Inter-simple Sequence Repeat Polymerase Chain Reaction Research Articles

Spatial pattern of genetic diversity in field populations of Fusarium incarnatum-equiseti species complex.

Fusarium is associated with a number of wilt, blight, scab, and rot diseases in a range of economically important staple food crops worldwide. An assessment of the genetic structure and population stratification of Fusarium incarnatum‐equiseti species complex (FIESC) pathogen populations is important to understand the evolutionary potential of such populations in adapting to environmental change. Based on intersimple sequence repeat polymerase chain reaction (ISSR‐PCR), it was found that the pathogen population was structured into three genetic clusters for which genetic differentiation was higher within than among populations. There was high intrapopulation genetic diversity for population 1 (94.63%) which consisted largely of isolates collected from North Trinidad. Populations 2 and 3 had a low level of admixture among the populations based on overall population differentiation. Population 1 accounted for the highest amount of genetic variation (95.82%) followed by populations 2 and 3. Population stratification was reflected in the dendrogram topology, which consisted of three main genetic clusters and which coincided with the outcome of Bayesian and PCoA analyses. The populations were isolated by distance, and Voronoi tessellations indicated physical or structural barriers to gene flow which contributed to restricted admixture between two of three populations. These findings suggest a high evolutionary potential for this FIESC pathogen population, the implications of which directly affect disease management strategies.

Identification and Genetic Diversity Analysis of Edible and Medicinal Malva Species Using Flow Cytometry and ISSR Molecular Markers

The Malva genus contains species that reveal therapeutic properties and are mostly important in medicine and the functional food industry. Its breeding, cultivation, and utilization are based on proper germplasm/plant identification, which is difficult using morphological features. For this reason, we applied flow cytometry and inter simple sequence repeat polymerase chain reaction (ISSR-PCR) for fast and accurate species identification. Genome size estimation by flow cytometry was proposed as the first-choice method for quick accession screening. Out of the 12 tested accessions, it was possible to identify six genotypes based on genome size estimation, whereas all species and varieties were identified using ISSR markers. Flow cytometric analyses revealed that Malva species possessed very small (1.45–2.77 pg/2C), small (2.81–3.80 pg/2C), and intermediate (11.06 pg/2C) genomes, but the majority of accessions possessed very small genomes. Additionally, this is the first report on genome size assessment for eight of the accessions. The relationships between the investigated accessions showed the presence of two clusters representing malvoid and lavateroid group of species. Flow cytometry and ISSR molecular markers can be effectively used in the identification and genetic characterization of Malva species.

Ameliorating Effect of Boric Acid Against Vanadium Toxicity in Wheat (Triticum aestivum L.)

Heavy metal pollution, which is one of the most important environmental problems, has a significant effect on plant productivity. Vanadium (V) is considered one of the most important elements of the twenty-first century due to its high consumption in industries. Morphological and physiological measurements have been performed in V studies, but there are deficiencies in molecular studies. The purpose of the present work was to elucidate the effects of V on enzymes activity, DNA damage levels and genomic template stability (GTS%) in Triticum aestivum L., as well as to investigate whether boric acid (BA) has preventive effects on these changes. Antioxidant enzyme activities were determined by SOD (superoxide dismutase), POD (peroxidase) and MDA (malondialdehyde) level. The inter-simple sequence repeats polymerase chain reaction (ISSR-PCR) assay was used to determine the genotoxic effects of V on DNA. According to the obtained results, while V stress increased MDA level and POD enzyme activity, SOD enzyme activity decreased. When V and BA were applied together, MDA level decreased and SOD and POD enzyme activities increased. All doses of V (4.4, 6.6, 8.8 mM) resulted in DNA damage and decreased GTS%. On the other hand, different concentrations of BA (4 and 8 mM) combined with V decreased the toxic effects of V. The results suggested that V could also have negative effects on wheat plants by increasing DNA damage and lipid peroxidation, and BA has an antagonistic effect against V toxicity. BA may be an alternative to reduce genetic damage in plants.

