Showing papers on "RAPD published in 2006"

An overview of molecular marker methods for plants

Abstract: The development and use of molecular markers for the detection and exploitation of DNA polymorphism is one of the most significant developments in the field of molecular genetics. The presence of various types of molecular markers, and differences in their principles, methodologies, and applications require careful consideration in choosing one or more of such methods. No molecular markers are available yet that fulfill all requirements needed by researchers. According to the kind of study to be undertaken, one can choose among the variety of molecular techniques, each of which combines at least some desirable properties. This article provides detail review for 11 different molecular marker methods: restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), inter-simple sequence repeats (ISSRs), sequence characterized regions (SCARs), sequence tag sites (STSs), cleaved amplified polymorphic sequences (CAPS), microsatellites or simple sequence repeats (SSRs), expressed sequence tags (ESTs), single nucleotide polymorphisms (SNPs), and diversity arrays technology (DArT).

The random amplified polymorphic DNA (RAPD) assay and related techniques applied to genotoxicity and carcinogenesis studies: a critical review.

Abstract: More than 9000 papers using the random amplified polymorphic DNA (RAPD) or related techniques (e.g. the arbitrarily primed polymerase chain reaction (AP-PCR)) have been published from 1990 to 2005. The RAPD method has been initially used to detect polymorphism in genetic mapping, taxonomy and phylogenetic studies and later in genotoxicity and carcinogenesis studies. Despite their extensive use, these techniques have also attracted some criticisms, mainly for lack of reproducibility. In the light of their widespread applications, the objectives of this review are to (1) identify the potential factors affecting the optimisation of the RAPD and AP-PCR assays, (2) critically describe and analyse these techniques in genotoxicity and carcinogenesis studies, (3) compare the RAPD assay with other well used methodologies, (4) further elucidate the impact of DNA damage and mutations on the RAPD profiles, and finally (5) provide some recommendations/guidelines to further improve the applications of the assays and to help the identification of the factors responsible for the RAPD changes. It is suggested that after proper optimisation, the RAPD is a reliable, sensitive and reproducible assay, has the potential to detect a wide range of DNA damage (e.g. DNA adducts, DNA breakage) as well as mutations (point mutations and large rearrangements) and therefore can be applied to genotoxicity and carcinogenesis studies. Nevertheless, the interpretation of the changes in RAPD profiles is difficult since many factors can affect the generation of RAPD profiles. It is therefore important that these factors are identified and taken into account while using these assays. On the other hand, further analyses of the relevant bands generated in RAPD profile allow not only to identify some of the molecular events implicated in the genomic instability but also to discover genes playing key roles, particularly in the initiation and development of malignancy. Finally, to elucidate the potential genotoxic effects of environmental contaminants, a powerful strategy could be firstly to use the RAPD assay as a screening method and secondly to apply more specific methods measuring for instance DNA adducts, gene mutations or cytogenetic effects. It is also envisaged that these assays (i.e. RAPD and related techniques), which reflect effects at whole genome level, would continue to complement the use of emerging technologies (e.g. microarrays which aim to quantify expression of individual genes).

Ready-to-use DNA extracted with a CTAB method adapted for herbarium specimens and mucilaginous plant tissue

Abstract: This report summarizes major changes in previously published protocols for DNA extraction to improve the quality of DNA extracted from plants. Here, we highlight the critical modifications in the original protocols. The efficiency of these changes results in high-quality DNA ready to use in a variety of phytogenetically distant plant families, in particular species with mucopolysaccharides. The DNA obtained can be used without further purification in various molecular biology assays, including direct sequencing and AFLP and RAPD (random-amplified polymorphic DNA) analyses. The effectiveness of this method is proven by the amplification and sequencing of PCR products of up to 1 kb with DNA extracted from herbarium tissue ≥60 years old. This versatility is not usually found in DNA extraction protocols. In addition, this method is quick, adaptable to standard laboratories, and most important, safer and more cost-effective.

