ISSR Method Research Articles

Molecular and Phenotypic Markers from Many Okra Genotypes were Used to Estimate the Genetic and Phenotypic Distances

The experiment was conducted in the spring of 2021 in an open field in the Al-Mahmudiya district/Baghdad to study the genetic and phenotypic distance of a number of genotypes in okra using a randomized complete block design (RCBD) and three replications. As replication contained 18 genotypes of okra, distributed randomly on 18 experimental units, with ten cultivars giving the symbols (O1-O10) and eight hybrids giving the symbols (O11-O18), and at the third true leaf Samples were taken from (18 genotypes) extract DNA and the ISSR method was used to estimate the genetic distance, and the plants agricultural service operations continued and traits of vegetative, flowering and yield growth of genotypes were studied to study the phenotypic distance between them. The results of the genetic distance based on ISSR technology demonstrated that there is a genetic distance between the genotypes of okra under study, with the highest genetic distance between the two genotypes O6 and O15 reaching 0.735. The O6 cultivar displayed a high genetic distance with the most of the other genotypes, as the O10 cultivar which displayed a high genetic distance with most of the other genotypes, which advises plant breeders to use them to create development single hybrids. The hybrids O11, O12, O13, O14, O15, O16, and O17 also demonstrated a high genetic distance with the cultivars O6, O7, O8, O9, and O10, which plant breeders can use it in breeding program to development three-way hybrids. The ISSR results divided into two groups A and B in the genetic Cluster dendogram. the findings of the statistical analysis of the phenotypic variables also revealed that there were significant differences between the genotypes in the measured traits. As the O4 cultivar produced the greatest values for plant height and leaf number, flowers number, pods number (169 pods ) and pod set percentage, The O10 cultivar produced the highest pod weight (5.29 g) and yield per plant (691 g), and the results of the genetic distance based on phenotypic traits demonstrated that there is a phenotypic distance between the genotypes, with the two genotypes O10 and O15 exhibiting the highest genetic distance based on phenotypic traits (371.96 ).as The genotypes were divided into two groups within the cluster analysis depended on phenotypic traits.

Determination of genetic diversity of Astragalus L. species distributed in Erzincan and its surroundings by ISSR method

Bu çalışmada, 2021 yılının vejetasyon döneminde Erzincan’daki 42 farklı lokaliteden Astragalus L. cinsine ait bitki örnekleri toplanmıştır. Yapılan bitki tayini çalışmaları sonucunda 12 takson (A. pennatulus, A. compactus, A. aduncus, A. xylobasis, A. angustifolius subsp. angustifolius, A. karamasicus,A. kurdicus, A. cancellatus, A. eriocephalus subsp. eriocephalus, A. lycius, A. microcephalus, A. lagurus) belirlenmiştir. Bunlardan 3’ü (Astragalus pennatulus, Astragalus karamasicus, Astragalus lycius) endemiktir. Belirlenen türler arasındaki genetik ilişkiler ISSR moleküler markırları ile araştırılmıştır. ISSR (kısa dizi tekrarları arası) yönteminde 16 primer (UBC868, UBC817, UBC807, UBC812, UBC816, UBC825, UBC824, UBC830, UBC836, UBC840, UBC848, UBC855, UBS856, UBC841, UBC842, UBC61) kullanılmıştır. PCR sonuçlarına gore, çalışılan primerler 100-2000 bç uzunluğunda bantlar vermiştir. ISSR primerlerine göre polimorfizm % 90.8 hesaplanmıştır. ISSR verileri ile UPGMA yöntemi kullanılarak dendrogramlar oluşturulmuştur. En yüksek genetik benzerlik A. kurdicus ile A. angustifolius supsp angustifolius taksonları arasında gözlenirken, en düşük genetik benzerlik A. angustifolius subsp angustifolius ile A. pennatulus taksonları arasında gözlenmiştir. Çalışılan bu markerlar Astragalus türleri arasındaki genetik benzerliği karakterize etmede uygun markırlar olduğunu ortaya çıkarmıştır.

