Inter-simple Sequence Repeat Patterns Research Articles

Pathogenicity, anastomosis groups, host range, and genetic diversity of Rhizoctonia species isolated from soybean, pea, and other crops in Alberta and Manitoba, Canada

Root rot is a common disease in soybean (Glycine max) and field pea (Pisum sativum), which restrain increased production in Canada. Sixty-seven isolates of Rhizoctonia were recovered from various diseased plants in Alberta, Canada along with three isolates from diseased soybean plants in Manitoba, Canada. According to their anastomosis behavior, 23 (32.9%) of the isolates were identified as anastomosis group (AG) 4 (AG4), 7 (10.0%) were AG2-1, 10 (14.3%) were AG2-2, 7 (10.0%) were AG5, 3 (4.3%) were AG-E and the AGs of the remaining 20 (28.6%) isolates could not be determined. Isolates belonging to AG4 produced typical symptoms of stem rot and root rot on seedlings of soybean and pea and were more aggressive than the AG2-1, AG2-2, AG5 and AG-E isolates. Selected isolates of AG4, AG2-1, AG2-2, AG5 and AG-E were to some degree able to infect common crops in Alberta, which included barley, canola, corn, faba bean, flax, lupin, lentil, pea, potato, soybean, and wheat. The genetic variability among these isolates was evaluated using phylogenetic analysis based on the rDNA ITS sequences and inter-simple sequence repeat (ISSR) markers. For the ITS sequence analysis, a neighbour-joining tree was constructed using the PAUP program, which clustered the Rhizoctonia isolates into five groups (Groups I to V). However, no correlation was observed between AGs, locations, aggressiveness or host origins. For the ISSR analysis, 54 polymorphic ISSR patterns were identified, indicating a high level of diversity among the isolates.

Hydrangea DNA Methylation Caused by pH Substrate Changes to Modify Sepal Colour is Detected by MSAP and ISSR Markers

The hydrangea (Hydrangea macrophylla (Thunb). Ser.) is an ornamental species with great market potential. It is known for its ability to change the colour of its inflorescence, according to the pH of the culture substrate. The molecular mechanisms that underlie these changes are still unclear. It is known that epigenetic mechanisms, such as DNA methylation, play an important role in genetic expression, so they could be responsible for this phenomenon in hydrangea. In the present study, the molecular markers ISSR (Inter Simple Sequence Repeat) and MSAP (Methyl-Sensitive Amplification Polymorphism) were used to detect molecular changes in the genome of hydrangea plants that were cultivated under different pH levels to modify the colour of the sepals. The results showed a correspondence between the methylation signal measured with MSAP and amplification ISSR patterns when compared before and after the modification of pH culture substrates. These results suggest that DNA methylation might be involved as a molecular mechanism underlying the colour change of hydrangea sepals in response to a differential pH in the substrate. In addition, the results pave the way to study the relationship between DNA methylation and ISSR marker profiles.

Genetic relatedness and taxonomy in closely related species of Hedysarum (Fabaceae)

A multidisciplinary study, engaging morphological, carpological and molecular data, has been performed to investigate the genetic relatedness and taxonomic boundaries of the close species Hedysarum gmelinii, H. setigerum and H. chaiyrakanicum (Fabaceae) with overlapped distribution areas in southern Siberia. The diagnostic features of these legume species are analyzed and discussed, including their macro- and micromorphological characteristics, seed coat ornamentation and inter-simple sequence repeat (ISSR) profiles. The morphometric features, pod and seed microsculpture traits of H. chaiyrakanicum and the ISSR patterns of the three species have been determined for the first time. Sprout, leaf, calyx, corolla, and stem rachis measurements, leaflet indumentum type and ISSR patterns significantly discriminate H. chaiyrakanicum from the other two species, whereas plant height, lengths of stem and leaf, and length and width of leaflet show opposite ranges of variation for H. gmelinii and H. setigerum though none of them is reliable in species identification. Ornamentation of seed coat and ISSR patterns does not differ significantly in the species. Therefore, our study supports the separate taxonomic treatment of H. chaiyrakanicum and the subordination of the cryptic species H. setigerum within H. gmelinii.