Similarity between Human and Chicken Escherichia coli Isolates in Relation to Ciprofloxacin Resistance Status

Abstract: Background. The food supply is suspected to be a source of fluoroquinolone-resistant Escherichia coli that cause disease in humans, but supporting molecular data are lacking. Methods. We performed a molecular-epidemiological comparison, in Barcelona, Spain (1996-1998), of 117 contemporaneous, geographically matched E. coli isolates from humans (35 blood isolates and 33 fecal) or chickens (49 fecal) that were either susceptible (n = 57) or resistant (n = 60) to ciprofloxacin and analyzed them by phylogenetic group, virulence genotype, and O antigens using random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). Results. When analyzed by phylogenetic distribution, virulence profiles, and O antigens, resistant human isolates were distinct from susceptible human isolates but were largely indistinguishable from chicken isolates, whereas resistant and susceptible chicken isolates were similar. Susceptible human isolates contained more virulence-associated genes and more frequently expressed virulence-associated O antigens than did resistant human or any chicken isolates. Certain resistant human isolates closely resembled chicken isolates by RAPD and PFGE analysis. Conclusions. Ciprofloxacin-resistant E. coli may arise de novo in poultry from susceptible progenitors, be transmitted to humans via the food supply, and go on to cause potentially life-threatening infections. If confirmed, these findings would mandate efforts to eliminate this reservoir of drug-resistant pathogens and/or to block their transmission to humans.

One group of genetically similar Listeria monocytogenes strains frequently dominates and persists in several fish slaughter- and smokehouses.

Abstract: Contamination of foods with the human pathogen Listeria monocytogenes may occur during processing, and the purpose of this study was to determine whether genetically similar strains colonize different processing plants or whether specific persistent strains are unique to each processing plant. We hypothesized that specific L. monocytogenes strains may be better adapted to specific environmental niches in the processing environment. L. monocytogenes contamination patterns were identified by the collection of 686 and 267 samples from the processing environments: raw fish and products of four fish smokehouses and four fish slaughterhouses, respectively. Samples were collected both during production and after cleaning and disinfection. Typically, these samplings were separated by 1 to 3 months. Sampling sites were targeted toward areas likely to harbor the bacterium. L. monocytogenes was isolated from 213 samples, and one strain from each positive sample was typed by RAPD (random amplified polymorphic DNA) analysis with four different primers. The 213 strains were divided into 37 RAPD types. One RAPD type was predominant; 86 of 213 strains belonged to this type. This type was found in three smokehouses and two slaughterhouses and was predominant in three of these plants. A subset of 35 strains was also analyzed by amplified fragment length polymorphism typing, which confirmed the genetic similarity of the groups. Moreover, strains of the dominant RAPD type were indistinguishable from strains isolated frequently from smoked fish products 10 years ago. One smokehouse was surveyed for a year and a half, and the dominant RAPD type persisted throughout the survey period and accounted for 94 of 118 isolates. Our study indicates that strains of L. monocytogenes that are genetically very closely related may be especially adapted to colonizing the processing equipment or especially resistant to cleaning and disinfection.

An SSR-based linkage map of Capsicum annuum

Abstract: There are five cultivated species of pepper, of which Capsicum annuum is the most widely cultivated as a vegetable or spice and the main experimental material of most pepper breeding programs. However, the number of simple sequence-repeat (SSR) markers known for C. annuum is limited. To develop SSR markers for Capsicum species, we constructed four SSR-enriched libraries from the genomic DNA of C.␣annuum, sequenced 1873 clones, and isolated 626 unique SSR clones. A higher percentage of these SSR markers were taken from dinucleotide motif libraries than from trinucleotide motif libraries. Primer pairs for the 626 SSR clones were synthesized and tested for polymorphisms; 594 amplified products were detected with the expected size. However, only 153 products were polymorphic between the parents of our mapping population. Using 106 highly reproducible pairs from the primer pairs, we constructed a linkage map of C. annuum in an intraspecific doubled haploid population (n=117) that contains nine previously reported SSRs as well as AFLP, CAPS, and RAPD markers and the trait of fruit pungency. The map contains 374 markers, including 106 new SSR markers distributed across all 13 linkage groups, and covers 1042 cM. The polymorphism information content (PIC) of these new SSR markers was calculated using 14 lines of Capsicum species. The average number of alleles per locus was 2.9 and the average PIC value was 0.46, even within C. annuum. The SSR markers developed in this study will be useful for mapping and marker-assisted selection in pepper breeding, and the linkage map provides a reference genetic map for Capsicum species.

