Analysis of genetic diversity for wild and captive green peafowl populations by random amplified polymorphic DNA technique

The genetic diversity of the populations for 14 wild green peafowls (Pavo muticus) and 18 captive green pea-fowls was investigated by using the technology of random amplified polymorphic DNA (RAPD). Totally 161 and 166 ampli-fied bands were obtained by using 23 arbitrary primers to amplify the genomic DNA of wild and captive green peafowls re-spectively. The results showed that the average relative genetic distance of the wild and captive green peafowls popula-tions was 0.0555 and 0.1355, respectively, and difference of the average relative genetic distances between the two popu-lations was 0.1635. The Shannon diversity index for the wild and captive green peafowl populations was 0.4348 and 1.0163, respectively, which means that there exists significant difference in genetic diversity between the two populations, and the genetic diversity of wild green peafowl was low. The two populations originated from two different families according to analysis by the UPGMA method. This research can provide the theoretical basis for supervising genealogies management of peafowl populations.

Use of random amplified polymorphic DNA （RAPD） analysis to detect jujube witches＇ broom phytoplasmas

Jujube witches＇ broom is a devastating disease of Ziziphusjujube that occurs in various jujube regions of China. Nucleic acid extracted from midribs of samples collected from three jujube varieties （＂Suanzao＂, ＂Lajiaozao＂ and ＂Langzao＂） from symptomatic and asymptomatic shoots were tested by random amplified polymorphic DNA analyses. Using 13 different 10 and 11-bp random primers the amplification of jujube DNA was achieved from all the samples; AMI4 primer provided amplification of specific DNA fragments of about 400 bp, only from samples collected from symptomatic plants. No genetic variations in these varieties were identified using the other 11 arbitrary primers; only with primer AL07 it was possible to differentiate ＂Langzao＂ from the other two varieties tested. All the experiments were repeated 2 times and the results were consistent. Compared with PCR analyses with phytoplasma-specific primers, RAPD techniques resulted to be an alternative rapid and sensitive method for detecting jujube phytoplasmas presence in different jujube varieties.

Genetic instability in cancer tissues analyzed by random amplified polymorphic DNA PCR

OBJECTIVE: To detect DNA and chromosomes instabilities during the progression of tumors and screen new molecular markers coupled to putative or unknown oncogenes and/or tumor suppressor genes. METHODS: Five kinds of tumors, in a total of 128 specimens, were analyzed by random amplified polymorphic DNA (RAPD) PCR. Bands representing instabilities were recovered, purified, and cloned, labeled as probes for Southern and Northern blot analysis and DNA sequencing. RESULTS: Sample 5 and 3 of the gastric cancer tissues showed the highest genomic changes and the average detectability in five cancers was up to at least 40% (42.2% - 49.4%). There were significant differences in the ability of each primer to detect genomic instability, which ranged from 27% (primer 8) to 68% (primer 2). Band B is a single copy fragment, and was found to be an allelic loss in gastric and colon cancers. It is a novel sequence and was registered in GenBank with Accession Number AF151005. Further analysis revealed that it might be part of a cis-regulatory element of a new tumor suppressor gene, containing a promoter of cis-action 'CACA' box, an enhancer of 'GATA' family and a start codon. CONCLUSIONS: It was impossible or difficult to get great achievements for cancer treatments with the procedure of gene therapy only to one oncogene or one tumor suppressor gene because the extensive DNA variations occurred during the progression of tumor. RAPD assay connected with other techniques was a good tool for the detection of genomic instabilities and direct screening of some new molecular markers related to tumor suppressor genes or oncogenes.

