ISSR Method Research Articles

Evaluation of yarrow (Achillea) accessions by phytochemical and molecular genetic tools

Yarrow ( Achillea ) species are known and utilized worldwide. In the recent study our primarily goal was to get information about the intraspecific diversity of A. collina in the Carpathian Basin. Five cultivated genotypes and six populations of wild origin were compared involving seven other species as control. Essential oil (EO) and proazulene (PA) contents were determined and the DNA samples were evaluated by RAPD (11 primers) and ISSR (12 primers) methods. The EO content varied between 0.010 ( A. distans ) and 0.365 ( A. collina ) ml/100g DW, the PA content was found between 0.021 and 0.173% DW. The used RAPD markers provided 140 bands (97.14% polymorphic). They distinguished primarily among species and less characteristically among the A. collina populations. With ISSR primers we detected 188 bands (97.34% polymorphic). ISSR markers and combined RAPD and ISSR method enabled an informative intraspecific evaluation of A. collina accessions. The largest genetic distances were found between A. ptarmica and the members of sect. Achillea (genetic distances 0.52-0.72). Similarity is highest (genetic distance 0.27) among the populations of lower geographical distances. Nei’s genetic distances of cultivated populations are also relatively low (0.23- 0.36). Some wild accessions may represent valuable biological resources for breeding.

Genetic variation, population structure, and differentiation in scots pine (Pinus sylvestris L.) from the northeast of the Russian plain as inferred from the molecular genetic analysis data

The DNA polymorphism in Pinus sylvestris from Severodvinsk, Upper Vetluga, and Vetluga-Vyatka populations, which were isolated earlier based on specific features of the geographic variation of allometric cone indices, was examined by the ISSR method. It was demonstrated that the Severodvinsk population of P. sylvestris differed from the chorologically adjacent Upper Vetluga population with respect to all of the examined genetic indices, and the Upper Vetluga population differed from the Vetluga-Vyatka population. It was suggested that the main the reason for the lack of statistically significant differences between Upper Vetluga and Vetluga-Vyatka samples of P. silvestris with respect to genetic variation indices (P95, H(E), n(a), n(e)) may be their formation based on the gene pools of two glacial refugia. It was demonstrated that the proportion of the interpopulation component of total genetic diversity (G(ST)), as calculated based on the ISSR marker polymorphism, reached a value of 0.488, which was an order of magnitude higher than the previous estimates obtained based on data from allozyme analysis. It was concluded that P. sylvestris cone allometric indices reflected the specificity of spatial population subdivision, like the genetic diversity and population genetic structure indices calculated based on ISSR-marker polymorphism. Population isolation and mapping based on two-step phenogenetic studies is suggested.

Allium canariense (Amaryllidaceae), a species endemic to the Canary Islands

A revision of the Allium species of the section Molium in the Canary Islands is presented. As part of the phylogenetic revision of the Eurasian representatives of the subgenus Amerallium we found large disagreements in the nomenclature and taxonomy of Allium roseum in the floristic publications on the flora of the Canary Islands. At least four species of the section Molium are mentioned for the flora of the Canary Islands: Allium roseum, A. subvillosum, A. subhirsutum and A. trifoliatum. To learn more about the phylogenetic relationships within a group of closely related species of the section Molium, we used maximum parsimony and Bayesian analyses of combined nuclear (ITS—internal transcribed and ETS—external transcribed spacers of rRNA genes) and three chloroplast (rpl32-trnL and trnL-trnF intergenic spacers and rps16 intron) datasets of 7 taxa. For comparison of the relationships of A. canariense populations between the islands we used the ISSR method. We found that only one species of the section Molium occurs in the Canary Islands—the endemic species Allium canariense, closely related to Allium subvillosum. Independence of this new species has been confirmed by morphological and molecular features. Discussion on phylogeny, origin and geographic distribution is provided.

Rhaponticum carthamoides regeneration through direct and indirect organogenesis, molecular profiles and secondary metabolite production

