条纹斑竹鲨基因组的RAPD分析初报

采用11种随机引物对4 条条纹斑竹鲨基因组进行了RAPD检测。结果表明, 11种引物在每条个体上扩增的 DNA片段总数在77～84之间, 单个随机引物扩增的DNA片段数目由1至11条不等,平均为7.5条 DNA 片段, 片段的大小在 300～2 800bp之间。个体之间的相似率在90％以上。 Abstracts　4 individuals of Chiloscyllium plagiosum were analyzed by RAPD method using 11 arbitrary primers. For the 11 arbitrary primers, each individual showed 77～84 bands corresponding to amplified products. Each primer gave 1～11 bands for each individual. On average, about 7.5 bands were obtained per primer per individual. The length of the fragment is 300～2 800 bp. The similarity between band profiles of the four individuals was over 90％.     

RAPD分析在绢丝昆虫亲缘关系研究中的应用Ⅱ.柞蚕品种间的遗传差异

采用RAPD技术,对5个柞蚕品种的遗传差异进行比较研究.结果表明,所采用的40个随机引物中,有27个引物扩增谱带清晰且重复性较好,扩增总片段数253条,单个引物的扩增片段数在4～16之间,片段大小在0.33～3.0kb之间.不同柞蚕品种间的遗传差异较小,遗传距离(D)在0.066～0.1659之间,根据D值,由UPGMA聚类分析软件绘制了它们的分子进化树。 Abstract:Random amplified Polymorphic DNA (RAPD) was used to analyze the genetic diversity among Antheraea pernyi.The genetic variance of five Antheraea pernyi was studied.The result showed that:27 of 40 arbitrary primers could amplify clear and repeating bands.A total of 262 fragments were obtained.Each primer gave 4~16 bands and the average was 9.7.The length of the band was 0.33~3.0kb.The D value between different breeds of Antheraea pernyi was 0.066~0.1659.The D value was used to construct a dendrogram by UPGMA.     

用超常的复性温度改善小麦RA PD分析的效果

用38～66℃不同复性温度处理,比较了18条随机引物的扩增结果.发现复性温度在40～50℃之间均有数量不等的扩增产物,不同引物的最高复性温度不同,有些引物用60℃以上的复性温度仍有扩增产物.一些在35～38℃的复性温度下非特异性产物较多的引物,通过大幅度提高复性温度,能提高扩增产物的特异性,获得清晰的RAPD带型。 Abstract:To decrease nonspecific products and obtain clear RAPD patterns,18 10- mer random primers were tested at different annealing temperatures.The results indicated that all the amplification can be performed when the annealing temperature is in the range of 40~50℃.There were a few primers with which the amplification was still performed when the annealing temperature is above 60℃.By using high annealing temperatures,some primers which produce more nonspecific product at the annealing temperatures of 35~38℃ could generate reproducible and distinct bands.     

RAPD分析在绢丝昆虫亲缘关系研究中的应用I.蓖麻蚕品种间的遗传差异

用RAPD技术对蓖麻蚕基因组DNA进行多态性研究,分析了5个蓖麻蚕品种间的遗传差异。结果表明,所采用的40个随机引物中,有27个引物扩增谱带清晰且重复性较好,扩增总片段数达243个,单个引物的扩增片段数在4～17之间,平均为9条,片段大小在0.33～3.0kb之间。不同蓖麻蚕品种间的遗传距离(D)在0.0683～0.1603之间,根据D值,由UPGMA聚类分析软件绘制了它们的聚类分子树。 Abstract：Random amplified Polymorphic DNA (RAPD) was used to analyze the genetic diversity among eri sickworm. The genetic variance of five erisickworm was studied. The result showed that: 27 of 40 arbitrary primers could amplify clearly with repeatable bands.243 fragments were obtained.Each primer gave 4～17 bands and the average was 9.The length of the band was 0.33～3.0kb. The genetic distance (D) value between different breeds of Eri Silkworm was 0.0683～0.1603. The D value was used to construct a dendrogram by UPGMA.     