Combined Effects of Cadmium and Butachlor on Microbial Activities and Community DNA in a Paddy Soil

Due to frequent soil Cd contamination and wide use of butachlor in China,there is a need to assess their combined toxicity to soil microorganisms.The combined effects of cadmium(Cd,10 mg kg-1 soil) and herbicide butachlor(10,50,and 100 mg kg-1 soil) on enzyme activities and microbial community structure in a paddy soil were assessed using the traditional enzyme assays and random amplified polymorphic DNA(RAPD) analysis.The results showed that urease and phosphatase activities were significantly reduced by high butachlor concentration(100 mg kg-1 soil).When the concentrations of Cd and butachlor added were at a ratio of 1:10,urease and phosphatase activities were significantly decreased whereas enzyme activities were greatly improved at the ratio of 1:5,which indicated that the combined effects of Cd and butachlor on soil urease and phosphatase activities depended largely on their addition concentration ratios.Random amplified polymorphic DNA(RAPD) analysis showed loss of original bands and appearance of new bands when compared with the control soil.Random amplified polymorphic DNA fingerprints suggested substantial differences between the control and treated soil samples,with apparent changes in the number and size of amplified DNA fragments.The addition of high concentration butachlor and the combined impacts of Cd and butachlor significantly affected the diversity of the microbial community.RAPD analysis in conjunction with other biomarkers such as soil enzyme parameters would prove a powerful ecotoxicological tool.Further investigations should be carried out to understand the clear link between RAPD patterns and enzyme activity.

Analysis of the Genetic Structure of Sclerotinia sclerotiorum （Lib.） de Bary Populations from Different Regions and Host Plants by Random Amplified Polymorphic DNA Markers

: The genetic diversity and genetic structure of a population of isolates of Sclerotinia sclerotiorum (Lib.) de Bary from different regions and host plants were investigated using the random amplified polymorphic DNA (RAPD) method with 20 random decamer primer pairs in order to provide some information on the phylogenetic taxa and breeding for resistance to sclerotinia stem rot. A minimum of three and a maximum of 15 unambiguously amplified bands were generated, furnishing a total of 170 bands ranging in size from 100 to 3 200 bp, corresponding to an average of 8.5 bands per primer pair. One hundred and four of these 170 bands (61.2%) were polymorphic, the percentage of polymorphic bands for each primer pair ranging from 0.0% to 86.7%. The genetic relationships among the isolates, based on the results of RAPD analysis, were examined. The genetic similarity of all selected isolates was quite high. At the species level, the genetic diversity estimated by Nei's gene diversity (h) was 0.197 and Shannon's index of diversity (I) was 0.300. The unweighted pair-group mean analysis (UPGMA) cluster analysis showed that most isolates from the same regions were grouped in the same cluster or a close cluster. The population of isolates from Hefei (Anhui Province, China) was more uniform and relatively distant to other populations. The Canadian population collected from carrot (Daucus carota var. sativa DC.) was relatively close to the Polish population collected from oilseed rape (Brassica napus L.) plants. There was no relationship between isolates from the same host plants. An analysis of molecular variance (AMOVA) revealed that the percentage of variance attributable to variation among and within populations was 50.62% and 49.38%, respectively. When accessions from China, Europe, and Canada were treated as three separate groups, the variance components among groups, among populations within groups, and within populations were ?0.96%, 51.48%, and 49.47%, respectively. The genetic differentiations among and within populations were highly significant (P < 0.001). Similarly, the coefficient of gene differentiation (Gst) in total populations calculated by population genetic analysis was 0.229 4, which indicated that the genetic variation among populations was 22.94%. The gene flow (Nm) was 1.68, which indicated that the gene permutation and interaction among populations was relatively high.

Assessment of Genetic Variation Within Indian Mustard (Brassica juncea) Germplasm Using Random Amplified Polymorphic DNA Markers

