Showing papers on "RAPD published in 2007"

Inter and intra-population variability of Jatropha curcas (L.) characterized by RAPD and ISSR markers and development of population-specific SCAR markers

Abstract: Jatropha curcas (Euphorbiaceae) is an oil-bearing species with multiple uses and considerable potential as a bioenergy crop. The present investigation has been undertaken to assess the extent of genetic diversity in a representative set of 42 accessions of J. curcas encompassing different crop growing regions in India along with a non-toxic genotype from Mexico as a prelude for utilization of promising and genetically divergent materials in the breeding programmes. Molecular polymorphism was 42.0% with 400 RAPD primers and 33.5% with 100 ISSR primers between accessions indicating modest levels of genetic variation in the Indian germplasm. The within-population variation based on RAPD polymorphism was 64.0% and was on par with the inter-population variation. Polymorphic ISSR markers have been identified that could differentiate the Indian accessions from the Mexican genotype and two of them were converted to SCAR markers. The SCAR primer pair ISPJ1 amplified a 543 bp fragment in all the Indian populations, while ISPJ2 with a specific amplicon of 1,096 bp was specific to the Mexican genotype. Population-specific bands have been identified for the accession from Kerala (2 RAPD markers), Neemuch-1 from Rajasthan (1 each of RAPD and ISSR markers) and the non-toxic genotype from Mexico (17 RAPD and 4 ISSR markers), which serve as diagnostic markers in genotyping. The study indicates an immediate need for widening the genetic base of J. curcas germplasm through introduction of accessions with broader geographical background.

Molecular diagnostic key for identification of single juveniles of seven common and economically important species of root‐knot nematode (Meloidogyne spp.)

Abstract: A molecular protocol is presented for distinguishing seven of the most common and economically important Meloidogyne spp DNA was extracted from individual second-stage juvenile (J2) nematodes of Meloidogyne spp and amplified by PCR (polymerase chain reaction) Fifteen PCRs including amplification of rDNA, specific SCAR (sequence characterized amplified region) and RAPD (random amplified polymorphic DNA) fragments were possible from the extracted DNA This enabled a molecular diagnostic key for M incognita, M javanica, M arenaria, M mayaguensis, M hapla, M chitwoodi and M fallax to be designed The key unifies published methods into a single logical schematic using primer combinations that were previously validated and shown to work reliably and specifically The protocol can be used with single juvenile or adult nematodes and the schematic can readily be expanded to accommodate more species The use of RAPD amplification to assist with identification of samples which do not yield diagnostic amplification products after the first three steps of the molecular key is also described

QTL mapping for economic traits based on a dense genetic map of cotton with PCR-based markers using the interspecific cross of Gossypium hirsutum · Gossypium barbadense

Abstract: A high-density molecular marker linkage map of cotton based entirely on polymerase chain reaction-based markers is useful for a marker-assisted breeding program. Four kinds of markers—simple sequence repeats (SSRs), sequence-related amplified polymorphism (SRAP), random amplified polymorphic DNA (RAPD), and retrotransposon-microsatellite amplified polymorphism (REMAP)—were used to assay an F2 population from a cross between “Handan208” (Gossypium hirsutum) and “Pima90” (Gossypium barbadense). Sixty-nine F2 plants were used for map construction using 834 SSRs, 437 SRAPs, 107 RAPDs, and 16 REMAPs. Linkage analysis revealed that 1,029 loci could be mapped to 26 linkage groups that extended for 5,472.3 cM, with an average distance between 2 loci of 5.32 cM. The corresponding 69 F2:3 families were grown, arranged in two replicates, and scored for eight phenotypes. Quantitative trait loci (QTL) analysis was performed by means of composite interval mapping using WinQtlCart ver 2.0. A total of 52 distinct QTLs were detected: 4 QTLs for lint index, 8 for seed index, 11 for lint yield, 4 for seed cotton yield, 9 for number of seed per boll, 3 for fiber strength, 5 for fiber length, and 8 for micronaire value. The present map and QTL analysis may provide a useful tool for breeders to transfer desirable traits from G. barbadense to the mainly cultivated species, G. hirsutum.