GENETIC DIVERSITY ANALYSIS AMONG SEVERAL APPLE GENOTYPES USING ISSR MARKERS

Apple (Malus domestica Borkh.) is one of the most cultivated fruit trees species worldwide, its fruits being consumed not only for their organoleptic attributes, but also for their nutraceutical properties. Therefore, the genetic variability of the species is extremely important to insure a large enough pool of cultivars to accommodate consumer demands for various fruit traits, such as taste, flavor, color, shape, etc., as well as to preserve cultivars that have less desirable organoleptic properties but are resistant/tolerant to biotic and/or abiotic stress and could be good genitors for these traits. The current study presents the use of ISSR method to assess the genetic variability among seven Romanian apple cultivars from the orchard collection of University of Agronomic Sciences and Veterinary Medicine of Bucharest, Romania: ‘Florina’, ‘Remar’, ‘Ciprian’, ‘Iris’, ‘Rebra’, ‘Generos’, and ‘Redix’. In addition, the method proved to be useful in identifying closely related individual genotypes, allowing the identification and elimination of duplicates from collections, without compromising the collection’s genetic variability.

Population genetic diversity of Huperzia chinensis (Herter ex Nessel) Ching. in Northwest region of Vietnam revealed by ISSR and SCcoT markers

Genetic diversity of natural Huperzia chinensis population in Northwest region of Vietnam was analyzed based on the DNA fingerprint data induced by ISSR and SCoT methods separately and combined data from both methods. The genetic diversity parameters and genetic distances among individuals based on SCoT technique were higher than ISSR technique [expected heterozygousity (HeS) was 0.2812; Shannon index (IS) was 0.4153; Percentage of polymorphic bands (PPBS) was 75.44%; Genetic similarity coefficients (GSCsS ) were in range of 0.561 - 0.947 with the average value (AGSCS) of 0.758 revealed by SCoT technique compared to HeI was 0.2394; II was 0.3515; PPBI was 60.87%, GSCsI were 0.551 - 0.986 and AGSCI was 0.860 revealed by ISSR technique. Combined data showed that genetic diversity level of investigated population (He was 0.2583; I was 0.3804; PPB was 67.46%, higher than genetic diversity level of Huperzia serrata population in the same distribution area which was previously evaluated by the same markers. Combined data also indicated the genetic similarities among individuals (GSCs) were 0.579 - 0.960 with the average (AGSC) of 0.814. ISSR method reflected more loci in the investigated plants than SCoT method but SCoT method could differentiate the tested samples better and reflects genetic diversity at higher level than ISSR method.

Determination of genetic divergence in some bread wheat varieties by IRAP and ISSR analyses

In this study, it was aimed to detect the differences of genotypes through the measurement of genetic distance in bread wheat genotypes and to show the usability of chromosome mapping for different aims such as breeding programs, elite seed productions. The genetic diversity of 12 bread wheat genotypes bread wheat genotypes (Bozkır, Harmankaya 99, Altay 2000, Yıldırım, Bezostaja 1, Ahmetağa, Müfitbey, Aldane, Es 26, Alperbey, Atay 85 ad Eraybey) was examined through ISSR and IRAP techniques. As fast molecular techniques, ISSR and IRAP methods can be effectively included in breeding programs both for genetic exploration and evaluation and for the protection of elite breeding and production materials. 12 genotypes were classified into two main clusters in the dendrogram produced by using the ISSR and IRAP markers. While Cluster I included Bozkır, Harmankaya 99, Altay 2000, Yıldırım, Bezostaja 1, Ahmetağa and Müfitbey, cluster II included Aldane, Es 26, Alperbey, Atay 85 and Eraybey. Furthermore, the results obtained in this study indicated that ISSR and IRAP methods were effective for the definition of bread wheat genotypes.