Effects of continuous cucumber cropping and alternative rotations under protected cultivation on soil microbial community diversity

Abstract: The diversity of soil microbial communities as affected by continuous cucumber cropping and alternative rotations under protected cultivation were evaluated using community level physiological profiles (CLPP) and random amplified polymorphic DNA (RAPD) analysis. The soils were selected from six cucumber cropping systems, which cover two cropping practices (rotation and continuous cropping) and a wide spectrum for cucumber cropping history under protected cultivation. Shannon–Weaver index and multivariate analysis were performed to characterize variations in soil microbial communities. Both CLPP and RAPD techniques demonstrated that cropping systems and plastic-greenhouse cultivation could considerably affect soil microbial functional diversity and DNA sequence diversity. The open-field soil had the highest Shannon–Weaver index (3.27 for CLPP and 1.50 for RAPD), whereas the lowest value occurred in the 7-year continuous protected cultivation soil (3.27 for CLPP and 1.50 for RAPD). The results demonstrated that continuous plastic-greenhouse cultivation and management can cause the reduction in the species diversity of the biota. Higher Shannon–Weaver index and coefficients of DNA sequence similarity were found in soils under rotation than those under continuous cropping. Cluster analysis also indicated that microbial community profiles of continuous cultivation soils were different from profiles of rotation soils. The reduction in diversity of microbial communities found in continuous cultivation soils as compared with rotation soils might be due to the differences in the quantity, quality and distribution of soil organic matter.

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

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

Genetic analysis of the cultivated potato Solanum tuberosum L. Phureja Group using RAPDs and nuclear SSRs

Abstract: The Solanum tuberosum L. Phureja Group consists of potato landraces widely grown in the Andes from western Venezuela to central Bolivia, and forms an important breeding stock due to their excellent culinary properties and other traits for developing modern varieties. They have been distinguished by short-day adaptation, diploid ploidy (2n = 2x = 24), and lack of tuber dormancy. This nuclear simple sequence repeat (nSSR or microsatellite) study complements a prior random amplified polymorphic DNA (RAPD) study to explore the use of these markers to form a core collection of cultivar groups of potatoes. Like this prior RAPD study, we analyzed 128 accessions of the Phureja Group using nuclear microsatellites (nSSR). Twenty-six of the 128 accessions were invariant for 22 nSSR markers assayed. The nSSR data uncovered 25 unexpected triploid and tetraploid accessions. Chromosome counts of the 102 accessions confirmed these nSSR results and highlighted seven more triploids or tetraploids. Thus, these nSSR markers (except 1) are good indicators of ploidy for diploid potatoes in 92% of the cases. The nSSR and RAPD results: (1) were highly discordant for the remaining 70 accessions that were diploid and variable in nSSR, (2) show the utility of nSSRs to effectively uncover many ploidy variants in cultivated potato, (3) support the use of a cultivar-group (rather than a species) classification of cultivated potato, (4) fail to support a relationship between genetic distance and geographic distance, (5) question the use of any single type of molecular marker to construct core collections.

Application of random amplified polymorphic DNA (RAPD) to detect the genotoxic effect of heavy metals

Abstract: This paper presents the results of a study on the influence of lead, copper, manganese and cadmium on DNA integrity in plant cells. Plants, as biological indicators, can measure the potential effects of pollutants when they are used to measure effects of heavy metals. The genotoxicity of heavy metals in kidney-bean (Phaseolus vulgaris) seedlings was subjected to RAPD (random amplified polymorphic DNA) analysis. An RAPD 'fingerprinting' technique was used to detect DNA damage in the kidney-bean seedlings treated with two selected heavy metals at concentrations of 150 and 350 mg x l(-1). Polymorphisms became evident as the presence and/or absence of DNA fragments in treated samples compared with the untreated one. At 350 mg x l(-1), a high number of both missing bands and new amplified fragment were observed. Results suggested that a qualitative measure reflecting changes in RAPD profiles were significantly affected at higher concentrations (350 mg x l(-1)) of the tested heavy metals. A total of 467 RAPD fragments in RAPD profiles were detected by using six random primers (decamers) and 224 of these fragments showed polymorphism. There was a distinct distance between the band patterns of treated plants and the control samples when the cluster method was applied. In addition, the result derived from numerical analysis revealed a considerable distance between the band pattern of the plant samples treated with 350 mg x l(-1) heavy metals and the control sample. Finally, a comparison between untreated and treated genomes shows that RAPD analysis can be used to evaluate how the environmental pollutants modify the structure of DNA in living organisms.