Rapid identification of Trichoderma koningiopsis and Trichoderma longibrachiatum using sequence-characterized amplified region markers

Molecular markers enable the detection and classification of fungi isolated from their natural environments. To develop species-specific markers for detecting Trichoderma koningiopsis and T. longibrachiatum, the sequence-characterized amplified region technique, using 20 inter-simple sequence repeat-polymerase chain reaction primers, was performed. The two specific markers for amplifying a single unique band consistent with T. koningiopsis and T. longibrachiatum, which were absent with other Trichoderma strains, were successfully identified. These fragments had no meaningful sequence homology with known sequences available in the National Center for Biotechnology Information and TrichOKEY databases. Compared with traditional identification techniques, these markers can facilitate more rapid and less complicated studies of Trichoderma population dynamics and evaluate their establishment after release into agricultural environments.

EFFICACY OF MOLECULAR DIAGNOSIS TO DETERMINE GENETIC DIVERSITY AMONG INDIGENOUS ISOLATES OF Erwinia carotovora UNDER EGYPTIAN CONDITION

Thirty-Four isolates of Erwinia carotovora subspecies carotovora (Ecc) causing soft rot of potato, were collected from tubers of potato plants grown in North and East Egypt. The frequency of the isolated species of soft rot-associated bacteria, being 10, 10, 8 and 6 isolates from Governorates of Sinai, Sharqia, Gharbia and Beheira, respectively. The isolates were identified as Ecc by biochemical method and molecular identity then was confirmed by polymerase chain reaction (PCR) using specific primers FEc1 and REc1 which amplified the Eca-specific band of 690-733 bp. Pathogenicity test divided these 9 isolates into 3 aggressiveness groups, High infection (2 isolates), moderate infection (5 isolates) and low infection (2 isolates), receptively. These isolates were also characterized using the technique of Inter Simple Sequence Repeats polymerase chain reaction (ISSR-PCR) found that 61 amplified fragments, 56 were polymorphic (91.8%) and the percentage of polymorphism ranged from 100% (844B) to 84.62% (HB12); the fragment sizes were 224bp to 1.36 kb. The unweighted pair-group method with arithmetical average (UPGMA) cluster analysis based on pairwise genetic similarity coefficient revealed that the similarity between the selected 9 soft rot bacterial isolates ranged from 0.125 between Ec6 and Ec9 to 0.875 between Ec1, Ec3 and Ec4. The average of similarity among genotypes was 0.500 and divided the soft rot isolates into three well-defined clusters showing a great level of genetic diversity. However, these clusters were not specific to aggressiveness groups, origin or special potato variety. Isolates with different aggressiveness levels, originated from different potato varieties participate between the same clusters. This means that the isolates likely derived from the same source population and got scattered from one area to another through their hosts.

Physiological, cytological and molecular analysis of PVYNTN-infected potato cultivars

The tuber necrotic strain of Potato Virus Y (PVYNTN) is considered the most aggressive strain of PVY. Effects of PVYNTN on some metabolic activities and cytological characteristics of three potato cultivars Lady Rosetta, Mondial and Santana were investigated. Virus concentration, infection percentage and disease severity were also estimated. Potato cultivars differed in their susceptibilities to PVYNTN infection, whereas various levels of apical necrosis with different disease severity 30, 60 and 100% were recorded for Mondial, Santana and Lady Rosetta cultivars respectively. The lowest amount of leaf pigments was found in PVYNTN-infected potato cv. Santana. Total phenolic and total soluble sugars contents were highly accumulated in PVYNTN-infected leaves compared to healthy ones. A noticeable increased level of sodium cation was detected in PVYNTN-infected leaves of potato cv. Lady Rosetta. Electron microscopy analysis showed changes in nuclei, mitochondria, cell walls, and chloroplasts in PVYNTN-infected cells of potato cultivars compared with healthy. Genetic variations among potato cultivars under PVYNTN infection were detected by Inter Simple Sequence Repeat-Polymerase Chain Reaction (ISSR-PCR) analysis. The ISSR primers amplified 57 bands, and 52 showed polymorphism (91.23%). According to our results, potato cvs. Lady Rosetta and Santana were highly susceptible to PVYNTN infection while, Mondial was moderately resistant.