Ecologiсal and genetic changes in populations of silver (Hypophtalmichthys molitrix) and bighead (Aristichthys nobilis) carp

A comparative characterization of the genetic structure of populations of silver carp and bighead carp by four primers (CTC)6C, (GAG)6C, (AGC)6G, (AGC)6C was given. The average value of (Na) in silver carp and bighead carp was at the level of 45.7 and 42.7. In the bighead carp, the highest number of amplicons and the highest value of the effective number of alleles per locus were observed for primer (AGC)6C and were equal to 62 and 15.4, respectively. The least polymorphic in number of amplicons per locus was primer (CTC)6C, where the values were 29. According to the studied four primers, the effective number of alleles per locus ranged from 6.3 to 15.4. In silver carp, the largest number of amplicons and the highest value of the effective number of alleles per locus were observed with primer (GAG)6C and were equal to 57 and 18.5, respectively. The least polymorphic in the number of amplicons per locus was primer (CTC)6C, where the values were 34. According to the studied four primers, the effective number of alleles per locus ranged from 6.3 to 18.5. The comparative analysis of ecological and genetic changes allowed studying the genetic variability of silver and bighead carp at the population level. In addition to specific amplicons, unique DNA fragments inherent in populations at the species level have been found. Specific «species» and «population» polymorphic ISSR-markers, identified in this work, allow to use them as a basis for further comprehensive research for molecular labelling on the population level and to establish phylogenetic relationships, genomic profile of the species, as well as monitoring the genetic structure of fish. The optimized ISSR method can serve as an effective tool for further genetic studies of populations of silver carp and bighead carp. The obtained results allow controlling the selection and breeding work in the process of reproduction of the gene pool of existing fish populations. To increase the efficiency of selection and breeding work in fish farming, it is advisable to use genetic markers that have a high specificity to individual DNA fragments of fish.

Молекулярно-генетический анализ и идентификация популяций Pinus sylvestris L. в Кировской области и Пермском крае

Molecular–genetic analysis and identification of 5 Pinus sylvestris L. hauls, collected in Kirov oblast and Perm krai was provided. Molecular–genetic analysis was provided with using ISSR–method of DNA polymorphism analysis. In 5 researched hauls of P. sylvestris 126 ISSR–markers were identified, 122 (P95=0.968) were polymorphic. Molecular–genetic identification of researched P. sylvestris populations was provided and molecular–genetic formulas and bar–codes were made. Identification ISSR–markers were identified. Genetic structure and differentiation analysis of P. sylvestris showed that hauls were distributed in three genetic populations.

Comparison of Molecular Markers ISSR, SSR and RFLP to Study the Genetic Variation of Strawberry Cultivars

Strawberry is grown in Indonesia because it has potential high economic value. The purpose of this study was to investigate relationship of phenetic lineage and genetic variations in 12 strawberry cultivars using three different molecular marker methods, including ISSR, SSR and RFLP. Five primers of ISSR markers were used and they generated 53 bands with 44 polymorphic DNA bands. Percentage of polymorphic using ISSR method was 83%. Five primers of SSR markers were used and they gave 41 DNA bands with 36 polymorphic DNA bands. Percentage of polymorphic using SSR method was 87%. Four primers of PCR-RFLP markers and 3 enzymes were used and they had 20 DNA bands with 9 polymorphic DNA bands. Percentage of polymorphic PCR-RFLP method was 45%. Three dendrogram results based on each method indicated that Stroberi Hitam has smallest similarity index compared with other eleven cultivars. 'Californica' cultivar had the closest similarity with Stroberi Hitam. Data analysis of molecular character requires complementary and supporting data of other characters such as morphology, biochemistry and cytology so this will strengthen the lineage data of the organisms to be investigated. Molecular marker can be used as a good method for genetic variation analysis if it has some advantages such as high polymorphism, codominance and specific characteristics and wide distribution.