Effect of subculture times on genetic fidelity, endogenous hormone level and pharmaceutical potential of Tetrastigma hemsleyanum callus

The effects of subculture frequency on genetic variations of Tetrastigma hemsleyanum callus under a long-term tissue culture were evaluated by means of flow cytometry (FCM), inter simple sequence repeat (ISSR), and sequence-related amplified polymorphism (SRAP) markers to clarify the variation pattern and frequency of somatic variation in the course of the subculture. The effect of subculture frequency on endogenous hormone content was also assessed by Enzyme-linked immunosorbent assay to explore the correlation within the differentiation rate, somaclonal variation frequency of callus and the endogenous hormone content. Moreover, the flavonoid content in the callus of different subculture times was compared and the antitumor activity of them was evaluated by using MTT cell proliferation and cytotoxicity assay. FCM confirmed similar ploidy level in mother plant and all callus, but some changes were observed in the ISSR and SRAP patterns, of which 10.3 and 6.06 % were polymorphic, respectively. The somatic variation frequency tended to increase with the increasing subculture time in the early stage of subculture, with a peak in the 4–6 times and then kept nearly at a constant after 8 times. High IAA content may be one reason for this variation by this period. The flavonoid content and antitumor potential increased and remained at high level during the 2nd and 6th subculture. Though there was a significant decline after the 6th subculture, they still had about the same flavonoid content and antitumor potential as those of mother plant. The results suggested that the in vitro callus of T.hemsleyanum is potentially useful for pharmaceutical uses. For supplying true-to-true seedlings, germplasm conservation and further genetic transformation, subculture frequency of callus should be controlled within 4 times to obtain clonally identical plantlets. However, for the development of germplasm improvement of the species, subculture frequency should be controlled in 6–8 times to make better use of somatic variations.

Inter-simple sequence repeat (ISSR) markers in the evaluation of genetic polymorphism of Egyptian Capsicum L. hybrids

DNA samples of six hybrids of Capsicum annuum and Capsicum frutescens were analyzed with ten inter-simple sequence repeat (ISSR) primers, which produced 52 polymorphic bands out of 87 bands with polymorphism average of 60%. ISSR patterns scored five distinguishable species-specific bands; two for C. frutescens and three for C. annuum and 16 unique bands for hybrids individually that were considered as molecular markers for Egyptian hybrids. The genetic dendrogram among the materials constructed by the unweighted pair-group method with arithmetic average based on the Dice coefficient showed that the six hybrids can be separated into two groups with genetic distance at 0.25. The first cluster included hybrids of C. frutescens and the other grouped those of C. annuum indicating narrow genetic base among the tested hybrids in both species. This study revealed considerable genetic diversity in the different Capsicum hybrids for conservation of genetic resources and efficient crop breeding programs.

No Indication of Strict Host Associations in a Widespread Mycoparasite: Grapevine Powdery Mildew (Erysiphe necator) Is Attacked by Phylogenetically Distant Ampelomyces Strains in the Field

Pycnidial fungi belonging to the genus Ampelomyces are common intracellular mycoparasites of powdery mildews worldwide. Some strains have already been developed as commercial biocontrol agents (BCAs) of Erysiphe necator and other powdery mildew species infecting important crops. One of the basic, and still debated, questions concerning the tritrophic relationships between host plants, powdery mildew fungi, and Ampelomyces mycoparasites is whether Ampelomyces strains isolated from certain species of the Erysiphales are narrowly specialized to their original mycohosts or are generalist mycoparasites of many powdery mildew fungi. This is also important for the use of Ampelomyces strains as BCAs. To understand this relationship, the nuclear ribosomal DNA internal transcribed spacer (ITS) and partial actin gene (act1) sequences of 55 Ampelomyces strains from E. necator were analyzed together with those of 47 strains isolated from other powdery mildew species. These phylogenetic analyses distinguished five major clades and strains from E. necator that were present in all but one clade. This work was supplemented with the selection of nine inter-simple sequence repeat (ISSR) markers for strain-specific identification of Ampelomyces mycoparasites to monitor the environmental fate of strains applied as BCAs. The genetic distances among strains calculated based on ISSR patterns have also highlighted the genetic diversity of Ampelomyces mycoparasites naturally occurring in grapevine powdery mildew. Overall, this work showed that Ampelomyces strains isolated from E. necator are genetically diverse and there is no indication of strict mycohost associations in these strains. However, these results cannot rule out a certain degree of quantitative association between at least some of the Ampelomyces lineages identified in this work and their original mycohosts.