Protocol for isolation of genomic DNA from dry and fresh roots of medicinal plants suitable for RAPD and restriction digestion

Abstract: A simple and efficient protocol for isolating genomic DNA from fresh and dry roots of medicinal plants was developed. It involves a modified CTAB procedure using 3%CTAB, 4% b-mercaptoethanol, 2 M NaCl and 5% PVP. The extraction was carried out at 70°C. A slight increase in the concentrations of these chemical components and temperature helped in the removal of secondary metabolites and polysaccharides from the DNA preparation. The quantity and purity of isolated DNA was higher when compared with DNA extracted by the methods of Dellaporta et al. (1983) and Doyle and Doyle (1990). The DNA yield ranged from 33 to 68 µg per g of root samples and it was 1.47 times greater in dried than fresh samples. The DNA samples were found suitable for analysis with restriction enzyme digestion and random amplification of polymorphic DNA (RAPD). The total duration for DNA extraction from roots of medicinal plants using this protocol was 135 min as compared to 225 min with existing protocols. Key words: DNA isolation, roots, medicinal plants, secondary metabolite, PCR amplification, restriction digestion.

PCR-based methods for fish and fishery products authentication

Abstract: This work intends to provide an updated and extensive overview on the PCR-based methods for fish and fishery products authentication. Various techniques such as PCR-sequencing, Multiplex-PCR, PCR-RFLP, PCR-SSCP, RAPD, Real-Time PCR and PCR lab-on-a-chip are described and discussed. Moreover, commercial PCR kits for fish species identification are provided in this review. These methods could allow consumers protection against fraudulent practices in the fishery industry and enforce national and trans-national laws and regulations.

Genetic diversity and antifungal activity of species of Pestalotiopsis isolated as endophytes from medicinal plants

Abstract: The genetic diversity of fungal endophytes in root, bark and twigs of four medicinally important plants, Azadirachta indica, Holarrhena antidysenterica, Terminalia arjuna and T. chebula were examined. Thirty isolates of Pestalotiopsis and two isolates of Bartalinia rohillardoides were genotypically compared by RAPD techniques and 241 reproducible polymorphic bands were obtained using 23 random primers. The data was subjected to unweighted pair-group (UPGMA) cluster analysis. The isolates grouped into four main clusters and subgroups, group I contained 12 isolates, group II contained 3 isolates of P. virgatula, group III contained 10 isolates including P. microspora, B. robillardoides, P. theae and Pestalotiopsis spp., group IV contained five isolates of P. microspora and finally one Pestalotiopsis spp. did not fall into any group. The ethyl acetate extracts of isolates from Terminalia arjuna showed greater antifungal activity than those from other medicinal trees against six test organisms viz., Alternaria carthami, Fusarium oxysporum, Fusarium verticilloides Macrophomina phaseolina, Phoma sorghina and Sclerotinia sclerotiorum, with the zone of inhibition ranging from 4 to 25 mm. in diameter. The results indicate that RAPD can be employed for detecting genetic diversity of Pestalotiopsis species from medicinal plants and for pre-selection of these isolates for bioactive screening programme.

Population structure and resistance genes in antibiotic-resistant bacteria from a remote community with minimal antibiotic exposure.

Abstract: In a previous study, we detected unexpectedly high levels of acquired antibiotic resistance in commensal Escherichia coli isolates from a remote Guarani Indian (Bolivia) community with very low levels of antibiotic exposure and limited exchanges with the exterior. Here we analyzed the structure of the resistant E. coli population from that community and the resistance mechanisms. The E. coli population (113 isolates from 72 inhabitants) showed a high degree of genetic heterogeneity, as evidenced by phylogenetic grouping (77% group A, 10% group B1, 8% group D, 5% group B2) and genotyping by randomly amplified polymorphic DNA (RAPD) analysis (44 different RAPD types). The acquired resistance genes were always of the same types as those found in antibiotic-exposed settings [ bla TEM , bla PSE-1 , catI , cmlA6 , tet (A), tet (B), dfrA1 , dfrA7 , dfrA8 , dfrA17 , sul1 , sul2 , aphA1 , aadA1 , aadA2 , aadA5 , aadB , and sat-1 ]. Class 1 and class 2 integrons were found in 12% and 4% of the isolates, respectively, and harbored arrays of gene cassettes similar to those already described. The cotransferability of multiple-resistance traits was observed from selected isolates and was found to be associated with resistance conjugative plasmids of the F, P, and N types. Overall, these data suggest that the resistance observed in this remote community is likely the consequence of the dissemination of resistant bacteria and resistance genes from antibiotic-exposed settings (rather than of an independent in situ selection) which involved both the clonal expansion of resistant strains and the horizontal transfer/recombination of mobile genetic elements harboring resistance genes.

