Inter-simple Sequence Repeat Polymerase Chain Research Articles

Molecular Characterization of Prunus spinosa L. (Rosaceae) Populations from the West Black Sea Region in Turkey Using Inter-simple Sequence Repeat Polymerase Chain Reaction

Molecular Characterization of Prunus spinosa L. (Rosaceae) Populations from the West Black Sea Region in Turkey Using Inter-simple Sequence Repeat Polymerase Chain Reaction

GENETIC ANALYSIS AND MOLECULAR CHARACTERIZATION OF THE WHEATGRASS (AGROPYRON CRISTATUM L. GAERTN.) IN SOUTH-EAST KAZAKHSTAN

Assembly, genetic analysis, and preservation of wheatgrass (Agropyron cristatum L. Gaertn.) diversity is an important task in improving the said crop under the environmental conditions of the Republic of Kazakhstan. The presented study evaluated and isolated various wheatgrass species and their populations for breeding and developing promising cultivars based on the genetic principles to improve the degraded pasture situation in South-East Kazakhstan from the perspective of global climate change. For the succeeding investigations, collection of the wheatgrass landraces from various regions of Kazakhstan ensued, as well as from accessions already conserved in the gene pool, and gained evaluation under the environmental conditions of South-East Kazakhstan. In performance-based screening, 20 wheatgrass cultivars identified underwent further genotyping by using inter-simple sequence repeat (ISSRs) polymerase chain reaction markers. The pertinent study significantly contributes to understanding the Agropyron species’ genetic structure and molecular characteristics. Using seven ISSR primers helped study the kinship and genetic polymorphism of the five Agropyron species collected from diverse locations. The ISSR primers were extremely informative for determining the Agropyron’s genetic diversity and interspecific similarity. Moreover, they come highly recommended for an in-depth study comparing the DNA sequencing of the Agropyron populations available in the National Center for Biotechnology’s database.

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.

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.

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.

Authenticity identification and leaf blight resistance evaluation of the F1 hybrids from two Lilium cultivars ‘Sorbonne’ and ‘Francia’

This study identified the genetic transmission pattern of the leaf blight resistance of Lilium species based on resistance distribution. A total of 133 hybrids of the ‘Sorbonne’ (female parent, highly resistant to Botrytis elliptica) and ‘Francia’ (male parent, highly susceptible to B. elliptica) cultivars were selected by inter-simple sequence repeat polymerase chain reaction. Among them, 17 were highly resistant, 42 moderately resistant, 46 moderately susceptible, and 28 susceptible. One F1 plant showed greater resistance than 'Sorbonne', and 17 showed greater susceptibility than 'Francia'. Disease resistance from 'Sorbonne' could be inherited and was a typical quantitative trait determined by polygenes.

Molecular identification of Trichinella isolates from wildlife animals of the Russian Arctic territories

Summary The parasitic nematodes of genus Trichinella are infective to a wide range of hosts, including humans, and have global distribution from tropic to arctic areas. Muscle samples from animals, collected in two areas of the Russian Federation, Chukotka Peninsula and Arkhangelsk Oblast, were tested for the presence of Trichinella spp. larvae. Trichinella spp. larvae were recovered from tissues of eleven wild and domestic animals: 1 polar bear, 1 wolverine, 3 arctic foxes, 1 ringed seal, 1 brown bear, 1 cat, 1 sled dog, 1 domestic pig and 1 northern sea lion. Two molecular methods, multiplex PCR and inter-simple sequence repeat polymerase chain reaction (ISSR PCR), were utilized for species identification. T. nativa was identified in the majority of tested animals and mixed infections with two Trichinella species were detected in four animals. T. spiralis/T. pseudospiralis mixed infection was found in a domestic pig and T. spiralis/T. nativa in two arctic foxes and a northern sea lion. Here, for the first time, Trichinella spp. infection in a northern sea lion and the northern sea lion as a new host for T. spiralis and T. nativa is reported. Discovery of Trichinella spp. infection in a new host, such as a northern sea lion, which is an opportunistic marine predator, mainly feeding on fish, indicates environmental contamination. Leaving of carcasses or waste from animals and improper management of livestock operations is important for spreading Trichinella spp. infection in free living animals. Therefore, further extensive epidemiological and environmental research and molecular studies are needed to investigate the local fauna.

