Inter-simple Sequence Repeat Marker System Research Articles

Bazı maydanoz (Petroselinum crispum Mill.) genotiplerinin moleküler karakterizasyonu

Parsley (Petroselinum crispum Mill.), a vegetable whose leaves are consumed, has many benefits for human health. The first step of parsley breeding is the characterization of existing genotypes. In this study, genetic diversity of 12 different parsley genotypes was determined by ISSR (Inter Simple Sequence Repeat) marker system. In the study, 41 of the 130 bands obtained from 16 ISSR primers were found to be polymorphic. The similarity coefficients ranged from 0.86–0.99 for ISSR. The average polymorphism was 31.5% and the number of bands varied between 4 and 14. While the genotypes that are genetically closest to each other were determined as the 5th and 6th genotypes, the most distant genotypes were the 3rd and 8th genotypes. Findings from the present study showed that there were genetic variations among the parsley genotypes examined. The obtained data will enable more effective utilization of the parsley genotypes, the genetic differences of which have been determined for the future breeding programs.

Genetic polymorphism detection in brazilian perennial cottons (Gossypium spp.) using an ISSR marker system and its application for molecular interspecific differentiation.

The semi-domesticated Brazilian perennial cotton (Gossypium spp.) germplasm is considered a source of variability for creating modern upland cotton varieties. Here we used Inter-simple Sequence Repeat (ISSR) markers to detect intra and interspecific genetic polymorphism in Gossypium hirsutum L. r. marie-galante and Gossypium barbadense L. and to use molecular data to assessing genetic diversity and molecular discrimination of these species. The sets contained 12 G. barbadense genotypes and 16 G. hirsutum genotypes from a Brazilian collection. The 11 ISSR primers were used for genotyping yielded 101 bands (polymorphism = 47.5%) and were classified as moderately informative (PIC = 0.304). The ISSR markers exposed a greater diversity in G. hirsutum (P = 24.72%; HE =0.071 and I = 0.111) as compared to G. barbadense (P = 17.98%, HE = 0.043 and I = 0.070). The AMOVA analysis showed that 89.47% of the genetic variation was partitioned within species which is supported by Nei's genetic differentiation (Gst = 0.598) and gene flow (Nm = 0.338), suggesting that strong reproductive barriers between species. The UPGMA Cluster Analysis, Principal Coordinate Analysis and Bayesian Model-Based Structural Analysis divided the 28 genotypes into two main clades consistent with the taxonomical delimitation. The ISSR marker system offers a new approach to determining molecular differences between two cotton species (G. hirsutum L. r. marie-galante and G. barbadense L.). This study can expand the molecular marker resources for the identification and improvement of our knowledge about the genetic diversity and relationships between perennial cotton genotypes.

Evaluation of accession structure and genetic diversity in Iranian milk thistle (Silybum marianum L.) by ISSR markers

The aim of this study was to investigate the genetic variations of Iranian Silybum marianum accessions using inter simple sequence repeat (ISSR) markers. Twenty-nine accessions from various Iran regions and a sample from Hungary were selected and evaluated by 19 ISSR primers. A clear banding pattern was produced by 9 primers and a total of 85 repeatable polymorphic bands were detected. ISSR7 and ISSR8 primers identified the most Polymorphic Information Content (PIC) with 100% among primers. In the tested accessions, polymorphism information content (0.45), polymorphic percentage (83 %), and Shannon?s information index (0.53) assessed a high level of genetic variation. Based on Jaccards distances, cluster analysis molecular traits with unweighted pair group method with arithmetic mean (UPGMA) method were taken into consideration, and accessions were grouped into nine clusters and confirmed by principal coordinate analysis. Four clusters were identified by a Bayesian structure analysis, and 12 individuals were maintained inside the admixed clusters. According to the findings, ISSR marker system can be considered as a powerful tool for detection of genetic diversity of accessions in S. marianum. The results indicate existence of a high variation among Iranian S. marianum accessions to start the breeding programs.

