Monitoring of cultivar identity in tissue culture-derived date palms using RAPD and AFLP analysis

In the present study, two polymerase chain reaction (PCR)-based methods namely, randomly amplified polymophic DNA (RAPD) and amplification fragment length polymorphism (AFLP) were employed to assess genetic variations, which may appeared, in tissue culture-derived date palm (Phoenix dactylifera) offshoots. Analysis of RAPD banding patterns generated by PCR amplification using 37 random primers gave no evidences for somaclonal variations and the percentage of polymorphic bands in a total of 259 scored bands was zero. Meanwhile, analysis of AFLP banding patterns generated using 13 primer combinations pointed to minor genetic variations in the AFLP banding patterns. The percentage of genetic variations (polymorphism) in tissue culture-derived date palm offshoots belonging to cultivars Sakkoty, Gandila and Bertamoda was 2.6, 0.79 and 1 %, respectively, as revealed by AFLP analysis. The low percentage of genetic variations confirms the genetic stability of tissue culture-derived dry date palm cultivars.     

Studies on somaclonal variation in Phalaenopsis

The morphological and genetic variations in somaclones of Phalaenopsis True Lady “B79-19” derived from tissue culture were evaluated. In 1360 flowering somaclones, no apparent difference was found in the shape of the leaves, whereas flowers in some somaclones were deformed. We have demonstrated that 38 selected random primers can be used to generate amplified segments of genomic DNA and to differentiate polymorphisms of somaclonal variations in Phalaenopsis. The random amplified polymorphic DNA (RAPD) data indicated that normal and variant somaclones are not genetically identical. We also studied the banding patterns of aspartate aminotransferase (AAT) and phosphoglucomutase (PGM) in young leaves of variant and normal somaclones of Phalaenopsis. With respect to AAT, three distinct banding patterns were found in normal somaclones and only two-banded phenotypes were detected in variant somaclones. In a comparison of the banding patterns of PGM isozymes, three to four bands were detected in normal somaclones and two to three bands in variant ones. Received: 15 August 1997 / Revision received: 16 February 1998 / Accepted: 1 May 1998     

Occurrence of chromosomal variations and plant regeneration from long-term-cultured citrus callus

Summary Embryogenic callus of Anliucheng sweet orange (Citrus sinensis Osbeck) is theoretically diploid. However, significant chromosomal variations occurred when the calluses were subcultured and preserved for a long time. Cytological observation revealed a variety of mitotic irregularities underlying the occurrence of chromosomal variations. Despite the ubiquitous existence of chromosomal variations, long-term-cultured calluses were still capable of producing somatic embryos and plants. Interestingly, chromosomal variants were selected against when somatic embryos and plants regenerated from the embryogenic callus. Randomly amplified polymorphic DNA (RAPD) analysis was also carried out to detect DNA sequence variation in regenerated plants derived from the embryogenic callus. No difference in banding patterns was detected. It was clear that the plant regeneration from long-term-cultured callus was inclined to select against somaclonal variations.     

Prospects for estimating nucleotide divergence with RAPDs

The technique of random amplification of polymorphic DNA (RAPD), which is simply polymerase chain reaction (PCR) amplification of genomic DNA by a single short oligonucleotide primer, produces complex patterns of anonymous polymorphic DNA fragments. The information provided by these banding patterns has proved to be of great utility for mapping and for verification of identity of bacterial strains. Here we consider whether the degree of similarity of the banding patterns can be used to estimate nucleotide diversity and nucleotide divergence. With haploid data, fragments generated by RAPD-PCR can be treated in a fashion very similar to that for restriction-fragment data. Amplification of diploid samples, on the other hand, requires consideration of the fact that presence of a band is dominant to absence of the band. After describing a method for estimating nucleotide divergence on the basis of diploid samples, we summarize the restrictions and criteria that must be met when RAPD data are used for estimating population genetic parameters.      