Molecular Epidemiology of Two Klebsiella pneumoniae Mastitis Outbreaks on a Dairy Farm in New York State

Abstract: Klebsiella spp. have become an important cause of clinical mastitis in dairy cows in New York State. We describe the occurrence of two Klebsiella mastitis outbreaks on a single dairy farm. Klebsiella isolates from milk, feces, and environmental sources were compared using random amplified polymorphic DNA (RAPD)-PCR typing. The first mastitis outbreak was caused by a single strain of Klebsiella pneumoniae, RAPD type A, which was detected in milk from eight cows. RAPD type A was also isolated from the rubber liners of milking machine units after milking of infected cows and from bedding in the outbreak pen. Predominance of a single strain could indicate contagious transmission of the organism or exposure of multiple cows to an environmental point source. No new cases with RAPD type A were observed after implementation of intervention measures that targeted the prevention of transmission via the milking machine as well as improvement of environmental hygiene. A second outbreak of Klebsiella mastitis that occurred several weeks later was caused by multiple RAPD types, which rules out contagious transmission and indicates opportunistic infections originating from the environment. The diversity of Klebsiella strains as quantified with Simpson's index of discrimination was significantly higher for isolates from fecal, feed, and water samples than for isolates from milk samples. Several isolates from bedding material that had the phenotypic appearance of Klebsiella spp. were identified as being Raoultella planticola and Raoultella terrigena based on rpoB sequencing.

A high-density, integrated genetic linkage map of lettuce (Lactuca spp.)

Abstract: An integrated map for lettuce comprising of 2,744 markers was developed from seven intra- and inter-specific mapping populations. A total of 560 markers that segregated in two or more populations were used to align the individual maps. 2,073 AFLP, 152 RFLP, 130 SSR, and 360 RAPD as well as 29 other markers were assigned to nine chromosomal linkage groups that spanned a total of 1,505 cM and ranged from 136 to 238 cM. The maximum interval between markers in the integrated map is 43 cM and the mean interval is 0.7 cM. The majority of markers segregated close to Mendelian expectations in the intra-specific crosses. In the two L. saligna x L. sativa inter-specific crosses, a total of 155 and 116 markers in 13 regions exhibited significant segregation distortion. Data visualization tools were developed to curate, display and query the data. The integrated map provides a framework for mapping ESTs in one core mapping population relative to phenotypes that segregate in other populations. It also provides large numbers of markers for marker assisted selection, candidate gene identification, and studies of genome evolution in the Compositae.

Low-cost media for in vitro conservation of turmeric ( Curcuma longa L. ) and genetic stability assessment using RAPD markers

Abstract: Up to 73% decrease in cost of media for plant regeneration and in vitro conservation was achieved in Curcuma longa cv Prathibha by using inexpensive carbon source and gelling agent. Laboratory reagent-grade sucrose was replaced by locally available commercial sugar (market sugar or sugar cubes) as carbon source and bacteriological grade agar by isabgol (also named isubgol) as gelling agent. No adverse effects on shoot regeneration and conservation on isabgol-gelled low-cost media were observed as compared to that on agar-gelled control medium (CM). Some 33–56% cultures of C. longa survived up to 12 mo. on isabgol-gelled medium in comparison to only 16% on CM. Genetic stability of 12-month-old in vitro-conserved plants was assessed using 25 random amplified polymorphic DNA (RAPD) primers; no significant variation was observed in RAPD profiles of mother plants and in vitro-conserved plantlets on CM and low-cost media.