Morphological and Molecular Assessment of Genetic Diversity of Seven Species of the Genus Artemisia L. (Asteraceae)

Species belonging to the genus Artemisia have high economic value and are used in folk and modern medicine. Analysis and identification of their genetic relationships can help in efficient conservation and further utilization of Artemisia genetic resources. This study was conducted to evaluate the genetic diversity among seven species of the genus Artemisia using their morphological characteristics and inter-simple sequence repeat (ISSR)-based molecular markers. The morphology of leaf and achene was examined by light microscopy and scanning electron microscopy. Morphological and molecular data were analyzed using the multivariate statistical package program. There were obvious variations in the morphology of leaf and achene among the Artemisia species. The ISSR results showed a total of 173 amplified loci, consisting of 132 polymorphic loci and 41 unique loci. Polymorphism percentage ranged from 62.5 to 88.8% for nine primers. Mean polymorphism information content for ISSR markers was 0.88, which suggested the efficacy of the ISSR marker system in determining polymorphisms. Significant correlation (r = 0.56, P = 0.004) was observed between similarity coefficients of morphological parameters and ISSR data based on the Mantel test. A wide range of diversity was observed at both morphological and molecular levels among the species. Genetic analyses using ISSR allowed for the detection of relationships between various Artemisia species than was apparent by the analyses of morphological characteristics of these species. Moreover, a mixed pattern of grouping in cluster analysis was found, indicating the close affinities of species with each other and also that the section Artemisia is polyphyletic.

Investigation of the genetic variation of anise (Pimpinella anisum L.) using RAPD and ISSR markers

Anise (Pimpinella anisum L.) is an annual spice and medicinal plant belonging to the family Apiaceae. Only a limited number of anise breeding studies are available. Therefore, genetic variation among anise landraces has not been extensively examined with molecular markers. In this study, the molecular diversity of 15 Turkish anise landraces collected from different regions of Turkey and four foreign landraces obtained from Cyprus, Syria and France were analyzed using randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers. The discriminatory power of these markers was assessed by calculating various marker parameters, namely percent polymorphism, polymorphism information content (PIC), resolving power (RP), and marker index (MI). The genomic DNA of 19 anise landraces was amplified with nine RAPD primers that generated 71 polymorphic bands and five ISSRs primers that produced 45 polymorphic bands. The percentage of polymorphic loci was 59.8% in RAPD profiling and 51.5% in ISSR profiling. The average PIC values obtained using RAPD and ISSR markers were 0.40 and 0.38, respectively. The mean RP (2.90 and 2.67) and MI values (1.23 and 1.05) of the RAPD assay were found to be somewhat higher than those of the ISSR assay, but ISSR was more favorable than the RAPD techniques in terms of presenting the phylogenetic relationships in anise landraces. A UPGMA cluster grouped the 19 anise landraces into two major clusters based on both markers. The RAPD and ISSR marker systems were found to be useful for determining genetic diversity in P. anisum L. and identifying variation among landraces.

Genetic variability in selected date palm (Phoenix dactylifera L.) cultivars of United Arab Emirates using ISSR and DAMD markers.

Nine (9) different date palm (Phoenix dactylifera L.) cultivars from UAE, which differ in their flower timings were selected to determine the polymorphism and genetic relationship between these cultivars. Hereditary differences and interrelationships were assessed utilizing inter-simple sequence repeat (ISSR) and directed amplification of minisatellite DNA region (DAMD) primers. Analysis on eight DAMD and five ISSR markers produced total of 113 amplicon including 99 polymorphic and 14 monomorphic alleles with a polymorphic percentage of 85.45. The average polymorphic information content for the two-marker system was almost similar (DAMD, 0.445 and ISSR, 0.459). UPGMA based clustering of DAMD and ISSR revealed that mid-season cultivars, Mkh (Khlas) and MB (Barhee) grouped together to form a subcluster in both the marker systems. The genetic similarity analysis followed by clustering of the cumulative data from the DAMD and ISSR resulted in two major clusters with two early-season cultivars (ENg and Ekn), two mid-season cultivars (MKh and MB) and one late-season cultivar (Lkhs) in cluster 1, cluster 2 includes two late-season cultivars, one early-season cultivar and one mid-season cultivar. The cluster analysis of both DAMD and ISSR marker revealed that, the patterns of variation between some of the tested cultivars were similar in both DNA marker systems. Hence, the present study signifies the applicability of DAMD and ISSR marker system in detecting genetic diversity of date palm cultivars flowering at different seasons. This may facilitate the conservation and improvement of date palm cultivars in the future.