English

A strain of Aspergillus niger PR-142 native to northern coast of Peru was subjected to successive processes of mutagenesis by ultraviolet light (UV) irradiation at 253.7 nm to increase the production of fructooligosaccharides (FOS). An initial selection was made by considering the mutants with increased invertase activity followed by the measurement of β-fructosyltransferase (FTase) activity both in mycelium and extracellular environment. Five selected mutants, which showed increased values of mycelium invertase activity (ranging from 101 to 128% as compared to the parent strain) at 40°C and sodium dodecylsulfate 0.15 (w/v), were grown in a fermentative medium in 50 mL conical tubes on a rotary shaker, and their FTase activity was determined. The 6-M69 mutant showed the most active mycelium activity of 1.5 fold as compared to the parent strain. When the same reaction was performed between 1 to 4 h, at the 3rd h, the mycelium FTase activity significantly increased up to 7 and 3 times in the mutant and parental strain, respectively. Finally, 4 mutants and the parental PR-142 were genetically characterized using inter simple sequence repeat polymerase chain reaction (ISSR-PCR) molecular markers. This analysis showed a significant 33% polymorphic bands between the parent and mutant markers, and 20 bands were unique to the mutants. Key words: Aspergillus, mutagenesis, β-fructosyltransferase, fructooligosaccharides, inter simple sequence repeat polymerase chain reaction (ISSR-PCR).

English

The management of colored cotton is an agricultural activity widely adopted by farmers located at Brazilian semiarid region. The fiber colors currently available are still limited to green and shades of brown, however, there is possibility to broaden the variability for this trait by using accessions from Gossypium Brazilian bank in breeding programs. Therefore, it is necessary to know the genetic diversity of available accessions in the collection. Here, the genetic divergence in colored fiber accessions was estimated in order to identify promising candidates for further use in hybridization procedures of cotton improvement. DNA of twelve accessions were extracted from leaves and used in inter simple sequence repeat-polymerase chain reaction (ISSR-PCR) assays, using commercial oligonucleotides. The genetic divergence was estimated by clustering-unweighted pair group method with arithmetic mean (UPGMA) method. Five groups were clustered among them, three were contributive results for further use in hybridization procedures, including Brazilian cultivars and Peruvian accessions. Based on level of divergence, we suggest that lines generated from these materials could generate news shades of fiber colors in further use for selection procedures in cotton breeding. Key words: Gossypium, molecular marker, variability, genetic improvement.

Genetic structure in two Phascolosoma species in the Pacific Ocean

ABSTRACTPhascolosoma agassizii, a commonly reported species of sipunculan worm, occurs at various depths throughout the north Pacific. However, previous studies have shown that this nominal species is actually comprised of two genetically divergent lineages. Phascolosoma agassizii is isolated to the eastern Pacific, whereas a second species, here referred to Phascolosoma sp., is located in the western Pacific and represents a unique, non-sister taxon to P. agassizii. Both species exhibit the same developmental mode with a long-lived, planktotrophic, pelagosphera larval stage, which may facilitate long-distance dispersal, although developmental timing differs. Using an inter-simple sequence repeat polymerase chain reaction (ISSR-PCR) genetic fingerprinting approach, this study examines non-coding polymorphic regions of the nuclear genomes of P. agassizii from four eastern Pacific populations and of Phascolosoma sp. from six western Pacific locations. Small-scale spatial genetic variation was analysed separately for each coastline using an Analysis of Molecular Variance. The data pointed to the potential presence of fine-scale genetic structure within P. agassizii, and recovered significant genetic structure within Phascolosoma sp. from the western Pacific. This study shows that ISSR-PCR is a relatively fast and cost-effective way to study fine-scale genetic diversity in marine invertebrates, although possible analyses and interpretations of the data are subject to the same limitations as other size polymorphism-based approaches.

