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.

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.

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.

Genetic diversity analysis of Penicillium marneffei isolated from AIDS patients in Guangdong, China using randomly amplified polymorphic DNA

Background Penicillium mameffei （P. marneffe~） is an emerging pathogenic fungus that can cause invasive mycosis in patients with AIDS. The epidemiological features of P. marneffeiinfection in AIDS patients in Guangdong province remain unclear so far. This study aimed to investigate the genetic diversity within a population of 163 P. mameffei isolates obtained from AIDS patients and search for the dominant clinical strains in Guanqdong province.

Genetic Diversity Caused by Environmental Stress in Natural Populations of Niupidujuan as Revealed by RAPD Technique

Multiplex environmental factors are generally expected to have significant effects on genetic diversity of plant populations.In this study,randomly amplified polymorphic DNA（RAPD） technique was used to reveal the genetic diversity in the same species of four populations collected from Niupidujuan（Rhododendron chrysanthum） at different altitudes,an endangered species,endemic to Northeast China.Initially,twenty informative and reproducible primers were chosen for final RAPD analysis.A total of 152 clear bands were obtained,including 143 polymorphic ones.With the help of POPGENE software,the poly rate was calculated to be 94.07% and the evenness of amplified bands for every primer was 6.8.Additionally,the mean observed number of alleles was 1.7265 with an effective number of 1.3608.An examination of the gene indicated a diversity of 0.2162 with an information diversity index of 0.3313.For these data,the clustering blurred analysis was performed with the aid of NTSYS-pc software to define the Nei＇s gene diversity and the Shannon information diversity index of the four plant populations.The relationships between the genetic diversity indexes on the one hand and the geographic and climatic factors on the other hand were estimated by the Pearson correlation with SPSS 11.0 software.The results of the correlation analysis show that there were significant（P〈0.05） or highly significant（P〈0.01） correlations between each of the genetic diversity indexes and the different temperature which were mainly caused by the altitude different populations located.These data highlight the importance of native populations in shaping the spatial genetic structure in Niupidujuan.

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

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.

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.

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.

Diversity Analysis in Selected Non-basmati Scented Rice Collection

Diversity analysis among 23 rice varieties including 16 non-basmati scented accessions, 5 basmati accessions and 2 non-scented accessions was performed by random amplified polymorphic DNA （RAPD） and inter-simple sequence repeat （ISSR） marker systems. The varieties analyzed by 11 RAPD and 8 ISSR primers yielded an average of 65% and 80% polymorphism, respectively. The average number of polymorphic bands generated per RAPD primer was 6 and per ISSR primer was 5.87. RAPD and ISSR data analysis individually could not segregate basmati and non-basmati scented rice accessions. However, the analysis using a combined data could group basmati and non-basmati scented rice accessions separately. The bands present specifically among three accessions of non-basmati scented rice were also identified. The study revealed a high genetic diversity among non-basmati scented rice accessions.

Genetic diversity and differentiation of pedunculate ( Quercus robur ) and sessile ( Q. petraea ) oaks

This study was conducted to determine the parent-off spring genetic structure of the pedunculate oak(Quercus robur L.),sessile oak(Q.petraea[Matt.]Liebl.)and their hybrids.Forty half-sib Quercus families and their maternal trees originating from one tree stand in southern Lithuania were analyzed using SSR and RAPD markers.Based on a preliminary study of leaf morphological traits,the individuals separated into six groups.The studied halfsib oak families were also compared for allelic diversity,including group variations;genotypic structure;genetic diversity;and the degree of genetic subdivision and diff erentiation.The level of genetic variation and subdivision was lower in the hybrid families than in the families of the parental species.Genotypic analysis of the half-sibling off spring showed the asymmetric nature of interspecifi c hybridization processes of pedunculate and sessile oaks in mixed stands.

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.

Quantitative Estimates of Outcrossing Rates in a Natural Population of Caldesia grandis (Alismataceae)

Outcrossing rate in a natural population of Caldesia grandis was estimated by the dominant random amplified polymorphism DNA （RAPD） marker using 10 open-pollinated progeny arrays of 24 individuals. The multilocus outcrossing rate estimated based on all 25 RAPD loci was 0.872 ±0.033 and the single-locus outcrossing rate was 0.795 ±0.032. Multilocus esti- mates did not differ significantly from the single-locus estimates. The fixation index, F, in the progeny estimated from RAPD data was -0.142 ±0.000. The estimates of multilocus outcrossing rates （tm） and single-locus outcrossing rates （ts） obtained from MLDT clearly indicate that outcrossing is predominant in the open-pollinated C. grandis population. An empirical analysis suggests that 15 should be the minimum number of dominant marker loci necessary to achieve robust estimates of tm.