ISSR에 의한 잔디속 식물의 DNA 다형성과 유전적 관계 평가

The genetic variability of four species of the genus Zoysia collected from South Korea was analyzed using an inter-simple sequence repeat (ISSR) marker system. Polymerase chain reactions (PCR) with eight ISSR primers generated 86 amplicons, 76 (87.1%) of which were polymorphisms. The polymorphism information content (PIC) value of the ISSR marker system was 0.848. The percentage of polymorphic loci (Pp) ranged from 41.2% to 44.7%. Nei’s gene diversity (H) ranged from 0.149 to 0.186, with an average overall value of 0.170. The mean of Shannon’s information index (I) value was 0.250. Total genetic diversity values (H T ) varied between 0.356 (ISSR-1) and 0.418 (ISSR-16), for an average overall polymorphic loci of 0.345. Interlocus variation in within-species genetic diversity (H S ) was low (0.170). On a per-locus basis, the proportion of total genetic variation due to differences among species (G ST ) was 0.601. This indicated that about 60.1% of the total variation was among species. Thus, about 39.9 of genetic variation was within species. The estimate of gene flow, based on GST, was very low among species of the genus Zoysia (N m = 0.332). The phylogenic tree showed three distinct groups: Z. macrostachya and Z. tenuifolia clades and other species were formed the separated clusters. In conclusion, the ISSR assay was useful for detecting genetic variation in the genus Zoysia, and its discriminatory power was comparable to that of other genotyping tools.

Determination of genetic diversity among some almond accessions

More recently the use of different molecular markers in fruit species to determine particularly genetic diversity, genetic relationships and cultivar identification has been gained more importance. In the study, 13 randomly amplified polimorfic DNA (RAPD) and 4 inter-simple sequence repeat (ISSR) markers were used to evaluate genetic relationships among 95 almong accessions (26 foreign cultivars and 69 national cultivars and selections). The all plant material found in Almond Germplasm Repository in Gaziantep, Turkey. Both RAPD and ISSR markers distinguished the almond cultivars and selections in various levels. 17 RAPD and ISSR markers yielded a total of 73 scorable bands, which 51 are polymorphic. The two marker system exhibited variation with regard to average band sizes and polymorphism ratio. The average polymorphism was higher in ISSR (88%) compared to RAPD (74%). RAPD and ISSR marker systems were found to be useful for determining genetic diversity among almong genotypes and cultivars. Combining of two dendrograms obtained through these markers show different clustering of 96 almond specimens without geographical isolation. These results supported that almonds in Turkey indicated considerable genetic diversity.

Efficiency of using RAPD and ISSR markers in evaluation of genetic diversity in sugar beet

Random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) markers were used for comparative analysis of genetic variation in 42 sugar beet accessions. A total of 24 polymorphic primers (12 RAPD and 12 ISSR) were used. The RAPD primers generated 204 amplification products and the ISSR primers produced 178 fragments, 190 and 173 of which were polymorphic, respectively. The average polymorphism level with the ISSR markers (97.2%) was higher than that with the RAPD primers (93%). High genetic diversity indices for both marker types (0.86 for RAPD and 0.91 for ISSR) suggested that these methods were equally effective in determining genetic variation in sugar beet accessions. Cluster analysis of the RAPD, ISSR, and combined datasets revealed similar grouping patterns. However, the dendrogram created from analysis of the combined RAPD+ISSR data was more similar to the RAPD-only dendrogram than the ISSR-only analysis, indicating that RAPD could determine genetic diversity with higher resolution than ISSR in the cultivars tested. High correlation between the RAPD and ISSR marker systems was shown using a Mantel test (r = 0.92). Screening a higher number of anonymous loci in sugar beet using these molecular markers will enable the selection of the best parent cultivars for the development of novel varieties.

Molecular characterization of the genus Papaver section Oxytona using ISSR markers

Papaver bracteatum Lindl., P. orientale L., and P. pseudo-orientale (Fedde) Medw., species of section Oxytona Bernh. in the genus Papaver L., are found widely in the native flora of Turkey. They are commercially important for their high alkaloid content. Because genus Papaver section Oxytona has similar morphological characters, it is not easy to distinguish these species. We used the inter-simple sequence repeat (ISSR) molecular marker system to determine the molecular characterisation of this section. In this study, 20 ISSR primers have been studied in 180 accessions collected from 5 different regions of Turkey. A total of 82 bands were obtained, of which 80 were polymorphic. Average genetic distance was found to be 0.35, the Shannon index was 0.50, and the polymorphism rate was 96.97%. The primer AT10 had the smallest number of bands, which was 3, while the primers AT19 and AT3 had the greatest number of bands, which was 5. In conclusion, the ISSR marker system can be used for the classification of section Oxytona.