Genetic diversity among 45 Indian mustard （Brassica juncea L.） genotypes comprising 37 germplasm collections, five advance breeding lines and three improved cultivars was investigated at the DNA level using the random amplified polymorphic DNA （RAPD） technique. Fifteen primers used generated a total of 92 RAPD fragments, of which 81 （88%） were polymorphic. Of these, 13 were unique to accession ‘Pak85559＇. Each primer produced four to nine amplified products with an average of 6.13 bands per primer. Based on pairwise comparisons of RAPD amplification products, Nei and Li＇s similarity coefficients were calculated to evaluate the relationships among the accessions. Pairwise similarity indices were higher among the oilseed accessions and cultivars showing narrow ranges of 0.77-0.99. An unweighted pair-group method with arithmetic averages cluster analysis based on these genetic similarities placed most of the collections and oilseed cultivars close to each other, showing a low level of polymorphism between the accessions used. However, the clusters formed by oilseed collections and cultivars were comparatively distinct from that of advanced breeding lines. Genetically, all of the accessions were classified into a few major groups and a number of individual accessions. Advanced breeding lines were relatively divergent from the rest of the accessions and formed independent clusters. Clustering of the accessions did not show any pattern of association between the RAPD markers and the collection sites. A low level of genetic variability of oilseed mustard was attributed to the selection for similar traits and horticultural uses. Perhaps close parentage of these accessions further contributed towards their little diversity. The study demonstrated that RAPD is a simple and fast technique to compare the genetic relationship and pattern of variation among the gene pool of this crop.

Identification of Random Amplified Polymorphic DNA Markers Linked to Sex Determination in Calamus simplicifolius C. F. Wci

The random amplified polymorphic DNA （RAPD） molecular marker technique was used to determine the sex of Calamus simplicifolius C. F. Wei In the present study, DNA samples were extracted individually from 10 male and 10 female plants. After a total of 1 040 decamer primers had been tested, an approximate 500-bp male-specific DNA fragment was generated with the S 1443 primer. It is feasible to identify sex at the early stages of plant life, which is beneficial for improving breeding programs of this dioecious species. In addition, we have obtained a proper RAPD protocol that is useful for other species of rattan.

Rapid detection of self-biting disease of mink by specific sequence-characterized amplified regions

Self-biting disease occurred in most farmed fur animals in the world. The mechanism and rapid detection method of this disease has not been reported. We applied bulked sergeant analysis （BSA） in combination with RAPD method to analyze a molecular genetic marker linked with self-biting trait in mink group. The molecular marker was converted into sequence-characterized amplified regions （SCAR） marker for rapid detection of this disease. A single RAPD marker A8 amplified a specific band of 263bp in self-biting minks, which was designated as SRA8-250, and non-specific band of 315bp in both self-biting and healthy minks. The sequences of the bands exhibited 75% and 88% similarity to Canis familiarizes major histocompatibility complex （MHC） class II region and Macaca mulatta MHC class I region, respectively. A SCAR marker SCAR-A8 was designed for the specific fragment SRA8-250 and validated in 30 self-biting minks and 30 healthy minks. Positive amplification of SCAR-A8 was detected in 24 self-biting minks and 12 healthy minks. χ2 test showed significant difference （p〈0.01） in the detection rate between the two groups. This indicated that SRA8-250 can be used as a positive marker to detect self-biting disease in minks. Furthermore, the finding that self-biting disease links with MHC genes has significant implications for the mechanism of the disease.

Panax ginseng-specific sequence characterized amplified region (SCAR) marker for testing medicinal products

To screen genetic polymorphisms of Panax ginseng, as well as those of Panax quinquefolium and Panax notoginseng, analysis of random amplified polymorphic DNA （RAPD） was performed using 120 random primers. Of the successful amplicons obtained, the Panax ginseng-specific RAPD marker C-12 was cloned into a TA vector and sequenced （Genl3ank access number KU553472）. Based on the sequence analysis results, a pair of primers specific to C-12 was designed. Finally, a SCAR marker-based identification system for Panax ginseng was developed after optimization of the reaction conditions. Using this method, two positive bands were stably observed at 300 bp and 130 bp in 33 batches of Panax ginseng samples tested, while negative results were obtained for another 101 batches of samples, including Panax quinquefolium, Panax notoginseng, adulterants, and other medicinal herbs. Thus, we successfully developed a PCR-based method for rapid and effective identification of Panax ginseng, which can be effectively used for the protection and utilization of germplasm resources and identification of the origins of Panax ginseng samples.