Diversity among landraces of Indian snapmelon ( Cucumis melo var. momordica )

Abstract: Diversity among 36 snapmelon landraces, collected from 2 agro-ecological regions of India (9 agro-climatic sub-regions), was assayed using RAPD primers, morphological traits of plant habit and fruit, 2 yield-associated traits, pest and disease resistance and biochemical composition (TSS, ascorbic acid, titrable acidity). Typical differences among accessions were observed in plant and fruit characteristics and snapmelon germplasm with high titrable acidity and possessing resistance to downy mildew, Cucumber mosaic virus, Zucchini yellow mosaic virus, Papaya ringspot virus, Aphis gossypii and Meloidogyne incognita was noticed in the collection. RAPD based grouping analysis revealed that Indian snapmelon was rich in genetic variation and region and sub-region approach should be followed across India for acquisition of additional melon landraces. Accessions of var. agrestis and var. momordica clustered together and there was a separate cluster of the accessions of var. reticulatus. Comparative analysis of the genetic variability among Indian snapmelons and an array of previously characterized reference accessions of melon from Spain, Israel, Korea, Japan, Maldives, Iraq, Pakistan and India using SSRs showed that Indian snapmelon germplasm contained a high degree of unique genetic variability which was needed to be preserved to broaden the genetic base of melon germplasm available with the scientific community.

Genetic relationships among pomegranate genotypes studied by fruit characteristics and RAPD markers

Abstract: SummaryThe aim of this research was to study 24 Iranian pomegranate genotypes and thereby reveal their genetic relationships. Measurements of 28 fruit characteristics together with Randomly Amplified Polymorphic DNA (RAPD) marker data were used for this purpose. Mean values of fruit characteristics were used for factor analysis which determined seven main factors. Grouping of pomegranate genotypes by these factors was performed by Ward’s method. Among 113 random decamer primers tested, 27 showed good amplification and polymorphism, and a total of 158 RAPD markers were produced. Estimates of genetic relationships, using Jaccard’s similarity coefficient, ranged from 0.30 – 0.88 for the RAPD data. Grouping by fruit characteristics was compared with the results from RAPD analysis, but did not produce a significant correlation. This means that information based on fruit characteristics is not sufficient for genetic discrimination in pomegranate; however, RAPD markers provided a useful technique to study geneti...

Characterization and transferability of microsatellite markers of the cultivated peanut (Arachis hypogaea).

Abstract: The genus Arachis includes Arachis hypogaea (cultivated peanut) and wild species that are used in peanut breeding or as forage. Molecular markers have been employed in several studies of this genus, but microsatellite markers have only been used in few investigations. Microsatellites are very informative and are useful to assess genetic variability, analyze mating systems and in genetic mapping. The objectives of this study were to develop A. hypogaea microsatellite loci and to evaluate the transferability of these markers to other Arachis species. Thirteen loci were isolated and characterized using 16 accessions of A. hypogaea. The level of variation found in A. hypogaea using microsatellites was higher than with other markers. Cross-transferability of the markers was also high. Sequencing of the fragments amplified using the primer pair Ah11 from 17 wild Arachis species showed that almost all wild species had similar repeated sequence to the one observed in A. hypogaea. Sequence data suggested that there is no correlation between taxonomic relationship of a wild species to A. hypogaea and the number of repeats found in its microsatellite loci. These results show that microsatellite primer pairs from A. hypogaea have multiple uses. A higher level of variation among A. hypogaea accessions can be detected using microsatellite markers in comparison to other markers, such as RFLP, RAPD and AFLP. The microsatellite primers of A. hypogaea showed a very high rate of transferability to other species of the genus. These primer pairs provide important tools to evaluate the genetic variability and to assess the mating system in Arachis species.