Intraspecies diversity of Macrophomina phaseolina in Iran

To study the pathogenic and genetic diversity of the Macrophomina phaseolina in Iran, 52 isolates of the fungus were isolated from 24 host plants across the 14 Iranian provinces. All isolates were confirmed to the species based on the species-specific primers. The aggressiveness of M. phaseolina isolates was evaluated on the common bean. Based on the pathogenicity tests, M. phaseolina isolates from the different hosts displayed different levels of aggressiveness on the common beans. The results showed that there was significant variation in the aggressiveness of the pathogen; however, there was no distinct pattern of differentiation based on the host or geographical origin linked to the virulence of the isolates, as frequently theisolates from the same host or geographical origin had different levels of aggressiveness. Inter-simple sequence repeat (ISSR) markers were used to assess the genetic diversity of the fungus. The unweighted pair-group method, using arithmetic mean clustering of data, showed that isolates did not clearly differentiate to the specific group according to the host or geographical origins; however, usually the isolates from the same host or the same geographical origin tend to group nearly. Our results did not show a correlation between the genetic diversity based on the ISSR and pathogenic patterns on common bean in the greenhouse. Similar to the M. phaseolina populations in the other countries, the Iranian isolates were highly diverse based on the pathogenic and genotypic characteristics.

Monitoring of cultivar identity in micropropagated olive plants using RAPD and ISR markers

Randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers were applied to assess the genetic stability of micropropagated olive (Olea europaea L. cv. Maurino) plants regenerated by axillary buds. Initial olive explants, isolated from one donor tree, were multiplied on Murashige and Skoog medium for 12 repeated subcultures. A total of 40 RAPD and 10 ISSR markers resulted in 301 distinct and reproducible band classes showing homogeneous RAPD and ISSR patterns. The amplification products revealed genetic stability among the micropropagated plants and between them and the donor plant. The results demonstrate the genetic stability of nine year old mature micropropagated olive plants cultured in field, and corroborated the fact that axillary multiplication is the safest mode for multiplication of true to type plants.

Monitoring of cultivar identity in micropropagated olive plants using RAPD and ISSR markers

Randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers were applied to assess the genetic stability of micropropagated olive (Olea europaea L. cv. Maurino) plants regenerated by axillary buds. Initial olive explants, isolated from one donor tree, were multiplied on Murashige and Skoog medium for 12 repeated subcultures. A total of 40 RAPD and 10 ISSR markers resulted in 301 distinct and reproducible band classes showing homogeneous RAPD and ISSR patterns. The amplification products revealed genetic stability among the micropropagated plants and between them and the donor plant. The results demonstrate the genetic stability of nine year old mature micropropagated olive plants cultured in field, and corroborated the fact that axillary multiplication is the safest mode for multiplication of true to type plants.

Genetic and epigenetic relationship in rye, Secale cereale L., somaclonal variation within somatic embryo-derived plants

In vitro regenerated plants of rye, Secale cereale L., Ailes and Merced cultivars, were studied to verify if genetic and/or epigenetic changes were promoted by in vitro conditions. Inter-simple sequence repeat (ISSR) fingerprints on HpaII/MspI-digested and uncut DNA were generated. DNA digested with methylation-sensitive isoschizomers revealed epigenetic modifications, while modification of ISSR patterns obtained with undigested DNA indicated genetic changes. With this technique, it was possible to study both genetic and/or epigenetic changes within the same DNA sequences. The frequency of plants with at least one variation was high: 73% and 30% of rye plants showed at least one genetic change, and 50% and 73% carried at least one methylation change, in the Ailes and Merced cultivars, respectively. Further analyses revealed that a considerable number of variable markers showed both types of modifications, indicative of both genetic and epigenetic changes. Moreover, genetic variation was related to the presence of the CCGG target in the analyzed bands. These results indicate the possible existence of a common mechanism connecting both types of variation.