INTRASPECIFIC VARIATIONS STUDIED BY ISSR AND IRAP MARKERS IN MASTIC TREE (Pistacia lentiscus L.) FROM TURKEY

In this study, intra-specific variations in naturally growing and cultivated mastic tree (Pistacia lentiscus L.) samples obtained from western parts of Turkey were examined using ISSR and IRAP marker techniques. Samples from Crete and Chios were also included in the study. Morphological measurements of some leaf characteristics of the samples were performed and the measured data was evaluated statistically with a Pearson Correlation analysis to reveal the correlations between character pairs. ISSR primers produced 81 bands between 161-1884bp with 96.3% polymorphism and IRAP primers produced 72 bands between 124-2027bp with 91.67% polymorphism. Polymorphism information content (PIC) values were 0.458 and 0.418 for ISSR and IRAP, respectively. Genetic similarity matrix was examined with Jaccard’s coefficient. Maximum similarity was found between the Cretan samples (LG2 and LG3) with the ISSR analysis (0.933) and between L25A (C1, Bodrum) and L29A (C1, Milas) with the IRAP analysis (0.593). Unweighted pair group method with arithmetic mean (UPGMA) dendrogram was divided into 12 and 4 groups by ISSR and IRAP methods, respectively. Specimens were segregated on 3 main different clusters by the Principal Component Analysis (PCA) based on the combined marker systems. The results showed that P. lentiscus has very high ratios of intraspecific variation. The present work is an original study in terms of large sampling including wild genotypes, cultivated specimen, Chios and Cretan varieties, use of ISSR and IRAP combination, determination of relations between culture and wild genotypes and the use of Bagy-1 retrotransposons in intraspecific polymorphism. This study may be considered as a reference study for studies on gene pools of P. lentiscus and phylogenetic relationships within the species and may contribute to species concept and agricultural breeding programs.

Morphological, chemical, and genetic diversity of Gypsophila L. (Caryophyllaceae) species and their potential use in the pharmaceutical industry

The aim of this study was to evaluate the morphological, chemical, and genetic similarity of species belonging to the genus Gypsophila L. The analysis included 7 Gypsophila species with potential use in the pharmaceutical industry for saponin production. In order to assess the variation of the morphological traits, a number of morphological characteristics have been determined (including the number of lateral roots, diameter and total root length, average plant height, number of shoots and their diameter, number of branches on the shoot, length and width of leaves, and fresh and air-dried weight of aerial and underground parts of plants), while prosaponin content was determined as the chemical trait. Gypsogenin 3-O-glucuronide and quillaic acid 3-O-glucuronide were the dominant prosaponins determined in the Gypsophila roots (amounting up to 13.5 mg g-1 dry extract in G. scorzonerifolia Ser. and 12.3 mg g-1 dry extract in G. acutifolia Spreng., respectively). Moreover, a variation analysis at the molecular level was carried out based on the RAPD and ISSR methods. The conducted analysis showed that the most distinct species at the morphological, chemical, and genetic levels were G. paniculata L. and G. oldhamiana Miq.

Estimation of the Genetic Structure of Helix pomatia L. Populations (Mollusca, Pulmonata) by Capillary Electrophoresis of ISSR DNA Fragments

Using the capillary electrophoresis of ISSR DNA fragments, the genetic structure of the adventitious populations of Helix pomatia L. (Mollusca, Pulmonata) inhabiting the territory of Eastern Europe was studied. A significant decrease in genetic heterogeneity (He = 0.095 ± 0.002) and population differentiation level (Fst = 0.074) was revealed in comparison with similar data obtained using the ISSR method with electrophoretic detection in agarose gel and allozymes in polyacrylamide gel. Similar to other methods, an effect of spatial isolation (RM =–0.476) was found on the basis of regression analysis between genetic and geographical distances. The effective population size, calculated with the help of M. Slatkin’s model, turned out to be the largest of the previously calculated values (Ne = 41.8), which indicates the high viability of the studied groups of Roman snails in newly developed territories.