Micropropagation in banana using high levels of cytokinins does not involve any genetic changes as revealed by RAPD and ISSR markers

Abstract: Use of high levels of growth regulators during micropropagation results in undesirable clonal variability in important commercial crops such as banana. The present study investigated the effects of high levels of cytokinins on micropropagation in banana (genotype AAB), and the genetic stability of plantlets was assessed using RAPD and ISSR markers. Cytokinins, such as BA and kinetin were added to the routine shoot multiplication medium at concentrations up to 10 mg l−1. After 12 weeks of culture involving three subcultures, the maximum number of shoot buds were produced in cultures receiving either 5 mg l−1 BA (80 shoot buds) or 4 mg l−1 kinetin (62 shoot buds). Certain morphological abnormalities observed during proliferation of shoot buds in vitro were not observed during acclimatization ex vitro. To check the genetic stability, RAPD and ISSR profiles of micropropagated plantlets obtained from different cytokinin-treatments were compared with control microplants maintained on MS medium as well as the field-grown mother plant. A total of 50 RAPD and 12 ISSR primers resulted in 625 distinct and reproducible bands. Thus a total of 17,400 bands were generated showing homogeneous RAPD and ISSR patterns. Band intensity histogram of each gel confirmed their monomorphic nature with no genetic variation in all the plantlets analysed. Based on these results a protocol for high rate shoot multiplication was worked out leading to uniform shoot production.

Genetic differentiation of Vigna species by RAPD, URP and SSR markers

Abstract: Seventy genotypes belonging to 7 wild and cultivated Vigna species were genetically differentiated using randomly amplified polymorphic DNA (RAPD), universal rice primer (URP) and simple sequence repeat (SSR) markers. We identified RAPD marker, OPG13 which produced a species-specific fingerprint profile. This primer characterized all the Vigna species uniquely suggesting an insight for their co-evolution, domestication and interspecific relationship. The cluster analysis of combined data set of all the markers resulted in five major groups. Most of the genotypes belonging to cultivated species formed a specific group whereas all the wild species formed a separate cluster using unweighted paired group method with arithmetic averages and principle component analysis. The Mantel matrix correspondence test resulted in a high matrix correlation with best fit (r = 0.95) from combined marker data. Comparison of three-marker systems showed that SSR marker was more efficient in detecting genetic variability among all the Vigna species. The narrow genetic base of the V. radiata cultivars obtained in the present study emphasized that large germplasm collection should be used in Vigna improvement programme.

Development of SCAR markers linked to powdery mildew (Uncinula necator) resistance in grapevine (Vitis vinifera L. and Vitis sp.)

Abstract: Sequence-characterized amplified regions markers (SCARs) were developed from six randomly amplified polymorphic DNA (RAPD) markers linked to the major QTL region for powdery mildew (Uncinula necator) resistance in a test population derived from the cross of grapevine cultivars “Regent” (resistant) × “Lemberger”(susceptible). RAPD products were cloned and sequenced. Primer pairs with at least 21 nucleotides primer length were designed. All pairs were tested in the F1 progeny of “Regent” × “Lemberger”. The SCAR primers resulted in the amplification of specific bands of expected sizes and were tested in additional genetic resources of resistant and susceptible germplasm. All SCAR primer pairs resulted in the amplification of specific fragments. Two of the SCAR markers named ScORA7-760 and ScORN3-R produced amplification products predominantly in resistant individuals and were found to correlate to disease resistance. ScORA7-760, in particular, is suitable for marker-assisted selection for powdery mildew resistance and to facilitate pyramiding powdery mildew resistance genes from various sources.

RAPD and ISSR molecular markers in Olea europaea L.: Genetic variability and molecular cultivar identification

Abstract: Thirty Portuguese and eight foreign olive (Olea europaea L.) cultivars were screened using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) markers. Twenty RAPD primers amplified 301 reproducible bands of which 262 were polymorphic; and 17 ISSR primers amplified 204 bands of which 180 were polymorphic. The percentage of polymorphic bands detected by ISSR and RAPD was similar (88 and 87%, respectively). The genetic variability observed was similar in the Portuguese and foreign olive cultivars. Seven ISSR and 12 RAPD primers were able to distinguish individually all 38 olive cultivars. Twenty specific molecular markers are now available to be converted into Sequence Characterised Amplified Region (SCAR) markers. Relationships among Portuguese and foreign cultivars is discussed.