Use of Random Amplified Polymorphic DNA Analysis for Economically Important Food Crops

The objective of this review is to summarize numerous studies on the use of the random amplified polymorphic DNA （RAPD） technique on rice, corn, wheat, sorghum, barley, rye, and oats to examine its feasibility and validity for assessment of genetic variation, population genetics, mapping, linkage and marker assisted selection, phylogenetic analysis, and the detection of somaclonal variation. Also we discuss the advantages and limitations of RAPD. Molecular markers have entered the scene of genetic improvement in different fields of agricultural research. The simplicity of the RAPD technique made it ideal for genetic mapping, plant and animal breeding programs, and DNA fingerprinting, with particular utility in the field of population genetics.

Intra-specific relationships among Tibetan Eared-pheasants based on randomly amplified polymorphic DNA(RAPD)analysis

The Tibetan Eared-pheasant Crossoptilon harmani is a rare species native to China.A captive population has been established in the Beijing Zoo since 1999.In order to determine the kinship of the offsprings in 2001,randomly amplified polymorphic DNA(RAPD)was used to examine the parenthood of seven Tibetan Eared-pheasants in the Beijing Zoo.To amplify the genomic DNA of each individual,53 arbitrary primers were selected.The results of amplifications showed that 14 primers had clear and distinct RAPD patterns.Totally,226 amplified fragments were generated by RAPD in this study.Cluster analysis of the seven Tibetan Eared-pheasants indicated that all the four young birds had the same father(No.5 male).This study provides a practical method to determine the relationship of offsprings whose parents are unknown in birds.

RAPD Markers and Genetic Information Entropy in Environmental Monitoring: A Case Study with Wild Mushrooms

Mushrooms have a remarkable scientific value due to their nutritional, medicinal properties and industrial applications in enzyme production, so that effort in the maintenance of native wild mushroom varieties is increasing. The present study focuses on the use of Random Amplified Polymorphic DNA (RAPD) markers for biodiversity measure of wild mushroom species of the Northwest mountainous region of Greece. Data mining of similarity matrices from RAPD analysis was used to extract measurable entropy parameters for mushroom biodiversity monitoring based on Shannon’s information entropy. Shannon information index provides an easy assessment of the entropy of the genetic information of the germplasm per mushroom species while the total equitability index (EH) = 0.871 offers an overall estimation of the genetic variation evenness of all species in the population of the studied mushrooms. Application of RAPDs with parallel entropy analysis is an easily applicable and low-cost valuable technology in environmental monitoring, using genetic information of wild mushroom species as an indicator that can lead to future actions in biodiversity maintenance and germplasm protection. The provided methodology can serve as a pilot procedure enriched with other environmental factors to monitor and protect wild mushroom communities native to the Greek countryside or in any part of the world and provide comparable results about biodiversity from different regions using common entropy indices.

Cytological and Molecular Identification of Alien Chromatin in Giant Spike Wheat Germplasm

Alien chromosomes of twelve giant spike wheat germplasm lines were identified by C-banding, genomic in situ hybridization (GISH), sequence characterized amplified region (SCAR), and random amplified polymorphic DNA (RAPD). All lines showed a chromosome number of 2n = 42, five of them carried both a pair of wheat-rye (Triticum aestivum-Secale cereal) 1BL/1RS translocation chromosomes and a pair of Agropyron intermedium (Ai) chromosomes, three carried a pair of Ai chromosomes only, three others carried a pair of 1BL/1RS chromosomes only, and one carried neither 1BL/1BS nor Ai chromosome. Further identification revealed that the identical Ai chromosome in these germplasm lines substituted the chromosome 2D of common wheat (T aestivum L.), designated as 2Ai. The genetic implication and further utilization of 2Ai in wheat improvement were also discussed.

Analysis and identification of SCAR molecular markers associated with birch fiber length trait

The fiber length trait （FLT） of 538 individuals from nature birch population in Maorshan region, Heilongjang, China were measured, of which 100 individuals were selected as representative variety of correlated fragments screening with random amplified polymorphism DNA （RAPD） technique. In total of 20 RAPD primers were tested through multiple regression analysis between amplified strip and the character behaviors, and a correlative segment BFLR-16 was obtained. The correlation coefficient between BFLI-16 and FLT was 0.6144, with the significant level of 1%. BFLI-16 was then cloned, sequenced and transformed into SCAR marker. The percentage of identifying long fiber birches by this SCAR was more than 92. The result indicates that the SCAR markers has high specificity for the long fiber individuals and is highly linked with the gene controlling the character of fiber length, and its existence is significantly correlative with the increase in the fiber length.