Ascertaining clonal fidelity of tissue culture raised plants of Bambusa balcooa Roxb. using inter simple sequence repeat markers

One of the major applications of plant tissue culture technology is that the regenerants are genetically identical to the mother plant. Although, of the various methods of in vitro propagation, axillary branching method is regarded as the safest, the possibility of genetic variation (somaclonal variation) cannot be ruled out even in this method. In the present study, inter simple sequence repeat (ISSR) marker assay was employed to validate the clonal fidelity of in vitro raised Bambusa balcooa plantlets multiplied by enhanced axillary proliferation up to 33 passages. Fifteen ISSR primers generated a total of 99 amplicons among the tissue-cultured progenies. Analysis of ISSR patterns revealed that the bands were shared by both the parent clump and the in vitro raised plants confirming the genetic stability in the latter.

Somatic hybrids obtained by asymmetric protoplast fusion between Musa Silk cv. Guoshanxiang (AAB) and Musa acuminata cv. Mas (AA)

To attempt to introduce genetic information of disease resistance from Musa acuminata cv. Mas (AA) to Musa silk cv. Guoshanxiang (AAB) and obtain somatic hybrids, we developed an asymmetric protoplast fusion with 20% (w/v) polyethylene glycol (PEG). The protoplasts derived from embryogenic suspension cultural cells of cv. Guoshanxiang (AAB) and cv. Mas (AA) were, respectively treated with 1.5 mM iodoacetamide (IOA) and with ultraviolet light (UV) at an intensity of 50 W/m2 for 120 s. A total of 47 regenerated green plants were obtained and eight of which were survived in greenhouse. Six of the survived plants were identified as hybrids by RAPD analysis and only three hybrids were retained vigorously in field. The hybrid nature of the three plants was further confirmed according to their ISSR (inter-simple sequence repeat) patterns and the results indicated that they were true somatic hybrids. Chromosome analysis revealed that the three hybrids possessed an aneuploid chromosome number (2n = 34).

Genetic and phenotypic diversity of geographically different isolates ofGlomus mosseae

In this work, we combined morphological taxonomy and molecular methods to investigate the intraspecific diversity of Glomus mosseae, whose global distribution has been reviewed by a survey of scientific literature and Web-available records from international germplasm collections (International Culture Collection of Vesicular Arbuscular Mycorrhizal Fungi and International Bank of Glomeromycota). We surveyed 186 publications reporting the occurrence of G. mosseae from at least 474 different sites from 55 countries throughout all continents, producing a geographical map of their distribution. The relationships among G. mosseae isolates originating from Europe (United Kingdom), the United States (Arizona, Florida, and Indiana), Africa (Namibia), and West Asia (Syria) were analyzed. The level of resolution of internal transcribed spacer (ITS) sequences strongly supports the morphological species definition of G. mosseae. An ITS - restriction fragment length polymorphism assay with the enzyme HinfI yielded a unique profile for all G. mosseae isolates, allowing a straightforward identification of this morphospecies. Genetic variability among G. mosseae isolates was revealed by the inter-simple-sequence repeat (ISSR) - polymerase chain reaction: the magnitude of genetic divergence shown by the investigated geographical isolates was higher than 50%, consistent with previous data on vegetative compatibility and functional diversity. The variability of ISSR patterns suggests that intraspecific diversity is much higher than that foreseen by morphology and rDNA regions, and should be further investigated by using other genes, such as those related to functional diversity.

Phylogeography and Demographic History of Chinese Black-Spotted Frog Populations (Pelophylax nigromaculata): Evidence for Independent Refugia Expansion and Secondary Contact