DNA Polymorphism among Rice Somaclones

Molecular markers were used to detect the influence of high concentrations of 2,4 dichlorophenoxyacetic acid (2,4-D) in the callusing media on DNA variations in regenerated rice plants. Restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and polymerase chain reaction (PCR) based RFLP analysis were carried out on 12 somaclones of Oryza sativa L. cv. B-370. In vitro culture induced DNA variations were detected in the regenerated plants but the effect of high auxin concentration in the medium could not be revealed. In a second study, fingerprinting of 15 semi-dwarf, high yielding somaclones of B-370 was carried out using RAPD technique. Amplification using 20 random primers produced a total of 167 DNA bands out of which 97 bands were polymorphic. A total of 32 unique DNA bands were detected across all the somaclones and they could be grouped based on their similarity to B-370. RAPD analysis helped to reveal similarity or differences among the somaclones while fingerprinting using additional RAPD markers was not successful.     

Use of RAPD markers for identifying Nelore bulls with early reproductive maturation onset

Random amplified polymorphic DNA (RAPD) markers were used for identifying animals with early (precocious) or late (non-precocious) reproductive maturation onset. Animals (n=34) were phenotyped according to spermatozoa appearance in ejaculates (group A, 20 animals) or to the expected progeny difference (EPD) values for scrotal circumference (group B, 14 animals). The RAPD markers were initially detected by amplifying two pooled samples of equimolar amounts of DNA from the eight precocious 12 and non-precocious animals of group A. Only 38 out of 320 random primers used for screening group A pooled samples detected polymorphisms. These polymorphic primers generated 443 distinguishable and reproducible bands, from which 174 were polymorphic and 269, monomorphic. These polymorphic primers were then used in RAPD reactions to amplify individual DNA samples from animals belonging to both groups, A and B. The dendrograms generated from RAPD patterns allowed phenotypic class differentiation in both cases. Therefore, RAPD markers can be used as a tool for identifying genotypes favoring early sexual maturation in Nelore breeding programs.     

Identification of cultivars and validation of genetic relationships in Mangifera indica L. using RAPD markers

Twenty-five accessions of mango were examined for random amplified polymorphic DNA (RAPD) genetic markers with 80 10-mer random primers. Of the 80 primers screened, 33 did not amplify, 19 were monomorphic, and 28 gave reproducible, polymorphic DNA amplification patterns. Eleven primers were selected from the 28 for the study. The number of bands generated was primer- and genotype-dependent, and ranged from 1 to 10. No primer gave unique banding patterns for each of the 25 accessions; however, ten different combinations of 2 primer banding patterns produced unique fingerprints for each accession. A maternal half-sib (MHS) family was included among the 25 accessions to see if genetic relationships could be detected. RAPD data were used to generate simple matching coefficients, which were analyzed phenetically and by means of principal coordinate analysis (PCA). The MHS clustered together in both the phenetic and the PCA while the randomly selected accessions were scattered with no apparent pattern. The uses of RAPD analysis for Mangifera germ plasm classification and clonal identification are discussed.     

Application of RAPD markers for characterization of γ-ray-induced rose mutants and assessment of genetic diversity

Six parent and their 12 gamma ray-induced somatic flower colour mutants of garden rose were characterized to discriminate the mutants from their respective parents and understanding the genetic diversity using Random amplification of polymorphic DNA (RAPD) markers. Out of 20 primers screened, 14 primers yielded completely identical fragments patterns. The other 7 primers gave highly polymorphic banding patterns among the radiomutants. All the cultivars were identified by using only 7 primers. Moreover, individual mutants were also distinguished by unique RAPD marker bands. Based on the presence or absence of the 48 polymorphic bands, the genetic variations within and among the 18 cultivars were measured. Genetic distance between all 18 cultivars varied from 0.40 to 0.91, as revealed by Jaccard’s coefficient matrix. A dendrogram was constructed based on the similarity matrix using the Neighbor Joining Tree method showed three main clusters. The present RAPD analysis can be used not only for estimating genetic diversity present in gamma ray-induced mutants but also for correct identification of mutant/new varieties for their legal protection under plant variety rights.     