利用RAPD分子标记对三组三系杂交水稻及亲本的遗传分析和鉴定

利用RAPD技术,从248个随机寡聚核苷酸（10bp）中筛选出13个引物能在供试的三组三系杂交水稻及亲本间扩增出43条稳定性较好的多态性片段,其中6个引物能在供试材料间扩增出20个强的多态性标记。利用这些标记能有效地区分各组合中不育系、保持系、恢复系和F1,并能看出各组合中不育系与保持系、不育系与恢复系、F1与亲本间的遗传关系。 Abstract:A total of 248 arbitrary 10-mer oligonucleotide primers were screened using RAPD (random amplified polymorphic DNA) techniques with the genome DNA of three groups of three-line hybrid rice and their parents.Thirteen primers produced 43 polymorphism fragments.Six primers of them produced 20 obviously repeatable polymorphic markers among rice lines tested.Using this RAPD markers,the hybrid rice combinations (sterile-line,maintainer-line,restorer-line and F1)can be effectively identified,and the genetic relationship among them can be shown.     

用RAPD技术检测野生鲫鱼的四个金鱼代表品种的基因组DNA多态性

利用随机扩增多态DNA技术检测了两个不同地区的野生鲫鱼(Carassius a uratus L.)和4个金鱼(Carassius auratus Var.)代表品种的基因组DNA的多态性。用26个随机引物对各品种实验鱼的基因组DNA进行扩增,平均每个品种观察到约134个标记,单个引物获得的标记在1-16个之间。实验结果经统计学分析表明,金鱼和鲫鱼的随机扩增多态DNA共亨度高,进一步证实了金鱼由野生鲫鱼演化而来,聚类结果表明,草金鱼形成后,首先演化为文种鱼,然后由文种再形成龙种和蛋种两个品系。 Abstract:Genomic DNA polymorphisms in the wild crucian from rwo different areas and in four representative varieties of goldfish were detected by using random amplified polymorphic DNA(RAPD)technique.The genomic DNA of each variety of fish was amplified with 26 primers.On average,about 134 RAPD markers were observed by each variety.The markers obtained by a single primer varied from 1 to 16.The statistical analysis of the experimental results indicated that random amplified polymorphic DNA of the goldfish and wild crucian have a high proportion of random amplified polymorphic DNA fragment shared.This further verified that goldfish is evolved from wild crucian.The cluster analysis suggested that after emerging,grass goldfish is evolved to wen goldfish,then through wen goldfish evolved to dragoneye goldfish and oval goldfish.     

RAPD标记在山葡萄种质鉴定中的应用

采用修改后的CTAB 法获得了高质量的基因组DNA 。利用RAPD 标记技术对山葡萄7 份种质进行鉴定, 用4 个引物(从30 个引物中筛选)对试材进行PCR 扩增, 共扩增出30 条谱带, 平均每条引物产生7.5 条谱带, 其中21 条谱带为多态性谱带, 占总谱带数的70%。不同引物扩增的谱带数不同, 范围在6~9 条之间。利用4 个引物扩增出的多态性谱带可以将7 份山葡萄种质区分。     

甜樱桃品种及其砧木的RAPD分析

利用RAPD技术,从130个随机引物中筛选出46个引物,对欧洲甜樱桃、欧洲酸樱桃、马哈利樱桃和野生中国樱桃4个类型樱桃种,以及欧洲甜樱桃与中国樱桃的种间杂交种共15个品种的基因组遗传变异进行分析。结果表明,46个随机引物均得到了稳定可重复的RAPD图谱,扩增出的DNA条带大小在100~2625bp之间,多态性位点数517个,多态性位点百分率为98.85%,每个随机引物扩增出的多态性DNA条带数在4~23条。品种间Nei遗传距离在0.166~0.479之间,平均遗传距离0.329;甜樱桃新品种‘秦樱1号’与‘秦岭玛瑙’、‘CDR-1’等10个樱桃砧木之间的遗传距离在0.248~0.376,并且根据遗传距离可以相互区分,所分析的15个樱桃品种均扩增出了特有的DNA条带,每个樱桃特有标记带在2~17个之间,共扩增出149个特有标记,据此可以进行樱桃品种及砧木的RAPD鉴定。研究认为利用RAPD技术可以在分子水平上对甜樱桃品种及其砧木进行快速鉴定。     