Phylogenetic relationships of some economically important cereal plants based on genome characterization using molecular markers

Genomes of 14 cereal germplasms (three barley cultivars: G124, G128, and G2000; two maize cultivars: Hybrid 10, and Hybrid 311; three rice cultivars: Sakha 101, Sakha 102, and G 182; two sorghum cultivars: white sorghum, and red sorghum; and four wheat cultivars: Sakha 95, Gemmizah 9, G 168, and Sakha 93) were characterized using two molecular markers: inter simple sequence repeat polymerase chain reaction (ISSR-PCR) and randomly amplified polymorphic DNA PCR (RAPD-PCR) to address the genetic relationships among them. Ten ISSR primers were used for fingerprinting the 14 cereal cultivars. A total of 109 ISSR markers of 400–3000 bp were scored as a result of ISSR-PCR markers. In addition, 130 RAPD markers were scored as a result of fingerprinting the same cultivars using 15 RAPD primers. ISSR-PCR and RAPD-PCR data were analyzed separately and together using the numerical taxonomy system of multivariate statistical (NTSYS-pc) program to address the genetic relationships among the 14 cereal cultivars under st...

Multilocus ISSR markers reveal two major genetic groups in Spanish and South African populations of the grapevine fungal pathogen Cadophora luteo-olivacea.

Cadophora luteo-olivacea is a lesser-known fungal trunk pathogen of grapevine which has been recently isolated from vines showing decline symptoms in grape growing regions worldwide. In this study, 80 C. luteo-olivacea isolates (65 from Spain and 15 from South Africa) were studied. Inter-simple-sequence repeat-polymerase chain reaction (ISSR-PCR) generated 55 polymorphic loci from four ISSR primers selected from an initial screen of 13 ISSR primers. The ISSR markers revealed 40 multilocus genotypes (MLGs) in the global population. Minimum spanning network analysis showed that the MLGs from South Africa clustered around the most frequent genotype, while the genotypes from Spain were distributed all across the network. Principal component analysis and dendrograms based on genetic distance and bootstrapping identified two highly differentiated genetic clusters in the Spanish and South African C. luteo-olivacea populations, with no intermediate genotypes between these clusters. Movement within the Spanish provinces may have occurred repeatedly given the frequent retrieval of the same genotype in distant locations. The results obtained in this study provide new insights into the population genetic structure of C. luteo-olivacea in Spain and highlights the need to produce healthy and quality planting material in grapevine nurseries to avoid the spread of this fungus throughout different grape growing regions.

Analysis of the genetic variability of Artemia franciscana Kellogg, 1906 from the Great Salt Lake (USA) based on mtDNA sequences, ISSR genomic fingerprinting and biometry

The genetic structure of the brine shrimp Artemia franciscana, an extremophile halophilic microcrustacean, from the Great Salt Lake (USA), was investigated by analysis of nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I (COI), genomic fingerprinting by ISSR-PCR (inter-simple sequence repeats polymerase chain reaction) and biometry. Samples from six different geographical localities of Great Salt Lake revealed ten distinct haplotypes with a mean high level of haplotype diversity (HD = 0.6). Phylogenetic and genetic fingerprinting analyses supported the null hypothesis, that the brine shrimps of this lake form a panmictic gene pool with a low level of genetic differentiation index (F ST = 0.05) and a high rate of gene flow (Nm = 2.8) between geographical areas. Moreover, morphological parameters revealed no population structure among all examined populations. Neutrality tests and mismatch distribution revealed that A. franciscana has undergone a recent population expansion. In spite of hypersaline conditions in the Northern (Gunnison Bay) regions of the Great Salt Lake, the genetic population structures of different areas of the lake are not linked to ecological or limnological conditions. Therefore, A. franciscana from the Great Salt Lake should be considered as a single management unit for conservation.