澳大利亚棉花黄萎病菌DNA多态性及其地理分布相关性研究(英文)

应用ＲＡＰＤ技术对澳大利亚东南部八个主要棉花种植区的９９个棉花黄萎病菌菌株进行了ＤＮＡ多态性分析。结果表明用１０个筛选的随机引物对供试菌株的全基因组ＤＮＡ扩增,共获得９２条ＲＡＰＤ谱带,其中５５．４％的谱带为多态带。经类聚分析,供试菌株类聚为１５个ＲＡＰＤ遗传指纹相似组,其中１０个指纹相似组的菌株与其采集区域有明显相关性,其余５个指纹相似组的菌株为普通分布的指纹类型。     

澳大利亚棉花黄萎病菌DNA多态性及其地理分布相关性研究

应用ＲＡＰＤ技术对澳大利亚东南部八个主要棉花种植区的９９个棉花黄萎病菌菌株进行了ＤＮＡ多态性分析。结果表明用１０个筛选的随机引物对供试菌株的全基因组ＤＮＡ扩增,共获得９２条ＲＡＰＤ谱带,其中５５．４％的谱带为多态带。经类聚分析,供试菌株类聚为１５个ＲＡＰＤ遗传指纹相似组,其中１０个指纹相似组的菌株与其采集区域有明显相关性,其余５个指纹相似组的菌株为普通分布的指纹类型。     

Genotyping of Ganoderma species by improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis

Species of Ganoderma are used in traditional medicines. An improved random amplified polymorphic DNA (RAPD) analysis, where the RAMP time is prolonged, has been used to characterize the genetic variation in some well known species of Ganoderma. The DNA materials were collected from ten Ganoderma strains, amplified with randomly selected 24 RAPD primers and evaluated by agarose gel electrophoresis. A cluster dendrogram was constructed for genetic analysis on the basis of amplification results. The improved RAPD amplified DNA with consistent and clear banding patterns. A total of 316 bands were found with 93% polymorphism. There was a significant genetic distance between the different strains of Ganoderma, with an index of similarity coefficient in the range of 0.52–0.74. The inter-simple sequence repeat (ISSR) analysis of the Ganoderma DNA samples showed similar trend results to the RAPD analysis with 0.49–0.81 similarity coefficients. This study reports the high level of genetic differences between different species or strains of a single species of Ganoderma and confirms the significance of the improved RAPD method in genetic characterization of organisms. Therefore, the improved RAPD combined with ISSR techniques might be used for the genetic characterization of organisms.     

RAPD typing for distinguishing species and strains in the genus Listeria

The randomly amplified polymorphic DNA (RAPD) technique was employed in the development of a typing protocol for Listeria isolates, particularly Listeria monocytogenes strains. A single strain of L. monocytogenes was used and 200 random decamer primers were screened for their discriminatory abilities by visualizing the amplification products electrophoretically. Three candidate primers displaying potentially useful banding patterns were selected and tested against 52 L. monocytogenes strains, encompassing 11 serotypes, and 12 other strains representing five other Listeria spp. Thirty-four banding profiles were obtained with one particular primer. RAPD analysis allowed differentiation between Listeria spp. and was found to further subdivide strains of the same serotype. Where only one primer was used strains from different serotypes were occasionally found to produce identical banding profiles. RAPD analysis, which in our hands proved to be reproducible, shows much promise as a molecular alternative to traditional L. monocytogenes typing protocols.     