Silver nanoparticles induced genotoxicity and oxidative stress in tomato plants

Among nanoparticles, silver nanoparticles (AgNPs) are intensively used in many materials owing to their antibacterial effects. In the present study different concentrations of AgNPs in Hoagland solution were applied to tomato seedlings. Total chlorophyll content, relative water content (RWC), antioxidant enzyme activities, and malondialdehyde content (MDA) as well as the genomic template stability (GTS) were analyzed. The intersimple sequence repeat polymerase chain reaction assay (ISSR-PCR) was used to determine the genotoxic effects of AgNPs on DNA. RWC did not change under AgNPs treatments; however, total chlorophyll content was significantly reduced by AgNPs applications. ISSR profiles demonstrated a consistent increase in polymorphic bands by the increase in the concentration of AgNPs. GTS value was also reduced depending on the concentration of AgNPs. SOD and APX activities were increased under low AgNPs treatments; however, these activities were decreased under high concentrations of AgNPs treatments. Tomato plants could be sensitive to AgNPs within the increase in MDA content in all of the AgNPs treatments. AgNPs nanotoxicity could be quite dose-dependent. AgNPs could also have negative effects on tomato plants by enhancing DNA damage and lipid peroxidation.

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.

Population development within the coffee wilt pathogen Gibberella xylarioides reflects host-related divergence

Variation of 45 Gibberella xylarioides cultures isolated from coffee trees in Africa was assessed by sequencing the partial translation elongation factor gene, by A + T rich DNA restriction fragment length polymorphism (RFLP) analysis and by inter-simple sequence repeat polymerase chain fingerprinting. Results were analysed in relation to host plant species and mating compatibilities to determined biological species (BS). Additionally, five “historical” strains isolated prior to the 1990s, and considered to belong to G. xylarioides, were obtained from internationally-recognised culture collections and a further 10 strains of various Gibberella/Fusarium spp., isolated from Coffea spp., were included to provide contextual comparisons. Ribosomal intergenic spacer (IGS) sequencing was undertaken for representatives of the suggested BS. Results confirmed all strains as G. xylarioides and that strains of sterile group 4 (SG4) may represent one or more additional “cryptic” species. ISSR fingerprints showed a single host correlated difference, suggesting stable clonal spread and this was supported by consistent A + T rich DNA band patterns for all strains. In contrast the IGS sequences identified potential phylogenetic events correlated to BS.

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.

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.

Sex identification and genetic variation of Saccharina (Phaeophyta) gametophytes as revealed by inter-simple sequence repeat (ISSR) markers

Inter-simple sequence repeat (ISSR) polymerase chain reaction (PCR) markers were utilized to investigate the genetic variation between male and female gametophyte populations of strains Rongfu and 901 of Saccharina. In total, 11 ISSR primers were able to generate 135 satisfactory and reproducible loci, of which 134 were polymorphic with 99.26 % polymorphism. The percentages of polymorphism of female gametophyte populations (60 and 62 % for their respective strains) were higher than those of the males (53 %), and the Nei’s genetic diversity and Shannon’s information index showed a similar tendency. The clustering of gametophytes of the same sex from each strain was well resolved by both an unweighted paired group method using the arithmetic average and a principal component analysis, suggesting that any male/female gametophyte pair could represent each strain. However, a single pair was not adequate for germplasm maintenance because the genetic variance among individuals within a population accounted for 57.45 % of the total (P < 0.0001), as shown by the analysis of molecular variance. The gametophyte sex could be identified by amplification with primer UBC809 because of a differential band present in the females. According to the sequence of this band, a pair of ISSR-derived sequence-characterized amplified region (SCAR) primers was designed. With the primers, one female-specific fragment was detected using PCR and Southern blot hybridization. This converted SCAR marker was localized on one unique chromosome of the female gametophytes of these two strains by use of fluorescence in situ hybridization, confirming that it was a female chromosome-specific marker.

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.

Molecular Markers Discriminate Closely Related Species &lt;I&gt;Encarsia diaspidicola&lt;/I&gt; and &lt;I&gt;Encarsia berlesei&lt;/I&gt; (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.