一种提高RAPD技术扩增效率的有效方法

RAPD技术是由Williams等于1990年首先创立的一种DNA分子标记技术。但是由于低的退火温度和短的引物序列等原因,常常使得RAPD技术的分辨率和可重复性低。本文介绍一种通过延长从退火到延伸的ramp时间,提高RAPD产物的分辨率和产量,由此提高RAPD技术的扩增效率的有效方法。 Abstract:The random amplified polymorphic DNA(RAPD)is a technique of DNA molecular marker,which was first established by Williams in 1990.The resolution and repetition is low because of the low annealed tempture and short primers in the RAPD.In this paper we introduce an improved method for increasing the efficiency of the technique of RAPD by prolonging the ramp time from annealing to extension and increasing the resolution and production.     

6个中外猪品种的RAPD分析

应用RAPD技术分析了梅山猪、淮南猪、通城猪、八眉猪、合作猪、大白猪的遗传变异。用276个随机引物对6个猪种混合DNA扩增筛选,24个引物产生多态性。此24个引物经重新优化反应条件体系后,对276个个体DNA进行扩增。结果6个猪种的遗传多样性指数分别为0.178672、0.17781、0.15995、0.14549、0.16949和0.14157,群体内平均遗传多样性指数为0.16216,总群体遗传多样性指数为0.2534。基于Rogers公式计算的遗传距离采用NJ和UPGMA法进行聚类分析,结果6个猪种的亲缘关系与它们的地理分布基本一致。 Abstract:The genetic variation of Meishan,Huainan,Tongcheng,Bamei,Hezuo and Largewhite pigs were analyzed by RAPD markers.Twenty-four single polymorphic primers were selected out of 276 primers by amplifying six pool DNA.The index of Shannon were 0.178672,0.17781,0.15995,0.14549,0.16949,0.14159 respectively;Hpop was 0.16216,Hsp was 0.2534.The phylogenetic tree was constructed using NJ and UPGMA.The results indicated that the phenylogenetic relationship of the six pig breeds was consistent with their distribution.     

Genetic characterization of Metapenaeus affinis (H. M. Edwards, 1837) using RAPD markers

Genetic structure of four populations of Metapenaeus affinis from Maharashtra, Orissa, Kerala and Tamil Nadu in India was studied using RAPD markers. Five selective primers provided distinct and consistent RAPD profiles in all the four populations. The bands in the range 225–1,900 bp were scored for consistent results. The RAPD profiles generated by all the five primers revealed varying degrees of polymorphism, ranging from 25.00% (primer E-03) to 65.00% (primer E-06). Nei’s (Nei M, Natl Acad Sci Proc USA 70:3321–3323, 1973) genetic diversity (h) among the four populations varied from 0.2565 ± 0.2146 (Orissa population) to 0.3576 ± 0.1897 (Maharashtra population).     

Stability of RAPD fingerprints in potato: Effect of source tissue and primers

Variations in random amplified polymorphic DNA (RAPD) profiles from leaf, stem, root, and tuber tissues were observed in case of two glasshouse grown potato cultivars using 40 decamer primers suggesting possible danger of cultivar misidentification. Genomic DNA extracted from the above four tissues of four in vitro grown potato cultivars, however, produced more uniform RAPD fingerprints. A significant effect of random primers on fingerprint uniformity was observed in case of both glasshouse and in vitro grown samples. A new concept of stability index for random primers based on homogeneity of RAPD profiles obtained from different tissues of a single plant have been introduced. It is concluded that RAPD analysis of genomic DNA extracted from any tissue of in vitro grown potato plants using 14 selected decamer primers could be used to develop RAPD fingerprints for identification of Indian potato cultivars.     