用RAPD技术检测不同生态类型蜘蛛代表品种的基因组DNA多态性

蜘蛛有3种生态类型,即洞穴型、结网型和游猎型。利用RAPD技术检测3种生态类型蜘蛛的代表品种,即白额巨蟹蛛(游猎型)、漏斗蛛(结网型)、虎纹捕鸟蛛和七纺器蛛(洞穴型)的基因组DNA的多态性。用11个随机引物对3种生态类型的代表蜘蛛的基因组DNA进行扩增,平均每个品种观察到约22.5个标记,单个引物获得的标记在4～13个之间。实验结果经统计学分析表明,L纺器蛛和虎纹捕鸟蛛的随机扩增多态DNA共享度(F)高,在分子水平上进一步证实了同生态类型蜘蛛的亲缘天系最近,聚类结果表明,3种类型蜘蛛的总体演化趋势为:洞穴型 结网型—游猎型,与以往的形态学研究相符。     

Genetic variability analysis among clinical Candida spp. isolates using random amplified polymorphic DNA

The patterns of genetic variation of samples of Candida spp. isolated from patients infected with human immunodeficiency virus in Vitória, state of Espírito Santo, Brazil, were examined. Thirty-seven strains were isolated from different anatomical sites obtained from different infection episodes of 11 patients infected with the human immunodeficiency virus (HIV). These samples were subjected to randomly amplified polymorphic DNA (RAPD) analysis using 9 different primers. Reproducible and complex DNA banding patterns were obtained. The experiments indicated evidence of dynamic process of yeast colonization in HIV-infected patients, and also that certain primers are efficient in the identification of species of the Candida genus. Thus, we conclude that RAPD analysis may be useful in providing genotypic characters for Candida species typing in epidemiological investigations, and also for the rapid identification of pathogenic fungi.     

Determination of paternity in dragonflies by Random Amplified Polymorphic DNA fingerprinting

We used Random Amplified Polymorphic DNA (RAPD) fingerprinting to address issues of paternity in two odonate species. Amplification artifacts of RAPD markers were controlled by assessing paternity patterns relative to the banding patterns generated by quantitative mixtures of DNA from putative parents ('synthetic offspring'). In the aeshnid dragonfly Anax parthenope , for which the mating histories of both males and females were unknown, we found strong evidence for complete paternity success for the contact guarding male. In the highly polygamous libellulid dragonfly Orthetrum coerulescens , we detected and quantified mixed paternity in sequentially produced offspring clutches and demonstrated that fertilization success is correlated with the duration of copulation. Our results suggest that RAPD fingerprinting is suitable to address issues of paternity in systems which are genetically uncharacterized and produce large offspring clutches.     

稻恶苗菌异核体三种核型核DNA的RAPD分析

利用RAPD技术,对稻恶苗菌TJ47-26异核体的紫、红、白三种核型(同核体)核DNA进行比较,用80种10bp长的随机引物进行DNA扩增,共检测了基因组中近400个位点,未发现三种核型的基因组间有明显差异,表明紫、红、白三种核型的DNA碱基序列高度相似。由此推测TJ47-26异核体的三种不同核型是来自同一个核的变异体。     

DNA polymorphism in various goose lines by RAPD-PCR

RAPD markers often primers were used to assess the polymorphism among pooled DNA of eight goose lines. The number of bands amplified by each primer ranged from 1 to 8, within a mean of 2.86. Some bands appeared specific for the line or genetic background. RAPD technique is an effective method for generating the polymorphic DNA marker in the goose. RAPD patterns from mixed DNA samples can reflect the genetic information of populations. The present study indicated that 10 generations selected for egg production and body weight under various pressure, resulted in genetic variation among goose lines as detected by RAPD. Selection for meat traits caused greater genetic diversity than selection for egg production.     