Neither gastric topological distribution nor principle virulence genes of Helicobacter pylori contributes to clinical outcomes

AIM: Studies on Helicobacter pylori (H pylon) and gastroduodenal diseases have focused mainly on the distal sites of the stomach, but relationship with the gastric cardia is lacking. The aim of this study is to determine if the gastric topology and genotypic distribution of Hpyloriwere associated with different upper gastrointestinal pathologies in a multiethnic Asian population. METHODS: Gastric biopsies from the cardia, body/corpus and antrum were endoscoped from a total of 155 patients with dyspepsia and/or reflux symptoms, with informed consent. H pylori isolates obtained were tested for the presence of 26kDa, ureC, cagA, vacA, iceA1, iceA2 and babA2 genes using PCR while DNA fingerprints were generated using random amplification polymorphic DNA (RAPD). RESULTS: Hpyloriwas present in 51/155 (33%) of patients studied. Of these, 16, 15 and 20 were isolated from patients with peptic ulcer diseases, gastroesophageal reflux diseases and non-ulcer dyspepsia, respectively. Of the Hpyloripositive patients, 75% (38/51) had Hpyloriin all three gastric sites. The prevalence of various genes in the H pylori isolates was shown to be similar irrespective of their colonization sites as well as among the same site of different patients. The RAPD profiles of H pylori isolates from different gastric sites were highly similar among intra-patients but varied greatly between different patients. CONCLUSION: Topographic colonization of H pylori and the virulence genes harboured by these isolates have no direct bearing to the clinical state of the patients. In multiethnic Singapore, the stomach of each patient is colonized by a predominant strain of H pylori，irrespective of the clinical diagnosis.

RAPD analysis of natural populations of Acanthopanax brachypus

Random Amplified Polymorphic DNA (RAPD) analysis is a new technology of molecular marking which has proved very powerful in detecting genetic diversity at the level of population. The genomic DNAs used in our experiment were extracted from fresh leaves taken from 59 individuals sampled from three natural populations in Yan An, Shanxi Province. Through more than 2,000 PCRs, deep-going RAPD analysis was carried out on DNA samples from 49 inviduals. The percentage of polymorphic RAPD loci found in these three populations were respectively 27.2 %, 18.6 % and 5.4 %; the average genetic distances within population, 0.055, 0.036 and 0.008; the average genetic distances between populations (Ⅰ-Ⅱ), (Ⅰ-Ⅲ) and (Ⅱ-Ⅲ), 0.105, 0.096 and 0.060. The genetic diversity of A. brachypus within and between populations was found, for the first time, to be rather poor,thus revealing innate factors as the cause contributing to its endangered status. In addition, our work also provides basic materials for elucidating the underlying cause of its endangerment and for its protection biology.

CYTOTOXICITY AND GENOTOXICITY OF POLYETHYLENIMINE AND NICKEL CHLORIDE IN RED SEA BREAM (Pagrosomus major) FIN CELL LINE RSBF

A continuous marine fish cell line RSBF (i.e. Red Sea Bream Fin) was utilized to screen the cytotoxicity and genotoxicity of polyethylenimine (PEI) and nickel chloride (NiCl 2) in this study on the deleterious effects of aquatic genotoxins on fish. At the 0.01 to 1 μg/ml concentration tested, PEI had acute toxicity to the treated RSBF cells (IC 50 =1.12, 0.92, 0.88 and 0.64 μg/ml PEI for time 0 h, 24 h, 48 h and 72 h after treatment, respectively) and markedly inhibited their proliferation in a dose dependent manner. At the 0.001 to 5 μmol/L concentration tested, NiCl 2 posed no acute toxicity but significantly stimulated their growth (107%-214% of control). Random amplified polymorphic DNA (RAPD) technique was used to detect the genotoxic effects of PEI and NiCl 2 by comparing the RAPD banding patterns of the control and treated cells. RAPD analysis indicated that at the concentrations tested, PEI was more genotoxic than NiCl 2 to RSBF cells; that there was a slight dose dependent response in the genotoxic effect of PEI but not NiCl 2; and that RAPD technique might provide a sensitive, non specific genotoxic endpoint. And the potent cytotoxicity and genotoxicity of PEI on fish cells showed that we should be cautious in utilizing it as gene vector in fish gene transfer and human gene therapy.