Use of RAPD technique in evolution studies of four species in the family Canidae

The RAPD-PCR technique was applied to identify genetic markers able to distinguish between four canid species: the arctic fox (Alopex lagopus), red fox (Vulpes vulpes), Chinese raccoon dog (Nyctereutes procyonoides procyonoides) and six breeds of the domestic dog (Canis familiaris). A total of 29 ten-nucleotide arbitrary primers were screened for their potential use in the differentiation of these species. Ten primers amplified RAPD profiles that made it possible to distinguish between the investigated taxa. A number of species-specific bands was scored within RAPD profiles produced by these primers: 35.6% of all the polymorphic bands were unique to the Chinese raccoon dog, 29.6% were unique to the domestic dog, 21.2% were diagnostic for the red fox and 13.6% for the arctic fox. No breed-specific fragments were amplified from canine DNA; however, three primers produced bands characteristic for the dog, but not present in all of the investigated breeds. A Neighbor-Joining tree constructed on the basis of the analysis of RAPD profiles amplified by six primers revealed that the phylogenetic distance between the dog and the arctic fox is larger than the distance between the dog and the red fox. The phylogenetic branch of the Chinese raccoon dog was the most distinct on the dendrogram, suggesting that this species belongs to a different phylogenetic lineage. Obtained results make it possible to conclude that RAPD analysis can be a powerful tool for developing molecular markers useful in distinguishing between species of the family Canidae and for studying their phylogenetic relations.     

Use of Random Amplified Polymorphic DNA (RAPD) Analysis and Genetic Fingerprinting to Differentiate Isolates of Race O, C and T of Bipolaris maydis

Isolates of three races of Bipolaris maydis from China (races O, C and T) were compared using two techniques. Random Amplified Polymorphic DNA (RAPD) analysis using 24 primers indicated that race O and C isolates were more similar to one another than to the race T isolate. Twenty of the primers produced RAPD profiles that were similar for the race O and C isolates but differed for the race T isolate (four primers did not amplify products in any of the isolates). Four primers produced profiles which differed for all three races and two of these (A-09 and B-18) clearly differentiated the race O and race C isolates. Genetic fingerprinting of B. maydis using M13 DNA as a probe differentiated race O and C isolates from the race T with all four restriction enzymes used. Furthermore, when DNA was digested with Hind III, the hybridization profiles of the race O and C isolates differed from one another.     

Genetic characterization of two hill stream fish species Barilius bendelisis (Ham.1807) and Barilius barna (Ham.1822) using RAPD markers

Genetic structure of four wild populations of two hill stream fishes Barilius bendelisis (Ham.1807) and B. barna (Ham. 1822) from Uttarakhand, India, was studied using RAPD markers. Eight selective primers provided distinct and consistent RAPD profiles in both the species, producing a total of 47 and 35 scorable bands in B. bendelisis and B. barna respectively. The bands in the range 666–4,830?bp were scored for consistent results. The RAPD profiles generated by all the eight primers revealed varying degrees of polymorphism (25.00–50.00?%). The average genetic diversity (h) was estimated as 0.1661 and 0.1606 among the four populations of B. bendelisis and B. barna respectively.     

Use of conserved randomly amplified polymorphic DNA (RAPD) fragments and RAPD pattern for characterization of Lactobacillus fermentum in Ghanaian fermented maize dough.

The present work describes the use of randomly amplified polymorphic DNA (RAPD) for the characterization of 172 dominant Lactobacillus isolates from present and previous studies of Ghanaian maize fermentation. Heterofermentative lactobacilli dominate the fermentation flora, since approximately 85% of the isolates belong to this group. Cluster analysis of the RAPD profiles obtained showed the presence of two main clusters. Cluster 1 included Lactobacillus fermentum, whereas cluster 2 comprised the remaining Lactobacillus spp. The two distinct clusters emerged at the similarity level of <50%. All isolates in cluster 1 showed similarity in their RAPD profile to the reference strains of L. fermentum included in the study. These isolates, yielding two distinct bands of approximately 695 and 773 bp with the primers used, were divided into four subclusters, indicating that several strains are involved in the fermentation and remain dominant throughout the process. The two distinct RAPD fragments were cloned, sequenced, and used as probes in Southern hybridization experiments. With one exception, Lactobacillus reuteri LMG 13045, the probes hybridized only to fragments of different sizes in EcoRI-digested chromosomal DNA of L. fermentum strains, thus indicating the specificity of the probes and variation within the L. fermentum isolates.     