ISSR Analysis of Variability of Cultivated Form and Varieties of Pomegranate (Punica granatum L.) from Azerbaijan

The article presents the results of a study of genetic polymorphism for the first time carried out on pomegranate varieties and forms of Azerbaijan origin using molecular markers. In total, 102 PCR fragments were identified, of which 80 were polymorphic. The high level of polymorphism (75.5%) and the rich genetic diversity were identified among the studied pomegranate collection. As a result of data analysis and on the basis of the values of the basic parameters (PIC, EMR, MI, RP, MRP) determining informativeness of markers, all 14 ISSR primers were suitable for genotyping pomegranate accessions. The most effective markers (UBC808, UBC811, UBC834, and UBC840) were identified among the set of primers tested. A dendrogram was constructed on the basis of the data obtained, which made it possible to group genotypes into 16 major clusters. The genetic similarity index ranged from 0.032 to 0.94. The study of the genetic relationship of different pomegranate varieties confirms the effectiveness of the ISSR method, which makes it possible to determine the level of genetic diversity, as well as to establish the relationship among the studied pomegranate accessions.

Genetic diversity of Geoffroea decorticans , a native woody leguminous species from Atacama Desert in Chile

SUMMARY: Geoffroea decorticans (Fabaceae), chanar, is one of the few native tree species that are adapted to the ecologically limiting conditions from Atacama Desert. Despite its considerable value as a crop species, and important medicinal and food properties, the genetic variability of this plant has not yet been evaluated. The aim of this study was to analyze genetic diversity and degree of fragmentation in G. decorticans populations from six provinces in northern Chile. Genetic variability was assessed using eight ISSR and three RAPD primers, generating 97 % and 81 % polymorphic bands, respectively. AMOVA results based on nine populations showed high genetic diversity within populations (65 %) and moderate levels among populations (35 %). Moreover, genetic relationships among individuals provided evidence for the existence of two well defined clusters in the northern and southern regions of the Atacama Desert. A Mantel test showed a significant positive correlation between geographic and genetic distances (r = 0.58) in seven populations, indicating significant isolation-by-distance. Average Shannon Weaver diversity indices showed significantly lower values associated with Pachica, Copiapo river-mouth, Alto del Carmen and Vicuna populations relative to populations from San Pedro, Calama, Azapa and Copiapo. In this work, a combination of ISSR and RAPD methods provided high quality information to evaluate genetic variability, and could be used as an alternative method to identify and distinguish G. decorticans genotypes of interest. This work represents the first attempt to molecular characterization of G. decorticans in Chile, and its significance to future genetic research is discussed.

Evaluation of Genetic Similarity of Micropropogated Plants of Ventilago Maderaspatana Geartn using RAPD and ISSR Markers

Numerous scientific studies showed that tissue culture induced mutation has been detected in all species studied. Therefore, it is very important to study the degree of genetic similarity by comparing the gene sequence of regenerated plants with the mother plant. In this study, a Polymorphic Chain Reaction (PCR) based methods such as RAPD and ISSR marker techniques were used to study the genetic similarity of in-vitro cultured plants of Ventilago maderaspatana Geartn. Fourteen individuals were randomly tagged from the population of regenerated plants for the genetic similarity analysis. ISSR method carried using 22 primers from which a total of 98 clear and reproducible bands was scored. Out of 98 bands, 72 were monomorphic bands (73.47%) and 26 were polymorphic bands (26.53%). Dendrogram cluster analysis of data generated from 22 ISSR primers showed an estimated genetic distance of 0.07. RAPD method carried using 21 primers from which a total of 194 clear and reproducible bands was scored. Out of 194 bands, 126 were monomorphic bands (64.94%) and 68 were polymorphic bands (35.05%). Dendrogram cluster analysis of data generated from 21 RAPD primers showed an estimated genetic distance of 0.065. ISSR and RAPD data analyzed using NTSYS-pc showed that micro propagated plants exhibit maximum similarity with the mother plant.