Analysis on Genetic Relationship of Oxya chinensis and Oxya japonica from Xuzhou and Pingshan, China

Genetic relationship among Oxya chinensis populations and Oxya japonica populations collected from Xuzhou City, Jiangsu Province and Pingshan County, Hebei Province, China were analyzed by random amplified polymorphic DNA （RAPD） markers. A total of 125 DNA bands ranging from 200 to 2 200 bp were amplified by 10 random primers in DNA samples from 43 grasshopper individuals. One hundred and twenty-three （99%） of these bands were polymorphic. Shannon＇s index showed that the genetic, diversity within O. chinensis （0.3432） was higher than that of O. japonica （0.2781）. Nei＇s genetic distance between O. chinensis population and O. japonica population from the same area was less than that between populations from two different areas. The dendrogram based on Nei＇s genetic distance of RAPD markers was constructed using the unweighted pair group method with the arithmetic average （UPGMA） and Neighbor-Joining （NJ） methods. Cluster analysis indicated that all the individuals were grouped into two main clusters. O. chinensis populations from Xuzhou and Pingshan formed one cluster, and O. japonica populations from the two regions belonged to another cluster. The results demonstrated that RAPD can detect within species, and among closely related species. polymorphisms to distinguish minor difference among individuals

Genetic diversity of the endangered species Rosa rugosa Thunb.in China and implications for conservation strategies

Rosa rugosa Thunb. is one of the dominant and important shrub species in estuary dunes and shingle beaches of northern China. However, its area of distribution, the number of populations, and the size of each population have decreased rapidly in the past two decades because of habitat degradation and loss. Random amplified polymorphic DNA markers were used to determine the genetic diversity of four remaining large natural populations of R. rugosa and to discuss an effective conservation strategy for this endangered species in China. High genetic variations were detected in R. rugosa populations in China. The mean percentage of polymorphic loci （P%） within four local populations was 57.99%, with the P% of the total population being 75.30%. Mean Shannon＇s information index （H0） was 0.2826, whereas total Ho was 0.3513. The genetic differentiation among populations was 0.1878, which indicates that most genetic diversity occurs within populations. Population Tumenjiang （TMJ） showed the highest genetic diversity （P% = 66.27%; H0 = 0.3117） and contained two exclusive bands. Population Changshandao （CSD） showed higher genetic diversity （P% =59.04%; H0 = 0.3065）. Populations TMJ and CSD contained 95.33% and 99.33%, respectively, of loci with moderate to high frequency （P〉0.05） of the total population. These results indicate that populations TMJ and CSD should be given priority for in situ conservation and regarded as seed or propagule sources for ex situ conservation. The results of the present study also suggest that R. rugosa in China has become endangered as a result of human actions rather than genetic depression of populations; thus, human interference should be absolutely forbidden in R. rugosa habitats.

RAPD Analysis of Rosa laevigata Michx. from Different Origins

[ Objective] This study aimed to investigate the genetic diversity and phylogenetic relationship of 30 populations of Rosa laevigata Michx. from different origins. [ Method] Using RAPD molecular marker technique, eight effective primers were screened from 35 RAPD random primers for amplification. UPGMA clus- ter analysis was performed using NTSYSpc. ver. 2.02 software. [ Result] A total of 86 loci were amplified using the screened primers, including 84 polymorphic loci, indicating an average polymorphic percentage （PPB） of 97.67%. The similarity coefficients of 30 populations of R. laevigata Michx. ranged from O. 11 to O. 58, and these populations were clustered into groups A, B and C. [ Conclusion ] R. laevigata Michx. exhibits multiple genetic polymorphism. R. laevigata Michx. populations from Guizhou Province have closer genetic relationships.