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

Abstract: 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.

Identification and validation of molecular markers linked to the leaf rust resistance gene Lr19 in wheat

Abstract: A leaf rust resistance gene Lr19 on the chro- mosome 7DL of wheat derived from Agropyron elonga- tum was tagged with random ampliWed polymorphic DNA (RAPD) and microsatellite markers. The F2 pop- ulation of 340 plants derived from a cross between the leaf rust resistant near-isogenic line (NIL) of Thatcher (Tc + Lr19) and leaf rust susceptible line Agra Local that segregated for dominant monogenic leaf rust resis- tance was utilized for generating the mapping popula- tion. The molecular markers were mapped in the F2 derived F 3 homozygous population of 140 seedlings. Sixteen RAPD markers were identiWed as linked to the alien gene Lr19 among which eight were in a coupling phase linkage. Twelve RAPD markers co-segregated with Lr19 locus. Nine microsatellite markers located on the long arm of chromosome 7D were also mapped as linked to the gene Lr19, including 7 markers which co- segregated with Lr19 locus, thus generating a saturated region carrying 25 molecular markers linked to the gene Lr19 within 10.2 § 0.062 cM on either side of the locus. Two RAPD markers S265 512 and S253 737 which Xanked the locus Lr19 were converted to sequence characterized ampliWed region markers SCS265 512 and SCS253 736 , respectively. The marker SCS265512 was linked with Lr19 in a coupling phase and the marker SCS253 736 was linked in a repulsion phase, which when used together mimicked one co-dominant marker capable of distin- guishing the heterozygous resistant seedlings from the homozygous resistant. The molecular markers were validated on NILs mostly in Thatcher background iso- genic for 44 diVerent Lr genes belonging to both native and alien origin. The validation for polymorphism in common leaf rust susceptible cultivars also conWrmed the utility of these tightly linked markers to the gene Lr19 in marker-assisted selection.

RAPD analysis of a variant of banana ( Musa sp.) cv. grande naine and its propagation via shoot tip culture

Abstract: A morphological variant obtained from in vitro corm-derived plants of banana (Musa sp.) cv. Grande Naine (AAA) was evaluated up to harvest, and the genetic basis of variation was confirmed by the random amplified polymorphic DNA (RAPD). The corms formed during the multiplication phase of shoot tip-derived cultures of the cv. Grande Naine grown on Murashige and Skoog (MS) medium enriched with 13.3 μM N6-benzyladenine (BA) developed numerous morphological variants after transfer to MS medium with 6.66 μM BA. The variant designated as CUDBT-B1, with distinct morphological features, was further evaluated. The morphological features of CUDBT-B1 were variegated leaf, pseudostem, bracts, ovary of the male flower and fruits, reduced height, decreased lamina length and breadth, and early flowering. These features were also manifested in the second-cycle progeny of CUDBT-B1. RAPD assay showed a marker DNA band of 1650 bp, and differential band intensity between the CUDBT-B1 and normal clone. CUDBT-B1 was m...

Detection of two quantitative trait loci for resistance to ascochyta blight in an intra-specific cross of chickpea (Cicer arietinum L.): development of SCAR markers associated with resistance.