Genetic groups of Clavibacter michiganensis subsp. michiganensis identified by DNA fingerprinting and the effects of inoculation methods on disease development

Clavibacter michiganensis subsp. michiganensis (CMM) strains collected in nine different prefectures in Japan were fingerprinted by inter-simple sequence repeats–polymerase chain reaction (PCR) and repetitive sequence–based PCR. Forty-three CMM strains corresponded to 22 DNA fingerprinting patterns, indicating that the CMM population in Japan has high genetic diversity. Tomato plants were inoculated with CMM by two different methods: defoliation using infected scissors and planting in soil containing infected plant debris. Defoliation resulted in a larger number of diseased plants, disease developed earlier, and plants survived for significantly fewer days. Upon planting in the infected soil, 33.3 % of plants were infected and 6.7 % were diseased, i.e., 20.1 % of infected plants developed disease symptoms. These results indicate that some plants are infected with CMM through plant debris in the soil as the primary inoculum and most infected plants maintain latent infection. Thus, growers may unwittingly carry out disbudding and defoliation of latently infected plants, resulting in secondary inoculum.

Anthocyanin profiling of wild maqui berries (Aristotelia chilensis[Mol.] Stuntz) from different geographical regions in Chile

Maqui (Aristotelia chilensis) is a Chilean species which produces small berries that are collected from the wild. Anthocyanins, because of their health benefits, are the major focus of interest in maqui fruit. For this study, we examined anthocyanin and phenolic content of maqui fruits from individuals that belonged to four geographical areas in Chile, and used DNA marker analysis to examine the genetic variability of maqui populations that had distinctly different fruit anthocyanin content. Twelve primers generated a total of 145 polymorphic inter simple sequence repeat-polymerase chain reaction (ISSR-PCR) bands. ISSR-PCR showed different banding patterns for the individuals evaluated, confirming that maqui populations belonged to different genotypes. Maqui fruit from four different geographical regions during two consecutive growing seasons showed high total anthocyanin (6.6-15.0 g cy-3-glu kg⁻¹ fresh weight (FW)) and phenolic (10.7-20.5 g GAE kg⁻¹ FW) contents and different anthocyanin profiles. Three maqui genotypes exhibited significantly higher anthocyanin content than the others, as measured by pH differential method and high-performance liquid chromatography-mass spectrometry. Significant genetic diversity was noted within each ecological population. ISSR-PCR analysis provided a fingerprinting approach applicable for differentiation of maqui genotypes.

Molecular and biochemical markers of some Vicia faba L. genotypes in response to storage insect pests infestation

Storage insect pests cause serious losses to all legume crops in both quality and quantity. This study was conducted to study some morphological characters and biochemical components in eight genotypes of faba bean to determine biochemical and molecular markers for resistance and susceptibility to infestation with stored insects. The results showed that chlorophyll content in leaves, phenol, tannin, peroxidase (POD), polyphenol oxidase (PPO), and protein content in leaves and seeds of faba bean plants either significantly or non significantly increased the effect of insect infestation in the resistance genotypes (L.551, L.512, L.153, NA112, and L.1) as compared with the susceptible genotypes (L.16 and T.W). Protein electrophoresis showed a wide variation between genotypes and determined some biochemical markers (sodium dodecyl sulfate poly acrylamide gel electrophoresis, SDS-PAGE). In addition, molecular genetic markers for stored insects' resistance were obtained using inter-simple sequence repeat polymerase chain reaction (ISSR-PCR) analyses.