The organogenic competence of different explants of Rhaponticum carthamoides was investigated on MS agar medium supplemented with BA, IBA or NAA at concentrations of 0.2 and 0.5 mg L−1. Adventitious shoot formation was obtained through direct organogenesis using leaves of in vitro cultures as explants and through indirect organogenesis when seedling explants (hypocotyl, cotyledon and root) were used for regenerative callus initiation. The shoots were rooted on half-strength MS medium (½ MS) without auxin or containing IBA (0.2–2.0 mg L−1). The plantlets regenerated through direct and indirect organogenesis were transferred into pots and grown in the greenhouse for 3 months. Significant differences in morphology, accumulation of chlorogenic acid and 20-hydroxyecdysone (20-HE) as well as in genetic profile were observed between these plants. UHPLC analysis showed that the highest level of chlorogenic acid (12 mg g−1 DW) was found in leaves of plants developed directly from explants, whereas leaves of plants developed via callus tissue accumulated the highest amount of 20-HE (7.4 mg g−1 DW). Its level exceeded that detected in leaves of 3-month-old plants obtained from seeds (2.4 mg g−1 DW). Genetic variations of R. carthamoides regenerated plants were evaluated by flow cytometry and RAPD and ISSR methods. Flow cytometry confirmed similar ploidy level in the mother plant and plants regenerated through direct and indirect organogenesis. Genetic similarity values calculated on the basis of RAPD and ISSR data among regenerated in vitro plants to the mother plant were ranged from 0.765 to 0.941 and 0.647 to 0.947, respectively.

Assessing the genetic diversity and resistance to Cucumber mosaic virus and Tobacco etch virus of tobacco cultivars in China

Control of Cucumber mosaic virus (CMV) and Tobacco etch virus is difficult (TEV) mainly because of the lack of resistant tobacco varieties. In this study, the resistance to CMV and TEV of 22 tobacco cultivars, grown under both greenhouse and field conditions, was evaluated from 2007 to 2012. The genetic relationships among these cultivars were also assessed using ISSR methods. Based on the disease index and DAS-ELISA results, one cultivar (Coker86) was characterized as resistant to CMV, and one cultivar (Shuangkang70) was characterized as resistant to TEV. A total of 145 repeatable amplified bands were generated with six ISSR primers, of which 139 were polymorphic. The tobacco cultivars were grouped into five major clusters according to phylogenetic analysis; the similarity coefficient between cultivars ranged from 0.476 to 0.855. This study provides an overall assessment of tobacco cultivars and the results should prove useful for both the control of tobacco virus diseases at present and the improvement of tobacco varieties in future breeding projects.

Genetic diversity of the endangered Faucaria tigrina (Aizoaceae) through ISSR “fingerprinting” using automated fragment detection

Faucaria tigrina (Haw.) Schwantes is a rare, threatened succulent found exclusively on the outskirts of Grahamstown, Eastern Cape, South Africa. There are three extant populations of the species; two large ones (approximately 300 and 620 adults) separated by under two kilometres, and a third very much smaller one slightly further away. Genetic theory expects that smaller, isolated populations face a loss in genetic variation through inbreeding and genetic drift and that as declining genetic variation is linked to a loss in fitness, this species may face extinction in the long term. This study used the ISSR-PCR genetic methods linked to an automated detection platform to determine if these populations are genetically distinct, and whether they are genetically depauperate. Two ISSR primers were used, and the automated detection system identified a total of 572 ISSR loci. An UPGMA clustering analysis showed each population to be genetically distinct, and that the genetic diversity of each population does not appear to be particularly low. F. tigrina is separable from the related Faucaria britteniae L. Bolus using the ISSR method, suggesting that this method may be appropriate for systematic studies in the Aizoaceae, which comprises many genera with closely related species.

Evaluation of genetic variability, rust resistance and marker-detection in cultivated Artemisia dracunculus from Iran

Artemisia dracunculus L. (tarragon), a small shrubby perennial herb, is cultivated for the use of its aromatic leaves in seasoning, salads, etc., and in the preparation of tarragon vinegar. In the present work, genetic analysis of 29 cultivated individuals of this species was carried out employing 12 ISSR and 11 SRAP markers. A total of 59 (71.64%) and 79 (83.14%) polymorphic bands were detected by 12 ISSR primers and 11 SRAP primer pairs, respectively. High similarity for patterns of genetic diversity and clustering of individuals was observed using two ISSR and SRAP marker systems and combined data. Range of genetic similarity by ISSR markers was 0.14 to 0.95, by SRAP markers was 0.14 to 0.90, while this range varied from 0.18 to 0.91 by ISSR+SRAP. In the UPGMA cluster analysis (ISSR, SRAP and ISSR+SRAP), we always found two clusters, the first cluster included 22 individuals and the second contained seven individuals. The results demonstrated that both ISSR and SRAP methods were suitable for discriminating among the studied individuals and the SRAP markers were more efficient and preferable. The results of multiple regression analysis revealed statistically significant association between rust resistance and some molecular markers that they can provide clues for identification of the individuals with higher rust resistance. The molecular marker-based study of genetic diversity suggests that the germplasm studied representing the kind of variability would be a valuable genetic resource for future breeding. In addition, in situ conservation measures are recommended to preserve the valuable A. dracunculus genetic resources as the most effective and economical approach.