Abstract: Two quantitative trait loci (QTLs), (QTL(AR1) and QTL(AR2)) associated with resistance to ascochyta blight, caused by Ascochyta rabiei, have been identified in a recombinant inbred line population derived from a cross of kabulixdesi chickpea. The population was evaluated in two cropping seasons under field conditions and the QTLs were found to be located in two different linkage groups (LG4a and LG4b). LG4b was saturated with RAPD markers and four of them associated with resistance were sequenced to give sequence characterized amplified regions (SCARs) that segregated with QTL(AR2). This QTL explained 21% of the total phenotypic variation. However, QTL(AR1), located in LG4a, explained around 34% of the total phenotypic variation in reaction to ascochyta blight when scored in the second cropping season. This LG4a region only includes a few markers, the flower colour locus (B/b), STMS GAA47, a RAPD marker and an inter-simple-sequence-repeat and corresponds with a previously reported QTL. From the four SCARs tagging QTL(AR2), SCAR (SCY17(590)) was co-dominant, and the other three were dominant. All SCARs segregated in a 1:1 (presence:absence) ratio and the scoring co-segregated with their respective RAPD markers. QTL(AR2) on LG4b was mapped in a highly saturated genomic region covering a genetic distance of 0.8 cM with a cluster of nine markers (three SCARs, two sequence-tagged microsatellite sites (STMS) and four RAPDs). Two of the four SCARs showed significant alignment with genes or proteins related to disease resistance in other species and one of them (SCK13(603)) was sited in the highly saturated region linked to QTL(AR2). STMS TA72 and TA146 located in LG4b were described in previous maps where QTL for blight resistance were also localized in both inter and intraspecific crosses. These findings may improve the precision of molecular breeding for QTL(AR2) as they will allow the choice of as much polymorphism as possible in any population and could be the starting point for finding a candidate resistant gene for ascochyta blight resistance in chickpea.

Development and application of RAPD-SCAR marker for identification of Phyllanthus emblica LINN.

Abstract: Correct genotype identification of medicinal plant material remains important for botanical drug industry. Limitations of chemical and morphological approaches for authentication have generated need for newer methods in quality control of botanicals. The present study was carried out to develop DNA based marker for identification of Phyllanthus emblica LINN. A putative marker (1.1 kb) specific for P. emblica was identified by Random Amplified Polymorphic DNA (RAPD) technique. Sequence Characterized Amplified Region (SCAR) marker was developed from the RAPD amplicon. The SCAR marker was found useful for identification of P. emblica in its commercial samples and Triphalachurna, a multi-component Ayurvedic formulation.

Assessment of agronomic, chemical and genetic variability in common basil (Ocimum basilicum L.)

Abstract: In the present work, agro-morphological characteristics, essential oil composition and randomly amplified polymorphic DNA (RAPD) markers were studied to estimate the relationships among 12 basil (Ocimum basilicum L.) genotypes, belonging to nine known cultivars grown in Italy. The basil cultivars were distinguished on the basis of agro-morphological determinations and constituents of essential oil. Chemical compounds of essential oils were found variable in the various basil cultivars. As a consequence, the plants were classified into main phenotypes and chemotypes. RAPD markers were used in order to assess the genetic relatedness among the basil cultivars. On the basis of their genetic similarities, RAPD analysis allowed to group the samples into two main clusters. One of these included cultivars suitable for food industry, which were also correlated via agro-morphological features. However, the same cultivars produced distinct essential oil profiles, which did not match with results obtained by agronomic and genetic analysis. This fact, maybe, is due to a different genic expression of the key enzymes involved in biosynthetic pathways that produce chemical compounds.

Evaluation of genetic diversity in jute (Corchorus species) using STMS, ISSR and RAPD markers

Abstract: Jute is an important fibre crop that has dominated the packaging sector for over one and a half centuries in India For sustenance of the trade in the face of tough competition from synthetics, there is an urgent need to redesign the ongoing breeding strategy to improve both the yield and quality of jute fibre It is therefore, essential to understand the pattern of diversity in this important commercial crop species In the present study, genetic diversity analysis of 20 exotic germplasm lines and 20 commercial varieties of the two cultivated species (Corchorus olitorius and C capsularis) and two wild relatives of jute (C aestuans and C trilocularis) was carried out using sequence tagged microsatellite site (STMS), inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) markers The first set of six STMS markers developed from the genomic sequence of C olitorius was not fully transferable to the related species C capsularis The level of intraspecific polymorphism revealed by these markers was very low The four ISSR and 22 RAPD primers employed in the study revealed 9844% and 100% polymorphism, respectively, across all the species, while the level of polymorphism was significantly low within a species The commercial varieties, particularly those of C capsularis, had an extremely narrow genetic base that demands immediate effort for diversification The germplasm accessions in both the cultivated species showed considerably higher levels of diversity and thus should be used in broadening the base of the varieties All the accessions of C olitorius together with the wild species C aestuans clustered separately from those of C capsularis and C trilocularis, suggesting a polyphyletic origin of the two cultivated species