Genetic Structure ofColletotrichum gloeosporioidessensu lato Isolates Infecting Papaya Inferred by Multilocus ISSR Markers

Colletotrichum gloeosporioides sensu lato is widely distributed throughout temperate and tropical regions and causes anthracnose disease in numerous plant species. Development of effective disease management strategies is dependent on, among other factors, an understanding of pathogen genetic diversity and population stratification at the intraspecific level. For 132 isolates of C. gloeosporioides sensu lato collected from papaya in Trinidad, inter-simple-sequence repeat-polymerase chain reaction (ISSR-PCR) generated 121 polymorphic loci from five ISSR primers selected from an initial screen of 22 ISSR primers. The mean percentage of polymorphic loci was 99.18%. Bayesian cluster analysis inferred three genetic subpopulations, where group 1 consisted exclusively of isolates collected in the southern part of Trinidad whereas groups 2 and 3, although genetically distinct, were mixtures of isolates collected from both the northern and southern parts of Trinidad. Principal coordinates analysis and unweighted pair-group method with arithmetic mean phylogeny were concordant with Bayesian cluster analysis and supported subdivision into the three subpopulations. Overall, the total mean gene diversity was 0.279, the mean within-population gene diversity was 0.2161, and genetic differentiation for the Trinidad population was 0.225. Regionally, northern isolates had a lower gene diversity compared with southern isolates. Nei's gene diversity was highest for group 1 (h = 0.231), followed by group 2 (h = 0.215) and group 3 (h = 0.202). Genotypic diversity was at or near maximum for all three subpopulations after clone correction. Pairwise estimates of differentiation indicated high and significant genetic differentiation among the inferred subpopulations (Weir's θ of 0.212 to 0.325). Pairwise comparisons among subpopulations suggested restricted gene flow between groups 1 and 2 and groups 1 and 3 but not between groups 2 and 3. The null hypothesis of random mating was rejected for all three inferred subpopulations. These results suggest that pathogen biology and epidemiology as well as certain evolutionary factors may play an important role in population substructuring of C. gloeosporioides sensu lato isolates infecting papaya in Trinidad.

Identifi cation of Species-Diagnostic Inter Simple Sequence Repeat Markers for Ten Phyllanthus Species

Phyllanthus has been widely used in traditional medicine as an antipyretic, a diuretic, and to treat liver diseases and viral infections. Correct genotype identification of medicinal plant material remains important for the botanical drug industry. Limitations of chemical and morphological approaches for authentication have generated the need for newer methods in quality control of botanicals. In the present study, attempts were made to identify species-diagnostic markers for ten Phyllanthus species using the inter simple sequence repeat-polymerase chain reaction (ISSR-PCR) fingerprinting method. PCR amplification using seven ISSR primers resulted in significant polymorphism among the populations from different species. P. angustifolius and P. urinaria showed monomorphic frequency of maximum (63.88%) and minimum (20.64%), respectively. Seventeen species-diagnostic markers were identified for seven species (P. acidus, P. emblica, P. fraternus, P. urinaria, P. rotundifolius, P. amarus, and P. angustifolius) while no marker was detected for P. reticulatus, P. nivosus, and P. virgulatus. A maximum of six species-diagnostic markers were identified for P. acidus and a minimum of only one of 755 bp was available for P. amarus. Among the seventeen markers, nine were present in all individuals of particular species. The species-specific differences in fragment numbers and sizes could be used as diagnostic markers to distinguish the Phyllanthus species quickly.

Comparative study of the phylogenetic structure in six Apodemus species (Mammalia, Rodentia) inferred from ISSR-PCR data

The utility of the Inter Simple Sequence Repeat-Polymerase Chain Reaction (ISSR-PCR) was explored in order to determine genetic variation in six species of the genus Apodemus (A. flavicollis, A. sylvaticus, A. uralensis, A. agrarius, A. mystacinus and A. epimelas) at the individual level, population level, in separate geographic samples and in the species as a whole. Six optimized primers produced highly reproducible and polymorphic DNA markers with 98.3% polymorphic bands on a total sample of 91 individuals from 32 localities in Europe and Asia. Moreover, each primer allowed for an exact diagnosis of each of the six Apodemus species and thus provides a simple and reliable tool for the hitherto problematic discrimination of species from the subgenus Sylvaemus. Genetic distances between species ranged from 0.079, among the closely related A. flavicollis and A. sylvaticus, to 0.203 between A. mystacinus and A. agrarius. A. flavicollis, A. uralensis and A. sylvaticus display a strong population substructure. The range of genetic distances among geographic samples within last two species reaches the values obtained for closely related species. ISSR markers proved to be a simple and reliable tool for species diagnosis, as well as for estimating genetic diversity below the species level and for closely related species, but they showed questionable reliability for larger genetic distances.