人体蠕形螨的DNA提取与随机引物PCR检测

【目的】探索人体毛囊蠕形螨和皮脂蠕形螨DNA的提取方法。【方法】采用液氮反复冻融研磨法破碎螨体细胞, 选用改良小昆虫DNA提取法、碱裂解法和试剂盒提取法, 分别提取冻存时间在5个月内和8~10个月的毛囊蠕形螨和皮脂蠕形螨基因组DNA, 并用随机引物PCR方法进行检测。【结果】蛋白核酸测定仪检测结果显示, 试剂盒法提取的DNA纯度较高、量较多, 明显优于改良小昆虫法和碱裂解法。随机引物扩增结果显示清晰的DNA指纹图谱, 两种人体蠕形螨DNA指纹具有明显差异。蠕形螨冻存时间影响DNA提取的量, 但对DNA提取的纯度和RAPD指纹图谱影响较小。不同DNA提取方法提取的同一种蠕形螨DNA指纹图谱基本相似, 试剂盒法和改良小昆虫法提取的DNA样本条带多而清晰, 碱裂解法提取的样本条带少而模糊。【结论】液氮反复冻融研磨法破碎蠕形螨细胞是有效的, 蠕形螨冻存时间不宜超过6个月, 试剂盒提取法是提取蠕形螨DNA的好方法。RAPD技术可以用于这两种人体蠕形螨DNA分子水平上的检测和分类。     

Randomly amplified polymorphic DNA PCR as a tool for assessment of marine viral richness

Recent discoveries have uncovered considerable genetic diversity among aquatic viruses and raised questions about the variability of this diversity within and between environments. Studies of the temporal and spatial dynamics of aquatic viral assemblages have been hindered by the lack of a common genetic marker among viruses for rapid diversity assessments. Randomly amplified polymorphic DNA (RAPD) PCR bypasses this obstacle by sampling at the genetic level without requiring viral isolation or previous sequence knowledge. In this study, the utility of RAPD-PCR for assessing DNA viral richness within Chesapeake Bay water samples was evaluated. RAPD-PCR using single 10-mer oligonucleotide primers successfully produced amplicons from a variety of viral samples, and banding patterns were highly reproducible, indicating that each band likely represents a single amplicon originating from viral template DNA. In agreement with observations from other community profiling techniques, resulting RAPD-PCR banding patterns revealed more temporal than spatial variability in Chesapeake Bay virioplankton assemblages. High-quality hybridization probes and sequence information were also easily generated from single RAPD-PCR products or whole reactions. Thus, the RAPD-PCR technique appears to be practical and efficient for routine use in high-resolution viral diversity studies by providing assemblage comparisons through fingerprinting, probing, or sequence information.     

Genomic instability in silica- and cadmium chloride-transformed BALB/c-3T3 and tumor cell lines by random amplified polymorphic DNA analysis

Our earlier studies using random amplified polymorphic DNA (RAPD) analysis have shown genetic instability in human lung cancer tissues. Here we have investigated the potential for genetic instability in silica- and cadmium chloride (CdCl2)-transformed BALB/c-3T3 cell lines. Non-transformed, transformed BALB/c-3T3 cells, and tumor cell lines (obtained by injecting nude mice with transformed cell lines) were analyzed for genomic changes. DNAs from 10 different transformed clones and their corresponding tumor cell lines were amplified individually by RAPD analysis using 10 arbitrary primers. DNA from non-transformed BALB/c-3T3 cells was used as a control to compare genetic alterations, if any, between non-transformed, transformed and tumor cell populations. PCR products from RAPD were electrophoretically separated on agarose gels and the banding profiles were visualized by ethidium bromide staining. Five of the 10 primers tested revealed genomic changes in silica-transformed cell lines when compared to non-transformed BALB/c-3T3 cells. Comparison of all 10 transformed and tumor cell lines showed varied degrees of genomic changes using all 10 primers. CdCl2-transformed cell lines displayed fewer genomic changes, only three of 10 primers showed a positive result. CdCl2-transformed cells and their corresponding tumor cell lines showed specific banding pattern differences in six of the 10 samples tested with six of the 10 primers. Changes in band intensity were the most commonly observed changes both in silica- and CdCl2-transformed and tumor cell lines. The results seem to indicate a progressive change in genomic rearrangements which may directly or indirectly be associated with progression of tumorigenesis.