Establishing Clonal Relationships between VIM-1-Like Metallo-β-Lactamase-Producing Pseudomonas aeruginosa Strains from Four European Countries by Multilocus Sequence Typing

Abstract: Ten multidrug-resistant Pseudomonas aeruginosa strains producing VIM-1-like acquired metallo-β-lactamases (MBLs), isolated from four European countries (Greece, Hungary, Italy, and Sweden), were analyzed for genetic relatedness by several methodologies, including fliC sequence analysis, macrorestriction profiling of genomic DNA by pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD), and multilocus sequence typing (MLST). The four approaches yielded consistent results overall but showed different resolution powers in establishing relatedness between isolates (PFGE > RAPD > MLST > fliC typing) and could usefully complement each other to address issues in the molecular epidemiology of P. aeruginosa strains producing acquired MBLs. In particular, the recently developed MLST approach was useful in revealing clonal relatedness between isolates when this was not readily apparent using RAPD and PFGE, and it suggested a common ancestry for some of the VIM-1-like MBL-positive P. aeruginosa strains currently spreading in Europe. The MBL producers belonged in three clonal complexes/burst groups (BGs). Of these, one corresponded to the previously described BG4 and included serotype O12 strains from Hungary and Sweden, while the other two were novel and included serotype O11 or nonserotypable strains from Greece, Sweden, and/or Italy. Comparison of the integrons carrying blaVIM-1-like cassettes of various isolates revealed a remarkable structural heterogeneity, suggesting the possibility that multiple independent events of acquisition of different blaVIM-containing integrons had occurred in members of the same clonal lineage, although a contribution of integrase-mediated cassette shuffling or other recombination mechanisms during the evolution of similar strains could also have played a role in determining this variability.

A genetic linkage map for hazelnut (Corylus avellana L.) based on RAPD and SSR markers.

Abstract: A linkage map for European hazelnut (Corylus avellana L.) was constructed using random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers and the 2-way pseudotestcross approach. A full-sib population of 144 seedlings from the cross OSU 252.146 x OSU 414.062 was used. RAPD markers in testcross configuration, segregating 1:1, were used to construct separate maps for each parent. Fifty additional RAPD loci were assigned to linkage groups as accessory markers whose exact location could not be determined. Markers in intercross configuration, segregating 3:1, were used to pair groups in one parent with their homologues in the other. Eleven groups were identified for each parent, corresponding to the haploid chromosome number of hazelnut (n = x = 11). Thirty of the 31 SSR loci were able to be assigned to a linkage group. The maternal map included 249 RAPD and 20 SSR markers and spanned a distance of 661 cM. The paternal map included 271 RAPD and 28 SSR markers and spanned a distance of 812 cM. The maps are quite dense, with an average of 2.6 cM between adjacent markers. The S-locus, which controls pollen-stigma incompatibility, was placed on chromosome 5S where 6 markers linked within a distance of 10 cM were identified. A locus for resistance to eastern filbert blight, caused by Anisogramma anomala, was placed on chromosome 6R for which two additional markers tightly linked to the dominant allele were identified and sequenced. These maps will serve as a starting point for future studies of the hazelnut genome, including map-based cloning of important genes. The inclusion of SSR loci on the map will make it useful in other populations.

Diversity of Phytophthora capsici in Northwest Spain: Analysis of Virulence, Metalaxyl Response, and Molecular Characterization.

Abstract: Phytophthora crown rot, caused by Phytophthora capsici, is potentially the most destructive disease of pepper in Spain. Phenotypic and genetic diversity of 16 P. capsici isolates collected from 11 farms in northwest Spain was characterized based on virulence, mating type, sensitivity to metalaxyl, and genetic analysis using random amplified polymorphic DNA (RAPD) methods. Low variability was observed among the isolates in their metalaxyl response, with 87.5% being highly sensitive. No isolates of the A2 mating type were detected. More variability was found in the virulence assay, and isolates were classified into two groups according to their pathogenicity on a set of four pepper cultivar differentials. Genetic variation examined with eight RAPD primers generated 92 polymorphic bands and revealed the existence of different patterns among isolates. Cluster analysis using the unweighted pair-group method with arithmetic averages (UPGMA) separated the Spanish isolates into three RAPD groups and esta...