Genetic analysis of Canarium album in different areas of China by improved RAPD and ISSR

To lay the foundation of the classification of Canarium album (C. album), and C. album from Terminalia Chebula (T. chebula) in different areas of China, improved RAPD and ISSR analysis were performed to analyze polymorphism and genetic relationship. Ten samples were collected from different locations in China. A total of 221 fragments were detected by improved RAPD, out of which polymorphic fragments accounted for 82.3% with average amplification bands of 10.05 per primer. ISSR markers revealed a total of 147 alleles, where polymorphic fragments accounted for 83.5%, with average amplification bands of 7.35 per primer. The sizes of fragments ranged from 200 to 2500bp and from 150 to 2000bp in RAPD and ISSR markers, respectively. The similarity coefficient ranged from 0.46 to 0.86 with RAPD markers and 0.36 to 0.89 with ISSR markers. The results indicated that improved RAPD and ISSR methods are useful for genetic diversity study of C. album. Thus, this study provides us the theoretical basis for the breeding and classification of C. album in South and Southwest China.

Molecular analysis of genetic diversity and population structure in Everniastrum cirrhatum (Fr.) Hale (Parmeliaceae) in India

Everniastrum cirrhatum is a medicinally important lichen used in Ayurvedic and Unani systems of medicine. In the present study, DAMD and ISSR methods were used to estimate the genetic variation and population structure of E. cirrhatum collected from different geographical regions of India. Four DAMD and ten ISSR primers detected 42 and 110 polymorphic bands and accounted for 95.65 and 94.24% polymorphisms, respectively. Cumulative band data generated for DAMD and ISSR markers resulted into 94.95% polymorphism across all the accessions of E. cirrhatum. The UPGMA dendrogram showed two major clusters. The clustering pattern in the UPGMA dendrogram revealed that the groupings are largely in congruence with the geographical distribution of the accessions. Clustering patterns in STRUCTURE revealed that geographical diversity is perfectly in congruence with the genetic diversity. The clustering pattern in STRUCTURE was also supported by PCoA. Mantel test for matrix correlation showed a weak but positive correlation between genetic and geographical distance. The hierarchical analysis of molecular variance revealed that maximum percentage of variation was found within a population (57%), followed by among regions (28%) and among populations (15%). The present study provides significant insight into the genetic variability and population structure of E. cirrhatum. Understanding population structure would provide baseline information for developing its sustainable management strategies. It would also be important to conserve populations of E. cirrhatum in different localities of the Himalayan regions to prevent population decline caused by anthropogenic and environmental stochastic effects.

AFLP, RAPD, and ISSR analysis of intraspecific polymorphism and interspecific differences of allotetraploid species Aegilops kotschyi Boiss. and Aegilops variabilis Eig

To evaluate genetic variation, 27 accessions of allotetraploid species Aegilops kotschyi and Ae. variabilis with the US genome were analyzed using the AFLP, RAPD, and ISSR methods. A total of 316 polymorphic RAPD fragments, 750 polymorphic AFLP fragments, and 234 polymorphic ISSR fragments were obtained. It was demonstrated that the analyzed species were characterized by a considerable level of nuclear genome variation. According to the data of ISSR and RAPD analysis, the average value of the Jaccard similarity coefficient for the accessions of Ae. variabilis from different geographical regions was slightly lower than that for the accessions of Ae. kotschyi. At the same time, AFLP analysis showed no considerable differences in the levels of intraspecific variation of the studied species. Analysis of the summarized RAPD, ISSR, and AFLP marking data in the Structure software program showed that most of the analyzed accessions with high degree of probability could be assigned to one of two groups, the first of which corresponded to Ae. kotschyi and the second corresponded to Ae. variabilis, thereby confirming the species independence of Ae. kotschyi and Ae. variabilis. Accessions k900, k907, k908, and v90 could not with a sufficiently high degree of probability be assigned to one of the species, which possibly was the result of interspecific hybridization. Analysis of the species diversity using different molecular markers made it possible to identify the accessions that were notably different from other accessions of its species.