The Use of ISSR and RAPD Markers for Genetic Diversity among South Tunisian Barley

Random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) were assayed to determine the genetic diversity of 80 barley specimens from South Tunisia. The ISSR primers showed variation in the percentage of polymorphism, band informativeness (Ib), and resolving power (Rp). The percentage of polymorphism is 66.67%, the average Ib ranged from 0.24 to 0.39, while Rp ranged from 0.74 to 1.16. In RAPD analysis, three primers yielded a total of 17 scorable bands, which are all polymorphic. The three polymorphic primers exhibited variation with regard to average band informativeness (AvIb) and resolving power (Rp). RAPD and ISSR marker systems were found to be useful for the genetic diversity among the barley specimens. The two dendrograms obtained through these markers show different clustering of 80 barely specimens, but we noted that some clusters were similar in some cases. A poor correlation () was found between both sets of genetic similarity data, suggesting that both sets of markers revealed unrelated estimates of genetic relationships. Therefore, the ISSR and RAPD molecular markers show two genetic grouping of studied barely specimens.

Analysis of indica- and japonica-specific markers of Oryza sativa and their applications

Asian rice, Oryza sativa L., is one of the most important crop species. Genetic analysis has established that rice consists of several genetically differentiated variety groups, with two main groups, namely, O. sativa ssp. japonica kata and ssp. indicakata. To determine the genetic diversity of indica and japonica rice, 45 rice varieties, including domesticated rice and Asia common wild rice (O. rufipogon Griff.), were analyzed using sequence-related amplified polymorphism, target region amplified polymorphism, simple sequence repeat, and intersimple sequence repeat marker systems. A total of 90 indica- and japonica-specific bands between typical indica and japonica subspecies were identified, which greatly helped in determining whether domesticated rice is of the indica or japonica type, and in analyzing the consanguinity of hybrid rice with japonica, which were bred from indica and japonica crossed offspring. These specific bands were both located in the coding and non-encoding region, and usually connected with quantitative trait loci. Utilizing the indica-japonica-specific markers, japonica consanguinity was detected in sterile hybrid rice lines. Many indica-japonica-specific bands were found in O. rufipogon. This result supports the multiple-origin model for domesticated rice. Javanica exhibited a greater number of indica-japonica-specific bands, which indicates that it is a subspecies of O. sativa L.

Study of relationships among twelve Phyllanthus species with the use of molecular markers

The present investigation was undertaken to describe the relationships among twelve species of Phyllanthus collected in India by help of molecular markers. In total, 259 marker loci were assessed, out of which 249 were polymorphic revealing 96.13% polymorphism. Nei's similarity index varied from 0.35 to 0.76 for RAPD (Random Amplified Polymorphic DNA) and from 0.31 to 0.76 for ISSR marker systems. Cluster analysis by the unweighted pair group method (UPGMA) of Dice coefficient of similarity generated dendrogram with more or less similar topology for both the analyses that offered a better explanation for diversity and affinities between the species. The phylogenetic tree obtained from both RAPD and ISSR (Inter Simple Sequence Repeat) markers has divided the 12 species into two groups: group I consisting of only one species Phyllanthus angustifolius (Sw.) Sw and group II with the rest of 11 species. Basically, these results were in compliance with notable morphological characterization. The present study revealed high variation among the species of Phyllanthus and will help to identify different Phyllanthus species.

Efficiency of RAPD and ISSR Markers in Differentiation of Homo- and Heterokaryotic Protoclones of Agaricus bisporus

Morphologically, nine different slow-growing protoclones were screened from regenerated protoplast of heterokaryotic Agaricus bisporus. The present study is the first report of fingerprinting on differentiating homo- and heterokaryotic protoclones using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Among 80 primers tested, the seven ISSR and seven RAPD primers selected for the analysis generated a total of 94 ISSR and 52 RAPD fragments, respectively. ISSR fingerprinting detected more polymorphic loci (38.29%) than RAPD fingerprinting (34.61%). Principal coordinate analysis (PCA) was employed to evaluate the resolving power of the markers to differentiate protoclones. The mean polymorphism information content (PIC) for each of these marker systems (i.e., 0.787 for RAPD and 0.916 for ISSR, respectively) suggests that the ISSR marker system was more effective in determining polymorphisms. The dendrograms constructed using RAPD, ISSR, and integrated RAPD, and ISSR marker systems were highly correlated with each other as revealed by the high Mantel correlation (r = 0.98). Pair-wise similarity index values ranged from 0.64 to 0.95 (RAPD), 0.67 to 0.98 (ISSR), and 0.67 to 0.98 (RAPD and ISSR), and mean similarity index values of 0.82, 0.81, and 0.84 for RAPD, ISSR, and combined data, respectively, were obtained. As there was a good correspondence between RAPD and ISSR similarity matrices, ISSR may be used as an alternative to replace RAPD in the genetic diversity assessment and accurate differentiation of homo- and heterokaryotic protoclones of A. bisporus.