Genetic Diversity and Pathogenicity Variation in Rhizoctonia solani Isolates from Rice in Sichuan Province, China

Fifty-five representative samples of Rhizoctonia solani isolates, which were collected and isolated from five different ecological regions in Sichuan Province, China, were purified and analyzed for the pathogenicity and molecular genetic variation. The hyphal fusion test revealed that almost all the isolates belonged to the AG-IIA group except the isolate D42. In addition, some of the isolates were ＇bridging isolates＇, which could fuse with several groups simultaneously. The pathogenicity analysis on in vitro leaves confirmed a significant pathogenicity variation in the tested isolates. The 55 isolates were then classified into 8 groups by further RAPD （randomly amplified polymorphic DNA） cluster analysis at the similarity coefficient of 0.941. The results suggest that under the certain ecological conditions in Sichuan Province, China, most of the R. solanistrains were genetically stable, but a few changed drastically.

Micropropagation of loblolly pine by somatic organogenesis andRAPD analysis of regenerated plantlets

Organogenesis was induced in callus derived from mature zygotic embryos of six families (J-56, S-1003, E-22, E-311, E-440, and Mc) of loblolly pine (Pinus taeda L.) within 24 weeks of culture. Elongation of adventitious buds was achieved on TE medium supplemented with 0.5 mg·L?1 indole-3-butyric acid (IBA) and 1 mg/l 6-benzyladenine (BA). The most suitable medium for root formation proved to be TE medium supplemented with 0.5 mg·L?1 IBA, 2mg·L?1 BA and 0.5 mg/l gibberellic acid (GA3). 169 regenerated plantlets were transferred to a perlite: peatmoss: vermiculite (1∶1∶1) soil mixture, and 98 plantlets survived in the field. Total DNA was extracted from the needles of the regenerated plantlets of the six families of loblolly pine. Analysis of random amplified polymorphic DNA (RAPD) using 20 arbitrary oligonucleotide 10-mers, show that amplification products were monomorphic for all the plantlets of family J-56, S-1003, E-22, E-311, E-440, and Mc of loblolly pine. These results suggested that organogenesis can be used for clonal micropropagation of some families of loblolly pine.

Tagging of Brown Planthopper Resistance Genes in F_2s of IR50 × Ptb33 of Rice by Using Bulked Segregant Analysis

Brown planthopper （Nilaparvata lugens Stal） is one of the most damaging pests causing hopper burn in rice, and thereby reducing the productivity and also the quality of the product. The effective management strategy to control this pest is the identification and transfer of desirable genes to local rice cultivars. The most important approach for developing resistant cultivars is the identification of markers, which can help in marker-assisted selection of more durable resistant genotype. The susceptible parent IR50 and the resistant parent Ptb33, and their F2 populations were used in bulked segregant analysis for identification of resistant genes with random amplified polymorphic DNA marker （RAPD） primers. The primers OPC7 and OPAG14 showed both dominant and susceptible specific banding pattern so called co-dominant markers. Moreover, OPC7697 and OPAG14680 showed resistant specific bands and thus being in coupling phase, whereas OPC7846 and OPAG14650 showed susceptible specific genotypic bands in bulked segregant analysis. Therefore, the coupling phase markers, OPC7697 and OPAG14680, are considered to be more useful in marker-assisted selection of rice genotypes in crop improvement.

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.

Application of Molecular Marker Technology in the Study of Forest Tree Species

Due to its unique advantages, molecular marker technology is widely applied in the research of forest tree species. This paper reviewed the application of molecular marker technology in tree species resource diversity, germplasm identification, genetic map construction, gene mapping and marker-assisted selection (MAS) breeding. In addition, it elaborated the great significance of molecular marker technology to promote the sustainable development of forestry production in China.

Effect of Development Stage on the Artemisinin Content and the Sequence Characterized Amplified Region （SCAR） Marker of High-Artemisinin Yielding Strains of Artemisia annua L.

The effects of development states on the artemisinin content of clone S1 of Artemisia anuua L. grown in a greenhouse were investigated in the present study. The artemisinin content increased gradually during the phase of vegetative growth and reached its highest level at 8-9 mg/g dry weight （DW） when the S1 was 6 months old on a long day （LD） photoperiod. Treatment with 9-18 d of short day （SD） photoperiod resulted in the artemisinin content reaching and being maintained at a higher level （2.059-2.289 mg/g DW）, twofold that of control plants and plants of S1 presented at the pro-flower budding and flower-budding stages. The artemisinin content varied in different parts of the plant. The artemisinin content of leaves was higher than that of florets and branches. The artemisinin content in middle leaves was higher than that of bottom leaves, and then top leaves. Different densities of capitate glands （the storage organ of artemisinin） located on the surface of leaves, florets, and branches explained the variations in artemisinin content in these parts of the plant. The correlation coefficient between artemisinin content and density of capitate glands on the surface of different organs was 0.987. The genetic marker for artemisinin content was screened using random amplified polymorphic DNA （RAPD） and sequence characterized amplified region （SCAR） techniques. The random primer OPAl5 （5＇-TTCCGAACCC-3＇） could amplify a specific band of approximately 1 000 bp that was present in all high-artemisinin yielding strains, but absent in all low-yielding strains in three independent replications. This specific band was cloned and its sequence was analyzed. This RAPD marker was converted into a SCAR marker to obtain a more stable marker.