Inter-simple sequence repeats (ISSR) molecular fingerprinting markers for authenticating the genuine species of rhubarb

Rhubarb is prescribed as the roots and rhizomes of Rheum officinale Baill., Rheum palmatum L., and Rheum tanguticum Maxim. ex Balf. in Chinese Pharmacopoeia. These three species are difficult to discriminate due to the morphological and anatomical similarity of the aerial parts and herbal medicines. In the present paper, inter-simple sequence repeats (ISSR) molecular fingerprinting markers have been employed to authenticate three genuine species of rhubarb using 15 primers to discriminate R. officinale, R. palmatum, andR. tanguticum. A total of 155 DNA fragments were amplified, of which 132 were polymorphic (85.2% of all bands). Four specific authentication markers have been found to authenticate three species of rhubarb. The UBC807 and UBC811 primers each generated one species-specific fragment that was clearly amplified in R. officinale (400bp from UBC807 and 248bp from UBC811). The application of the UBC807 primer also produced a 520bp DNA fragment from R. palmatum and R. tanguticum, and UBC816 primer generated a 620bp fragment that was specific for R. palmatum and R. officinale. Therefore, three loci combinations from primer UBC816 with any one of the primer UBC807and UBC811, that is UBC816-620bp with UBC807-520bp, UBC816-620bp with UBC807-400bp, and UBC816-620bp with UBC811-248bp, served the purpose of distinguishing three genuine species of rhubarb. To enhance the efficiency of authentication, ISSR fingerprinting codes have been constructed using four polymorphic bands for authenticating three genuine species of rhubarb. The present study could be applied to distinguish the genuine species of rhubarb at the molecular level. Key words: Rhubarb, inter-simple sequence repeats-polymerase chain reaction, molecular marker, authentication.

Molecular Markers Discriminate Closely Related Species <I>Encarsia diaspidicola</I> and <I>Encarsia berlesei</I> (Hymenoptera: Aphelinidae): Biocontrol Candidate Agents for White Peach Scale in Hawaii

We genetically characterized Encarsia diapsidicola Silvestri and Encarsia berlesei Howard (Hymenoptera: Aphelinidae) by two molecular methods: phylogenetic analysis of the cytochrome oxidase subunit I gene (COI) and intersimple sequence repeat-polymerase chain reaction (ISSR-PCR) DNA fingerprinting. These two closely related endoparasitoids are candidate biological control agents for the white peach scale, Pseudaulacaspis pentagona Targioni-Tozetti (Hemiptera: Diaspididae), in Hawaii. We developed species-specific COI molecular markers that discriminated the two species, and we tested the utility of the E. diaspidicola-specific COI marker to detect parasitism of white peach scale. The COI sequence data uncovered 46-bp differences between the two Encarsia spp. The level of COI genetic divergence between the two species was 9.7%, and the two clustered into their own clade on a parismonious phylogram. ISSR-PCR readily discriminated the two Encarsia spp. because each was observed with fixed species-specific banding patterns. The COI molecular markers were specific for each species because cross-reactivity was not observed with nontarget species. The E. diaspidicola-specific COI markers were successful at detecting parasitism of white peach scale by E. diaspidicola by 24 h. Both molecular marker types successfully discriminated the two Encarsia spp., whereas the COI markers will be useful as tools to assess levels of parasitism in the field and to study competitive interactions between parasitoids.