Identification and discrimination of thiobacilli using ARDREA, RAPD and rep-APD

A highly reliable procedure for fast identification and taxonomical categorization of thiobacilli is presented. The procedure includes RFLP analysis of PCR amplified 16S rDNA, 23S rDNA, and intergenic spacer rDNA between the 16S and the 23S rRNA-genes (amplified ribosomal DNA restriction enzyme analysis – ARDREA), as well as genomic fingerprinting using random primers (RAPD) and repetitive primers (Rep-APD). The taxonomic and discriminatory power of these three analytical approaches is compared. It is demonstrated that the RAPD and Rep-APD methods provide taxonomic results which are in agreement with the classification based on the RFLP analysis of the highly conservative ribosomal RNA ( rrn ) operons of the strains studied. Moreover, both kinds of genomic PCR fingerprinting, due to the fact that they derive information from different parts of the whole bacterial genome which may possess different degrees of variability, are more informative than ARDREA. By them, a discrimination of closely related Thiobacillus strains is possible. In addition, they are easier to perform and faster than the ARDREA. Using the method described, it was found that one Thiobacillus isolate recovered from uranium waste heaps described in the literature as Thiobacillus ferrooxidans ATCC 33020 is taxonomically neither closely related to the type strain of the species, Thiobacillus ferrooxidans ATCC23270^T, nor to any other strain of this species analysed in the present work.     

Genetic variation in Drechslera teres populations as indicated by RAPD markers

Random amplified polymorphic DNA (RAPD) was used to assess genetic variation among 48 isolates of Drechslera teres originating from different sites in Finland. RAPD profiles were generated with five arbitrary 10-mer primers and revealed polymorphisms suitable for screening differentiation in this fungal population. Using UPGMA clustering analysis, a similarity coefficient of approximately 63% was observed between all D. teres isolates studied. The variation was, however, distributed on a small scale as different genotypes were found from the same plant. The isolates could not be grouped according to geographic origin, aggressiveness, growth rate or morphological features, indicating that the primers used in this study were neutral markers for these characters. The primers were, however, able to differentiate between isolates of Helminthosporium species (D. teres, Drechslera graminea and Bipolaris sorokiniana).     

Random-amplified polymorphic DNA (RAPD) analysis shows intraspecies differences among Xanthomonas albilineans strains

Five strains of Xanthomonas albilineans , causal agent of leaf scald disease in sugarcane from various geographical regions, were compared using random amplification of polymorphic DNA (RAPD) to determine whether they could be differentiated at the DNA level. CsC1-purified genomic DNA from these strains were amplified by the polymerase chain reaction (PCR) using arbitrary 10-mer primers according to standard RAPD conditions and the amplification product profiles analysed by conventional agarose gel electrophoresis. Although most RAPD markers were common to all five strains, unique profiles for each strain were discernible using four 10-mer arbitrary primers individually. Reproducible DNA fingerprints indicate that RAPD analysis can be used to identify and differentiate the X. albilineans strains. This technique has the potential for use in monitoring the appearance of foreign strains of X. albilineans in various geographical regions and could be used for the construction of phylogenetic trees.     

Comparative Evaluation of RAPD and AFLP Based Genetic Diversity in Brinjal (Solanum melongena)

The present investigation was carried out with an objective of evaluating genetic diversity in brinjal (Solanum melongena) using DNA markers. A total of 38 brinjal accessions including one wild-species, Solanum sisymbrifolium were characterized using random amplified polymorphic DNA (RAP D) and amplified fragment length polymorphism (AFLP) techniques. Out of 45 primers employed to generate RAPD profiles, reproducible patterns were obtained with 32 primers and 30 (93.7%) of these detected polymorphism. A total of 149 bands were obtained, out of which 108 (72.4%) were polymorphic. AFLP analysis was carried out using four primer combinations. Each of these primers was highly polymorphic. Out of 253 fragments amplified from these four primer combinations, 237 (93.6%) were polymorphic. The extent of pair-wise similarity ranged from 0.264 to 0.946 with a mean of 0.787 in RAPD, in contrast to a range of 0.103 to 0.847 with a mean of 0.434 in AFLP. The wild species clustered separately from the brinjal genotypes. In the dendrogram constructed separately using RAPD and AFLP markers, the brinjal genotypes were grouped into clusters and sub-clusters, and the varieties released by IARI remained together on both the dendrograms. All the 30 RAPD primers in combination and each of the four primer pairs in AFLP could distinguish the brinjal accessions from each other. AFLP was thus found to be more efficient than RAPD in estimation of genetic diversity and differentiation of varieties in brinjal.