Evaluation of genetic diversity in Turkish melons ( Cucumis melo L.) based on phenotypic characters and RAPD markers

Abstract: The genetic relationships among 56 melon (Cucumis melo L.) genotypes collected from various parts of Turkey were determined by comparing their phenotypic and molecular traits with those of 23 local and foreign melon genotypes to investigate the taxonomic relationships and genetic variation of Turkish melon germplasm. Sixty-one phenotypic characters and 109 polymorphic RAPD markers obtained from 33 primers were used to define the genetic similarity among the melon genotypes by dendrograms or two and three dimensional scaling. There were high correlations (r ≥ 0.97) among the four resulting matrices used in molecular characterization. The correlations between phenotypic (Euclidean) and molecular Euclidean, Jaccard, Simple matching, and Nei analyses were r = 0.41, r = −0.40, r = −0.43 and r = −0.40, respectively. Related genotypes or genotypes collected from similar regions were partitioned to similar clusters. Both analyses (phenotypic and molecular) indicated that non-sweet melon types were dissimilar from sweet types and diversity of Turkish melon genotypes was higher than that of sweet foreign cultivars examined, but similar to that of the reference accessions employed. It was also observed that sweet Turkish melon genotypes belonging to groups inodorus and group cantalupensis were highly variable and could have intermated or have crossed with other non-sweet types.

An anchored linkage map for sugar beet based on AFLP, SNP and RAPD markers and QTL mapping of a new source of resistance to Beet necrotic yellow vein virus

Abstract: Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV), is an important sugar-beet disease worldwide and can result in severe losses of root yield and sugar content. We have identified a major QTL for BNYVV resistance from a new source in a segregating population of 158 individuals. The QTL explained an estimated 78% of the observed phenotypic variation and the gene conferring the partial resistance is referred to as Rz4. AFLP was used in combination with bulked segregant analysis (BSA) to develop markers linked to the resistance phenotype. AFLP marker analysis was extended to produce a linkage map that was resolved into nine linkage groups. These were anchored to the nine sugar-beet chromosomes using previously published SNP markers. This represents the first anchored sugar-beet linkage map to be published with non-anonymous markers. The final linkage map comprised 233 markers covering 497.2 cM, with an average interval between markers of 2.1 cM. The Rz4 QTL and an Rz1 RAPD marker were mapped to chromosome III, the known location of the previously identified BNYVV resistance genes Rz1, Rz2 and Rz3. The availability to breeders of new resistance sources such as Rz4 increases the potential for breeding durable disease resistance.

A SCAR marker for the sex types determination in Colombian genotypes of Carica papaya

Abstract: Sex type determination in papaya (Carica papaya L.) is very important for crop improvement processes because it accelerates the identification of the fruitful plants. The use of molecular technology provides a quick and reliable identification of sex types in plantlets growing in seedbeds. Random amplified polymorphic DNA (RAPD) markers were used to determine the sex types of Colombian cultivars of dioecious papaya genotypes. This species has three sex types (male, female and hermaphrodite) determined by a multiallelic locus. There are no morphological differences at the chromosome level; therefore the identification of sex types by chromosomal dimorphism is not possible. A RAPD marker of 900 bp was found in male plants, but not in females or hermaphrodites. From this RAPD marker a sequence characterized amplified region (SCAR) was developed and it was possible to amplify fragments from the genomes of male and hermaphrodite plants, but not the female ones. The results indicate that this new SCAR marker will be valuable to determine the sex type of papaya plants.

Development of molecular markers linked to the 'Fiesta' linkage group 7 major QTL for fire blight resistance and their application for marker-assisted selection.

Abstract: A fire blight resistance QTL explaining 34.3%–46.6% of the phenotypic variation was recently identified on linkage group 7 of apple cultivar ‘Fiesta’ (F7). However, markers flanking this QTL were AFLP and RAPD markers unsuitable for marker-assisted selection (MAS). Two RAPD markers bracketing the QTL have been transformed into SCAR (sequence-characterized amplified region) markers, and an SSR marker specific for the region was developed. Pedigree analysis of ‘Fiesta’ with these markers enabled tracking of the F7 QTL allele back to ‘Cox’s Orange Pippin’. Stability of the effect of this QTL allele in different backgrounds was analyzed by inoculating progeny plants of a cross between ‘Milwa’, a susceptible cultivar, and ‘1217’, a moderately resistant cultivar, and a set of cultivars that carry or lack the allele conferring increased fire blight resistance. Progenies and cultivars that carried both markers were significantly more resistant than those that did not carry both markers, indicating high stability ...