BackgroundPleistocene glaciations had considerable impact on phylogeographic patterns within and among closely related species of many vertebrates. Compared to Europe and North America, research on the phylogeography of vertebrates in East Asia, particularly in China, remains limited. The black-spotted frog (Pelophylax nigromaculata) is a widespread species in East Asia. The wide distribution of this species in China makes it an ideal model for the study of palaeoclimatic effects on vertebrates in East Asia. Our previous studies of P. nigromaculata revealed significant subdivisions between the northeast China populations and populations in other regions of the mainland. In the present study, we aim to see whether the deepest splits among lineages and perhaps subsequent genealogical divisions are temporally consistent with a Pleistocene origin and whether clade geographic distributions, with insight into expansion patterns, are similarly spatially consistent with this model.ResultsUsing 1143 nucleotides of the mitochondrial cytochrome b gene from 262 individuals sampled from 28 localities, two main clades (clade A and clade B) differing by c. 7.72% sequence divergence were defined from parsimony analyses. The corresponding timing of lineage divergence, 0.92 Mya, indicates a most likely Pleistocene split. The A clade is further subdivided into two sub-clades, A1 and A2 with 1.22% sequence divergence. Nested clade phylogeographical and population demographic analyses suggested that the current distribution of this frog species was the result of range expansion from two independent refugia during the last interglacial period. We discovered a population within which haplotype lineages A and B of P. nigromaculata coexist in the Dongliao area of China by nucleotide sequences, PCR-RFLP and ISSR (inter simple sequence repeat) patterns. The ISSR result in particular supported divergence between the mitochondrial clades A and B and implied introgressive gene flow between the two divergent lineages.ConclusionNested clade phylogeographical and population demographic analyses indicate that the current distribution of P. nigromaculata is the result of range expansion from two independent refugia during the last interglacial period in late Pleistocene. One refugium was in east China and the lower elevations of south-western plateau. The distribution of the other mitochondrial clade is consistent with the presence of a refugium in the Korean Peninsula. The gene flow as detected by ISSR markers suggests a range expansion of the two refugia and a secondary contact between the two highly divergent lineages in the Dongliao (DL) area of northeast China.

Genetic Diversity and Phylogenetic Relationships among Plum Germplasm Resources in China Assessed with Inter-simple Sequence Repeat Markers

Inter-simple sequence repeat (ISSR) markers were used to evaluate genetic similarity and interrelationship among 104 plum (Prunus L. spp.) and related accessions from the Chinese National Germplasm Repository for Plums and Apricots and the Tianshan Germplasm Repository for Wild Fruit Resources, including six plum species (Prunus salicina Lindl., Prunus simonii Carr., Prunus ussuriensis Kov. et Kost., Prunus domestica L., Prunus cerasifera Ehrh., and Prunus spinosa L.), two related species [apricot (Prunus armeniaca L.) and nanking cherry (Prunus tomentosa Thunb.)], eight putative hybrids between plum and apricot (plumcot), and six accessions of wild European plum (P. domestica). Out of the 42 ISSR primers, 12 were selected, which generated 103 markers in total, 99 of which were polymorphic. Possible accession-specific ISSR bands or patterns were also found. Some possible synonyms or homonyms were clarified or discussed, and closely related accessions such as bud mutants were discriminated. Based on the unweighted pair group method with arithmetic mean (UPGMA) analysis and principal coordinate analysis (PCoA) using the Jaccard coefficient, two different dendrograms were constructed—one including accessions grouped by species and one with all 104 accessions—and a two-dimensional plot was obtained. Three groups were formed in both dendrograms and PCoA plot: Group I including apricot (‘Yinxiangbai’) and plumcot types; Group II containing Asia-originated diploid species [e.g., P. cerasifera, P. ussuriensis, P. tomentosa, and Chinese plum-types (i.e., P. salicina and its hybrids)]; and Group III involving European-origin polyploid species (e.g., P. spinosa and P. domestica) and recently found wild European plum accessions in China. The dendrogram with accessions grouped by species implied that 1) plumcot types had closer relatedness with apricot than with plum; 2) P. simonii should be a variant of P. salicina while P. ussuriensis an independent species; 3) P. domestica was more closely related to P. spinosa than to P. cerasifera. Two accessions of European plum (‘89-7-3’ and ‘Wanhei’) were clustered into outgroups in the dendrogram with all 104 accessions, which could been grouped within Group III in the PCoA plot. The distribution of both European plum and Chinese plum-types across respective groups did not reflect the geographic origins. The present study also further confirmed that the wild plants found in Xinjiang of China were P. domestica.

Assessing the genetic diversity of tobacco germplasm using intersimple sequence repeat and inter‐retrotransposon amplification polymorphism markers

AbstractThe genetic diversity of 118 tobacco accessions, including flue‐cured tobacco, sun‐/air‐cured tobacco, burley tobacco, oriental tobacco and wild tobacco, was characterised using intersimple sequence repeat (ISSR) and inter‐retrotransposon amplification polymorphism (IRAP) markers. ISSR and IRAP banding patterns and genetic distance (GD) values showed the low level of genetic diversity within and among cultivated tobacco types. There was higher GD and average heterozygosity among wild tobacco types than those among cultivated tobacco. Genetic diversity of tobacco germplasm was low, with a high level of genetic identity (>0.77) between the different types. However, neighbour‐joining cluster analysis of marker‐based GDs showed that the accessions from the same tobacco type, as classified by manufacturing quality traits, were nearly clustered into the same group. These results will help in the formulation of appropriate strategies for variety improvement in tobacco, and ISSR and IRAP markers of the genetic diversity will contribute to further study and improvement of tobacco.

