinct subgroups in each MLEE group were distinguished with each of five primers, and 74 of the 75 isolates were distinguished when data obtained with five primers were combined. Thus, RAPD typing is far more sensitive than MLEE typing for discriminating among related strains of a species. Despite their different sensitivities, the same general relationships among strains were inferred from MLEE and RAPD data. Thus, our results recommend use of the RAPD method for studies of bacterial population genetic structure and evolution, as well as for epidemiology.     

RAPD标记在紫菜遗传多样性检测和种质鉴定中的应用

用ＲＡＰＤ技术对４类紫菜（Ｐｏｒｐｈｙｒａ ｙｅｚｏｅｎｓｉｓ,Ｐ．ｈａｉｔａｎｅｎｓｉｓ,Ｐ．ｋａｔａｄｎｉ ｖａｒ．ｈｅｍｉｐｈｙｌｌａ和Ｐ．ｏｌｉｇｏｓｐｅｒｍａｔａｎｇｉａ）的１５个无性系丝状体进行了遗传多样履分析,从５０个ＯＰＥＲＯＮ引物中经过初筛,其中６个引物可以扩增出稳定的可重复的图谱。这６个引物共扩增出了６０条带,多态性比例达９７．１％。根据ＲＡＰＤ结果将这１５个无性了紫菜的ＤＮＡ     

The Application of RAPD Markers in Diversity Detection and Variety Identification of Porphyra

RAPD (random amplified polymorphic DNA) markers were generated from filaments of 15 Porphyra lines representing four important groups, P. yezoensis, P. haitanensis, P. katadai var. hemiphylla and P. oligospermatangia. Among the total 69 fragments generated by 6 selected primers (among 50 primers), 67 appeared to be polymorphic (97.1%). Cluster analysis based on the RAPD results was performed. The 15 Porphyra lines were divided into 3 groups. This result was consistent with that from taxonomy analysis. A DNA fingerprinting based on 8 bands amplified with OPN-02 and OPJ-18 was constructed and might be used in Porphyra variety identification. Five specific RAPD fragments of 5 Porphyra lines were isolated and cloned into pGEM-T easy vector. These five RAPD fragments may be useful in germplasm identification and property protection of Porphyra.     

Biodiversity of Anabaena azollae isolates from different Azolla cultures

The random amplified polymorphic DNA (RAPD) profile of A. azollae strains isolated from four different Azolla cultures was studied by using different primers. The objective of this study was to determine whether polymerase chain reaction (PCR) with different primers could differentiate the isolated A. azollae strains from one another. The primers amplified specific sequences of the isolates and generated fingerprinting pattern characteristic of each isolate. Clear polymorphism was noticed among all the strains which depends on the primer sequence.     

RAPD Analysis of Germplasm in Ancient

By using 40 random primers and 8 anchor-SSR primers, RAPD and microsatellite DNA (SSR) polymorphism were detected in the ancient "Taizi lotus" and the modem Chinese red flower lotus (Nelumbo nucifera Gaertn. ) from Hebei, Harbin (wild population), Jiangxi and Hunan (cultivar). The polymorphism information could be provided with 13 random primers and 2 SSR primers. Total 135 loci were amplified, 71 loci of these were polymorphic (53%). Agarose electrophoresis showed high genetic identity without any genetic variation whithin "Taizi" and Harbin lotus by using the above-mentioned 15 primers. However, there were different extent of differentiation within Hebei, Jiangxi and Hunan lotus. According to the UPGMA analysis of MEGA program, "Taizi", Harbin and Hebei lotus were gathered to a branch in the dendrogram. The genetic distance of "Taizi" and Hebei lotus were very close (0.05). They shared a common ancestry. Comparing with the modem Chinese lotus ( N. nucifera ), the ancient "Taizi" lotus only lacked one locusOPM06-300, so it still belonged to N. nucifera. Hunan and Jiangxi lotus were close to gather and could be grouped in another branch. Their genetic distances from the ancient "Taizi" lotus were farthest (0.67).     

Use of RAPD-PCR fingerprinting to detect genetic diversity of soil Streptomyces isolates

Random amplified polymorphic DNA (RAPD) was used for identification and assessment of genetic diversity between isolates of Streptomyces from soil. Genomic DNA from 18 Streptomyces isolates and 2 reference strains were amplified using four different 10-mer primers. Different DNA fingerprinting patterns were obtained for all the isolates. Electrophoretic and cluster analysis of the amplification products revealed incidence of polymorphism among the isolates and none of them was identical to the reference strains although there were some common amplification bands. Two highly divergent groups were determined among the isolates. The results indicate that RAPD is an efficient method for discriminating and studying genetic diversity of Streptomyces isolates.     

Genetic stability of three economically important micropropagated banana (Musa spp.) cultivars of lower Indo-Gangetic plains, as assessed by RAPD and ISSR markers

An efficient micropropagation protocol produced large number of plants of the three elite banana (Musa spp.) cultivars Robusta (AAA), Giant Governor (AAA) and Martaman (AAB) from shoot tip meristem. The genetic relationships and fidelity among the cultivars and micropropagated plants as assessed by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers, revealed three somaclonal variants from Robusta and three from Giant Governor. A total of 5330 RAPD and 2741 ISSR fragments were generated with 21 RAPD and 12 ISSR primers in micropropagated plants. The percentage of polymorphic loci by RAPD and ISSR were found to be 1.75, 5.08 in Robusta and 0.83, 5.0 in Giant Governor respectively. Among the two marker systems used, ISSR fingerprinting detected more polymorphism than RAPD in Robusta and Giant Governor with most of the primers showing similar fingerprinting profile, whereas Martaman revealed complete genetic stability.