Genomic instability in phenotypically normal regenerants of medicinal plant Codonopsis lanceolata Benth. et Hook. f., as revealed by ISSR and RAPD markers

Abstract: Codonopsis lanceolata Benth. et Hook. f., commonly known as bonnet bellflower, is a high-valued herb medicine and vegetable. In this study, a large number of plants were regenerated via organogenesis from immature seed-derived calli in C. lanceolata by a simple and efficient method. Compared with the mother donor plant, the regenerated plants did not exhibit visible phenotypic variations in six major morphological traits examined at the stage of one-season-maturity under field conditions. To gain insight into the genomic stability of these regenerated plants, 63 individuals were randomly tagged among a population of more than 2,000 regenerants, and were compared with the single mother donor plant by two molecular markers, the inter-simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD). Apparent genomic variation was detected in the 63 regenerants, whereas preexisting heterozygosiy in the donor plant was deemed minimal by testing 30 seedlings germinated from selfed seeds of the same donor plant. The percentages of polymorphic bands (PPB) in the ISSR and RAPD analysis were respectively 15.7 and 24.9% for the 63 regenerated plants. Cluster analysis indicates that the genetic similarity values calculated on the basis of RAPD and ISSR data among the 64 plants (63 regenerated and one donor) were respectively 0.894 and 0.933, which allow classification of the plants into distinct groups. Nineteen randomly isolated bands underlying the changed RAPD or ISSR patterns were sequenced, and three of them showed significant homology to known-function genes. Detailed pairwise sequence comparison at one locus between the donor plant and a regenerant revealed that insertion of two short (24 and 19 bp) stretches of nucleotides in the regenerated plant relative to the donor plant occurred in an apparently stochastic manner.

Characterization of Macrophomina phaseolina, the charcoal rot pathogen of cluster bean, using conventional techniques and PCR‐based molecular markers

Abstract: Phenotypic and genetic diversity of 59 Macrophomina phaseolina isolates collected from various host species growing in or near cluster bean (Cyamopsis tetragonoloba) fields in four states of north and north-west India were characterized using RAPD and PCR–RFLPs of the ITS region. These isolates, and 11 from various hosts from culture collections, were classified into three mycelial phenotypes: dense, feathery and restricted, based on variable growth patterns on nutrient agar containing 120 mm chlorate. Pathogenicity of isolates was evaluated by measuring the length of stem lesions 21 days post-inoculation on the susceptible cluster bean genotype FS 277. Isolates showed considerable variation in aggressiveness, with the isolates from cluster bean with dense chlorate phenotype producing relatively higher lesion lengths on cluster bean plants. The results of the RAPD assay clearly distinguished the isolates on the basis of chlorate phenotype and host origin. Isolates from a single host were generally similar to each other, but differed distinctly from those from other hosts. Chlorate-sensitive isolates were distinct from chlorate-resistant isolates within a given host. A high degree of polymorphism in restriction patterns of the ITS region, including part of 25S rDNA, has been reported for the first time in the charcoal rot fungus.

Optimization of DNA isolation and PCR protocol for RAPD analysis of selected medicinal and aromatic plants of conservation concern from Peninsular India

Abstract: Genetic analysis of plants relies on high yields of pure DNA samples. Here we present the optimization of DNA isolation and PCR conditions for RAPD analysis of selected medicinal and aromatic plants of conservation concern from Peninsular India containing high levels of polysaccharides, polyphenols and secondary metabolites. The method involves a modified CTAB extraction employing polyvinyl pyrrolidone while grinding, successive long-term Chloroform : lsoamyalcohol extractions, an overnight RNase treatment with all steps carried out at room temperature. The yield of DNA ranged from 1-2 μg/μl per gram of the leaf tissue and the purity (ratio) was between 1.6-1.7 indicating minimal levels of contaminating metabolites. The technique is ideal for isolation of DNA from different plant species and the DNA isolated was used for randomly amplified polymorphic DNA (RAPD) analysis. RAPD protocol was optimized based on the use of higher concentration of MgCl 2 (3 mM), lower concentrations of primer (0.5 μM) and Taq polymerase (0.2 units), 50 ng of template DNA and an annealing temperature of 37°C, resulted optimal amplification. Reproducible amplifiable products were observed in all PCR reactions. Thus the results indicate that the optimized protocol for DNA isolation and PCR was amenable to plant species belonging to different genera which is suitable for further work on diversity analysis. Keywords: Vitex pubescens , Nervilia aragoana , Gymnema sylvestre , Withania somnifera , Origanum majorana , Boswellia serrata , Saraca asoca , Gloriosa superba , polysaccharides, PCR amplification