Detecting Genetic Diversity of <I>Dendrobium</I> Tissue Culture Seedlings with ISSR Method

Detecting Genetic Diversity of <I>Dendrobium</I> Tissue Culture Seedlings with ISSR Method

Methodology Analysis of genetic diversity in Larix gmelinii (Pinaceae) with RAPD and ISSR markers

Dahurian larch (Larix gmelinii), a deciduous conifer, is the northernmost tree, native to eastern Siberia and nearby regions of China. We used growth traits and molecular markers to assess genetic variation in different L. gmelinii growing regions; 105 individual samples were collected from seven regions of the Qingshan Forestry Centre, Heilongjiang Province, China. The greatest genetic regional variation was seen in the Youhao area, based on coefficients of variation for tree height, diameter and volume (14.73, 28.25, and 55.27%, respectively). Analysis using molecular markers showed rich genetic diversity. The RAPD and ISSR methods both indicated that most variation came from within populations. The seven regions were divided into two groups (Daxing'an and Xiaoxing'an Mountain ranges) by RAPD cluster analysis: Tianchi, Xiaojiuya, Yuanjiang, and Taiping regions were placed in the first group at a genetic distance of 0.08; while the other regions were in the second group. The correlation between RAPD markers and geographical distance was significant, with a correlation coefficient of 0.752.

In situ spine sampling and Inter-Simple Sequence Repeat (ISSR) PCRs proved relevant methods for molecular studies on the sea urchin Paracentrotus lividus

In France, there are raising concerns about the edible sea urchin Paracentrotus lividus, whose populations decline. In this context, molecular analyses contribute to the better understanding of processes that shape populations, which is required to adapt management and conservation programmes. The present study aimed at developing an in situ non-destructive spine sampling method and an ISSR-PCR approach, both dedicated to the genetic analysis of individuals and populations. Forty-five individuals of 3 close populations were studied. In situ spine sampling proved relevant, leading to the clear genotyping of all purified DNAs without any contamination. Molecular analysis revealed high genetic diversity with a large proportion of low-frequency ISSR markers. Six ISSR primers produced 154 scorable polymorphic markers of which 118 (77%) were present at a frequency lower than 25%. Based on genotyping profiles obtained with 3 ISSR primers, significant genetic differentiation between the studied populations was detected. Taken together, these results revealed that both in situ spine sampling and ISSR method are relevant for molecular analyses and that they will likely find their application in future conservation and management programmes.

Genetic diversity and gentiopicroside content of four Gentiana species in China revealed by ISSR and HPLC methods

Abstract G. macrophylla pall, G. dahurica Fisch, G. straminea Maxim, and G. crassicaulis Duthie ex Burk are the major cultivated Gentiana species in Gansu Province, China. The present study has collected 107 wild samples from 19 natural populations and focused on the analysis of their medicinal ingredients, genetic diversity, and relationship, concerning four species’s worrying situation and solutions. High performance liquid chromatography (HPLC) results show that their gentiopicroside contents are 9.75 ± 2.0%, 9.29 ± 2.6%, 8.18 ± 3.2%, and 7.96 ± 3.2% respectively and higher than the criterion standard for Radix Gentianae in Chinese Pharmacopoeia. Low genetic polymorphism was found among these four species with HE (expected heterozygosity) = 0.147, GST (interspecies coefficient of gene differentiation) = 0.3207, and Nm (gene flow) = 1.0593. The unweighted pair group method with arithmetic mean (UPGMA) analysis was performed with POPGENE 1.31 software and Nei’s method but did not clearly differentiate these four species. Their medicinal ingredients did not correlate with the genetic variants (r = −0.179, P = 0.586). The results indicate that there is no significant difference among four species in regard to the traits of medicinal qualities. All four Gentiana species can be widely cultivated to not only meet the demand of domestic and international market for Chinese traditional medicine, but also help the local conservation of the wild Gentiana resources in China.

Genetic diversity within and among the wild populations of Murraya koenigii (L.) Spreng., as revealed by ISSR analysis

Abstract Murraya koenigii (L.) Spreng., commonly known as curry leaf plant, is found in the different hilly regions of India. In the present study, fifty-nine accessions representing eight wild populations of M. koenigii were analyzed using thirteen ISSR primers. A total of 152 bands were amplified, out of which, 136 were polymorphic corresponding to 89.47% polymorphism across the accessions. The pairwise population genetic distances were calculated for all the populations that varied from 0.05 to 0.13 between the populations of M. koenigii . AMOVA and Nei’s genetic diversity analysis revealed higher genetic variations within populations than among the populations. The clustering of populations in the dendrogram was not in congruence with geographical affiliations. The results indicate that the ISSR method is sufficiently informative and powerful to estimate the genetic diversity in M. koenigii populations. As M. koenigii is an important wild plant genetic resource, therefore, information on genetic variability might be a potential source as breeding material for development of commercially valuable traits in M. koenigii plants.