Molecular characterization of Leishamania isolates from China by inter-simple sequence repeat polymerase chain reaction

Leishmania has distinct epidemiological and biological characteristics and causes a variety of clinical symptoms. To understand the genetic diversity and the phylogenetic relationships among Leishmania isolates from China, 29 Leishmania isolates from different geographic origins, vectors, and hosts were analyzed using 21 inter-simple sequence repeat polymerase chain reaction (ISSR-PCR) primers. A total of 864 polymorphic bands were obtained. According to the results of the neighbor-joining phylogenetic tree and principal component analysis, the 29 isolates studied clustered into six groups. Isolates of Leishmania donovani complex from China share the highest similarity with the reference strain of L. donovani (DD8). This study helps to elucidate the genetic relationship among Leishmania isolates from China and similarities between Chinese isolates and World Health Organization reference strains. Furthermore, ISSR-PCR could also be a quick, simple, and reliable method for Leishmania species identification.

GENOTYPIC ANALYSIS OF MUCOR FROM THE PLATYPUS IN AUSTRALIA

Mucor amphibiorum is the only pathogen known to cause significant morbidity and mortality in the free-living platypus (Ornithorhynchus anatinus) in Tasmania. Infection has also been reported in free-ranging cane toads (Bufo marinus) and green tree frogs (Litoria caerulea) from mainland Australia but has not been confirmed in platypuses from the mainland. To date, there has been little genotyping specifically conducted on M. amphibiorum. A collection of 21 Mucor isolates representing isolates from the platypus, frogs and toads, and environmental samples were obtained for genotypic analysis. Internal transcribed spacer (ITS) region sequencing and GenBank comparison confirmed the identity of most of the isolates. Representative isolates from infected platypuses formed a clade containing the reference isolates of M. amphibiorum from the Centraal Bureau voor Schimmelcultures repository. The M. amphibiorum isolates showed a close sequence identity with Mucor indicus and consisted of two haplotypes, differentiated by single nucleotide polymorphisms within the ITS1 and ITS2 regions. With the exception of isolate 96-4049, all isolates from platypuses were in one haplotype. Multilocus fingerprinting via the use of intersimple sequence repeats polymerase chain reaction identified 19 genotypes. Two major clusters were evident: 1) M. amphibiorum and Mucor racemosus; and 2) Mucor circinelloides, Mucor ramosissimus, and Mucor fragilis. Seven M. amphibiorum isolates from platypuses were present in two subclusters, with isolate 96-4053 appearing genetically distinct from all other isolates. Isolates classified as M. circinelloides by sequence analysis formed a separate subcluster, distinct from other Mucor spp. The combination of sequencing and multilocus fingerprinting has the potential to provide the tools for rapid identification of M. amphibiorum. Data presented on the diversity of the pathogen and further work in linking genetic diversity to functional diversity will provide critical information for its management in Tasmanian river systems.

Molecular Evidence Suggests That Populations of the Asian Citrus Psyllid Parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) from Texas, Florida, and Mexico Represent a Single Species

Abstract We genetically characterized Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) populations from Texas, Florida, and Mexico and the sister taxon Tamarixia triozae (Burks) by two molecular methods. T. radiata is an ectoparasitoid of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), nymphs. The populations were submitted to intersimple sequence repeat-polymerase chain reaction DNA fingerprinting with two primers. No fixed banding pattern differences were uncovered among the populations of T. radiata with either primer, whereas different patterns were observed in T. triozae, suggesting that there is no genetic differentiation among the populations. Support for these results was obtained by sequence analyses of the internal transcribed spacer region one and the mitochondrial cytochrome oxidase subunit one (COI) gene. In both genes, the intrapopulational variation range (percentage divergence, %D) fell within the interpopulational variation range. The %D at the COI gene between T. radiata and T. triozae was 9.0–10.3%. However, haplotype structure was uncovered among the populations. No haplotypes were shared between Florida and Texas/Mexico, whereas sharing was observed between Texas and Mexico. Two population-specific nucleotides were identified that allowed the discrimination of the Florida and Texas/ Mexico individuals. A neighbor-joining and a parsimonious tree clustered the populations into two distinct clades. The Florida population clustered into one clade, whereas the Texas/Mexico populations clustered into another clade. The COI phylogeographic analysis suggests that the population of T. radiata in Texas did not come from Florida. The data also suggest that the population in Texas came from Mexico, or vice versa, because the Mexico population showed less haplotype diversity.