Detecting DNA Polymorphism and Genetic Diversity in a Wide Pistachio Germplasm: Comparison of AFLP, ISSR, and RAPD Markers

Abstract: There are limited numbers of pistachio (Pistacia vera L.) cultivars in the world and their phenotypic appearance and productivity are variable. Understanding such variation would facilitate their use in cultivar breeding programs. Therefore, in this study, 69 pistachio cultivars and genotypes originating from seven countries were characterized by randomly amplifi ed polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR), and amplifi ed fragment-length polymorphism (AFLP) markers. The results showed that all three marker systems were able to reveal variability between pistachio cultivars and genotypes. The correlation coeffi cients for genetic distances were statistically signifi cant among all three molecular marker types. The correlation between RAPD and AFLP data was the highest (r = 0.73) and the value between RAPD and ISSR data was the lowest (r = 0.58). AFLP proved to be the best technique among them. ISSR and AFLP assays were reliable and produced reproducible bands. ISSR was preferred over RAPD, especially when fi nancial investment and technical knowledge are limited. The constructed unweighted pair group method with arithmetic averages (UPGMA) dendrogram obtained from combined data separated the genotypes into two main clusters: one cluster ("Iranian") included genotypes originating from Iran and the second cluster ("Mediterranean") contained most other genotypes. The "Mediterranean" cluster further divided into three subclusters: one ("Siirt") consisted of the cultivars Siirt and Hacireso with a few other selections; the second subcluster ("Turkish") included Turkish cultivars; and the third subcluster contained Syrian, Italian, and the remaining cultivars. The closeness of the clusters was "Iranian" - "Siirt" - "Turkish"/"Syrian." These fi ndings reveal a new understanding in the diffusion of pistachio cultivation from its center of origin, the Iranian-Caspian region, via southeastern Turkey to Syria, the Mediterranean region of Europe, and northern Africa.

CAPs markers to assist selection for low vicine and convicine contents in faba bean ( Vicia faba L.)

Abstract: The antinutritional factors (ANFs) present in Vicia spp. seeds are a major constraint to the wider utilization of these crops as grain legumes. In the case of faba bean (Vicia faba L.), a breeding priority is the absence vicine and convicine (v-c); responsible for favism in humans and for the reduced animal performance or low egg production in laying hens. The discovery of a spontaneous mutant allele named vc-, which induces a 10–20 fold reduction of v-c contents, may facilitate the process. However, the high cost and difficulty of the chemical detection of v-c seriously restricts the advances in breeding-selection. To identify random amplified polymorphic DNA (RAPD) markers linked to this gene, we have analysed an F2 population derived from a cross between a line with high v-c content (Vf6) and the vc- genotype (line 1268). Quantification of v-c was done by spectrophotometry on the parents and the F2 population (n = 136). By using bulked segregant analysis (BSA), two RAPD markers linked in coupling and repulsion phase to the allele vc- were identified and further converted into sequence characterized amplified regions (SCARs). Amplification of SCARS was more consistent, although the initial polymorphism between pools was lost. To recover the polymorphisms several approaches were explored. Restriction digestion with HhaI (for SCAR SCH01620) and RsaI (for SCAR SCAB12850) revealed clear differences between the parental lines. The simultaneous use of the two cleavage amplified polymorphism (CAP) markers will allow the correct fingerprinting of faba bean plants and can be efficiently used in breeding selection to track the introgression of the vc- allele to develop cultivars with low v-c content and improved nutritional value.