Use of ISSR fingerprints to detect microsatellites and genetic diversity in several related Brassica taxa and Arabidopsis thaliana.

Use of ISSR fingerprints to detect microsatellites and genetic diversity in several related Brassica taxa and Arabidopsis thaliana.

Inter-Simple Sequence Repeat fingerprints to assess genetic diversity in Tunisian fig (Ficuscarica L.) germplasm

The genetic diversity of 18 Tunisian fig cultivars was investigated at the DNA level using the Inter Simple Sequence Repeat (ISSR) associated with the Polymerase Chain Reaction (PCR). Using a set of primers, the most informative ones were selected that were characterized by an important Resolving power value of 29.6. A total of 47 discernible fragments were scored from samples, with a mean of 11.7 fragments per primer. The 90.4% of sample that were polymorphic were scored as molecular markers to examine the Tunisian fig germplasm polymorphism at DNA level. A large genetic diversity as related to ISSR patterns was found within the local Tunisian fig germplasm. An UPGMA dendrogram exhibits the unstructured variability in this crop. Moreover, the principal component analysis shows that the observed diversity was typically continuous. Our data provide a large number of ISSR markers that are useful in the fingerprinting of Ficus carica L. cultivars, and in the understanding of the genetic relationships among these accessions.

Statistical correlations of primer thermodynamic stability DeltaG degrees for enhanced flax ISSR-PCR cultivar authentication.

We revealed four statistically significant correlations related to inter-simple-sequence repeat (ISSR) patterns: (1) between thermodynamic free energy DeltaG degrees of ISSR primer sequence and PCR reamplification intensity (dA(i)), (2) between free energy DeltaG degrees of ISSR primer sequence and PIC coefficient quantifying the polymorphism of ISSR patterns, (3) and (4) between free energy DeltaG degrees of anchor sequence of primer and the number of total, and polymorphic bands in ISSR patterns, respectively. Methodological recommendations for effective ISSR primer design were inferred based on revealed correlations. In particular, free energy of ISSR primer sequence is recommended to be DeltaG degrees > 160 kJ/mol of interaction and free energy of flanking anchor sequence in primer to be around DeltaG degrees = 28 kJ/mol of interaction to produce ISSR patterns displaying maximum polymorphism of flax germplasm.

ISSR-based clustering of cultivated flax germplasm is statistically correlated to thousand seed mass.

Inter-simple sequence repeat (ISSR)-polymerase chain reaction (PCR) polymorphism was generated to provide useful markers for assessment of genetic diversity within flax germplasm collections. We used nine previously selected anchored ISSR primers for fingerprinting of 53 flax cultivars or genotypes and obtained 62 scorable bands, from which 45 bands (72.6%) were polymorphic. An efficient separation of 53 flax accessions into four groups and eight subgroups was achieved using unweighted pair group method with arithmetic means (UPGMA) clustering procedure based on genetic similarity expressed by the Jaccard similarity coefficient (JSC). Clustering procedure within both groups and subgroups successfully produced smaller homogenous clusters, whereas clustering between the main four groups of flax accessions displayed only a continuous decrease of similarity with a weak clustering effect. Statistical significance of grouping and subgrouping within a cluster dendrogram was estimated by calculation of the error flag and cophenetic correlation parameter for each branch. Principal coordinates (PCO) analysis mostly confirmed the separation by UPGMA clustering. We observed a statistically significant correlation between the number of total vs polymorphic bands in ISSR patterns. A one-way analysis of variance (ANOVA) test confirmed statistically significant differences in the average thousand seed mass (TSM) between eight subclusters of flax accessions from an ISSR-PCR-based UPGMA dendrogram, which indicate statistical correlation between flax ISSR polymorphism (the structure of ISSR-based clustering) TSM.