Genetic diversity of Poa pratensis L. depending on geographical origin and compared with genetic markers.

BackgroundPoa pratensis is one of the most common species of meadow grass in Europe. Most cultivars of the species found in Poland were originally derived from its ecotypes. We compared the effectiveness of the RAPD and ISSR methods in assessing the genetic diversity of the selected populations of P. pratensis. We examined whether these methods could be useful for detecting a possible link between the geographical origin of a given population and its assessed genetic variation.MethodsThe molecular markers RAPD and ISSR were used and their efficiency compared using, inter alia, statistical multivariate methods (UPGMA and PCA).ResultsThe low value of Dice’s coefficient (0.369) along with the significantly high percentage of polymorphic products indicates a substantial degree of genetic diversity among the studied populations. Our results found a correlation between the geographical origin of the studied populations and their genetic variations. For ISSR, which proved to be the more effective method in that respect, we selected primers with the greatest differentiating powers correlating to geographical origin.DiscussionThe populations evaluated in this study were characterized by a high genetic diversity. This seems to confirm the hypothesis that ecotypes of P. pratensis originating from different regions of Central Europe with different terrain structures and habitat conditions can be a source of great genetic variability.

Intraspecific diversity of Achillea collina Becker evaluated by molecular genetic markers

Achillea species has been known and utilized worldwide in folk medicine and in up-to date phytotherapy as well. In our recent study we wanted to evaluate the intraspecific diversity of A. collina and to look for reliable, relatively simple molecular method for differentiation of different accessions. Five cultivated genotypes and six populations of wild origin were investigated. Besides, for comparison and control six other species were involved into the trial. The DNA samples were evaluated by RAPD (11 primers) and ISSR (12 primers) methods. In the RAPD analysis 140 bands (97.14 % polymorph) were detected. They distinguished primarily among species and less characteristically among the A. collina populations. With ISSR primers we detected 188 bands (97.34 % polymorph). ISSR markers and combined RAPD and ISSR method enabled an informative intraspecific evaluation of A. collina accessions. The largest genetic distances were proven 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 where geographical distances of the original locations are only 52-55 km. Nei’s genetic distances of cultivated populations are also relatively low (0.23 0.36) and a common origin for the majority of these genotypes was assumed.

Effectiveness of ISSR markers for determination of the Aneura pinguis cryptic species and Aneura maxima

Aneura pinguis and Aneura maxima belong to the simple thalloid liverworts. Previous isozyme studies revealed that A. pinguis is a complex of cryptic species difficult to distinguished based on morphology. In the present study four cryptic species of the A. pinguis complex and A. maxima were examined by means of ISSR method to assess genetic variation and to develop species-specific markers. Eight ISSR primers used generated 460 bands, of which 453 were polymorphic. The highest values of resolving power 28.4 and marker index 18.1 were noted for primer 835 (AG)8-YC, while polymorphism information content for primer 842 (GA)7-AYG. The total gene diversity (HT) based on polymorphic loci was 0.284 for A. pinguis and 0.06 for A. maxima. ISSR markers supported existence of cryptic species in A. pinguis and showed genetic isolation between them. Species-specific bands were found for all studied cryptic species of A. pinguis and A. maxima, thus ISSRs can be used for their identification. A. maxima clearly differ from all the A. pinguis cryptic species in each amplified ISSR primer. The AMOVA conducted for the A. pinguis complex showed that most of genetic variation (ΦPT 0.586) was present among species.