Application of the ISSR method to estimate the genetic similarity of Dasypyrum villosum (L.) P. Candargy Greek populations to Triticum and Secale species

Application of the ISSR method to estimate the genetic similarity of Dasypyrum villosum (L.) P. Candargy Greek populations to Triticum and Secale species In the study, the genetic similarity between Dasypyrum villosum L. (P.) Candargy and Triticum L. and Secale L. species was studied on the basis of ISSR markers. As a very polymorphic, effective and reproducible method, ISSR can be successfully employed to evaluate polymorphism between and inside different species. The polymorphic information content values (PIC) of ISSR method ranged from 0.57 to 0.87, with the mean value of 0.7. The genetic similarity of the forms analyzed ranged from 0.27 to 0.97, with the mean value of 0.47, indicating their high diversity. A higher similarity of Dasypyrum villosum to Triticum species, in comparison with Secale was found - the mean Dice genetic similarity index between genera was calculated at 0.40 for Dasypyrum and Triticum, and at 0.31 for Dasypyrum and Secale.

Comparative genomic analysis of vibrio cholerae el tor preseventh and seventh pandemic strains isolated in various periods

To reveal sites of the donor genome in wheat crossed with Aegilops cylindrica, which acquired conferred resistance to fungal diseases, a comparative analysis of introgressive and parental forms was conducted. Two systems of PCR analysis, ISSR and SSR-PCR, were employed. Upon use of 7 ISSR primers in genotypes of 30 individual plants BC1 F9 belonging to lines 5/55-91 and 5/20-91, 19 ISSR loci were revealed and assigned to introgressive fragments of Aegilops cylindrica genome in Triticum aestivum. The 40 pairs of SSR primers allowed the detection of seven introgressive alleles; three of these alleles were located on common wheat chromosomes in the B genome, while four alleles, in the D genome. Based on data of microsatellite analysis, it was assumed that the telomeric region of the long arm of common wheat chromosome 6A also changed. ISSR and SSR methods were shown to be effective for detecting variability caused by introgression of foreign genetic material into the genome of common wheat.

Characterization of Czech hop (Humulus lupulus L.) genotypes by molecular methods

In the last year, new hybrid hop (Humulus lupulus L.) variety Agnus was released for cultivation in the Czech Republic. It has been necessary to prepare the quality system of Agnus identification from other Czech genotypes and characterise the germplasm of this variety by molecular methods. We proved that utilization of five STS primer combinations successfully and completely identified and determined Czech released varieties and new promising breeding materials. The use of STS method was also very effective and sensitive for control of authenticity and purity of variety Agnus in multipropagation cycle. The study of genetic diversity of 61 hop varieties by RAPD, STS, ISSR and AFLP methods confirmed, that germplasm of variety Agnus has ranked among high-alfa varieties. The results can be successfully used for identification, germplasm management, genetic studies and breeding purposes by breeders, multipropagators and hop growers.

Comparison of RAPD, STS, ISSR and AFLP molecular methods used for assessment of genetic diversity in hop (Humulus lupulus L.)

The cluster analysis of ten hop (Humulus lupulus L.) varieties and the comparison of amplified DNA polymorphism were assessed using RAPD, STS, ISSR and AFLP molecular methods. Thirty-five RAPD primers generated an average of 13.4 products with 5.7 polymorphic products (42.3% of polymorphism). Ten STS primer combinations generated an average of 9.3 products with 6.6 polymorphic products (71% of polymorphism). Seven ISSR microsatellite polymorphic primers and seven primer combinations generated an average of 12.9 products with 4.2 polymorphic products (32.6% of polymorphism) in agarose gels and 86.2 products with 24.3 polymorphic products (28.3% of polymorphism) in polyacrylamide gels. A total of 56 AFLP primer combinations generated an average of 91 products with 52.5 polymorphic products (57.6% of polymorphism). All molecular methods accurately distinguished all tested varieties, except for Osvald’s clones (31, 72 and 114), which were only distinguished by AFLP. The similarities between varieties were revealed by cluster analyses. These analyses were very similar for all molecular methods. Pairwise values of JCS (Jaccard’s similarity coefficient) ranged from 0.02 to 1.0 and the mean JCS values ranged from 0.395 (AFLP) to 0.462 (ISSR). The highest correlation coefficient was between ISSR and RAPD (0.966) and the lowest was between AFLP and STS (0.867). STS had the highest correlation of chemical data with molecular methods (0.592) and ISSR had the lowest (0.435).