Temporal dynamics and variation of multilocus ISSR-PCR DNA markers in the Uman’ population of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> over two decades (1984–2004)

The temporal dynamics of genomic variation in the Uman’ (Ukraine) population of Drosophila melanogaster over the period 1984–2004 was studied using multilocus ISSR markers. Considerable polymorphism of the genomic DNA fragments corresponding to ISSR markers was found in the D. melanogaster population studied: the values of average heterozygosity varied from 0.085 to 0.127 depending on the year. Significant differences in the frequencies of dominant alleles between the samples of different years were recorded for 12 of the 30 DNA fractions detected. These changes are nondirectional and random. The pattern of detected variation suggests the determining influence of gene drift and migration process on the variation of noncoding DNA sequences in the Uman’ population of D. melanogaster.     

Polymorphism and differentiation of multilocus DNA markers in natural populations of Drosophila melanogaster

Using multilocus (RAPD) markers, variation and divergence of genomic DNA was examined in two Drosophila melanogaster populations from Russia and three populations from Ukraine. The populations were found to exhibit high polymorphism at RAPD markers. Estimation of genetic distances between the populations showed low differentiation of geographically distant populations of D. melanogaster. Significant gene flow between the D. melanogaster populations was found, which depended on the geographical distance between them.     

Genetic variation within and among populations of a wild rice Oryza granulata from China detected by RAPD and ISSR markers

Genetic variation within and between five populations of Oryza granulata from two regions of China was investigated using RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat amplification) markers. Twenty RAPD primers used in this study amplified 199 reproducible bands with 61 (30.65%) polymorphic; and 12 ISSR primers amplified 113 bands with 52 (46.02%) polymorphic. Both RAPD and ISSR analyses revealed a low level of genetic diversity in wild populations of O. granulata. Furthermore, analysis of molecular variance (AMOVA) was used to apportion the variation within and between populations both within and between regions. As the RAPD markers revealed, 73.85% of the total genetic diversity resided between the two regions, whereas only 19.45% and 6.70% were present between populations within regions and within a population respectively. Similarly, it was shown by ISSR markers that a great amount of variation (49.26%) occurred between the two regions, with only 38.07% and 12.66% between populations within regions and within a population respectively. Both the results of a UPGMA cluster, based on Jaccard coefficients, and pairwise distance analysis agree with that of the AMOVA partition. This is the first report of the partitioning of genetic variability within and among populations of O. granulata at the DNA level, which is in general agreement with a recent study on the same species in China using allozyme analysis. Our results also indicated that the percentage of polymorphic bands (PPB) detected by ISSR is higher than that detected by RAPD. It seems that ISSR is superior to RAPD in terms of the polymorphism detected and the amplification reproducibility. Received: 29 March 2000 / Accepted: 15 May 2000     

Somaclonal variation at the nucleotide sequence level in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) as revealed by RAPD and ISSR markers,and by pairwise sequence analysis

The nature of somaclonal variation at the nucleotide sequence level was studied in rice cv. Nipponbare. First, we investigated genomic variations by using 2 molecular marker systems: RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat). This was followed by sequencing of selected bands that represented genomic variations, and pairwise sequence analysis taking advantage of the whole genome sequence of rice. In addition, transpositional activity of the active MITE, mPing, was analysed by locus-specific PCR amplifications. The 2-year-old calli and their regenerated plants, analysed with 24 RAPD and 20 ISSR primers, showed moderate levels of genomic variation (20.83% and 17.04%, respectively). To test whether DNA methylation plays a role in somaclonal variation, the calli were treated with 5-azacytidine, a chemical agent that reduces cytosine methylation by blocking the activity of DNA methyltransferase. Though dwarfism occurred in regenerants from treated calli (a hallmark of the drug treatment), there was only a slight increase in the frequency of somaclonal variation detected in the treated calli and their regenerated plants relative to untreated controls. The transposon mPing also remained immobile in both treated and untreated calli. Nevertheless, dendrograms constructed according to the Jaccard coefficient calculated by UPGMA of the ISSR bands revealed that the 5-azacytidine-treated and untreated somaclones were grouped into 2 distinct clusters, suggesting a possible indirect effect of the treatment on the genomic changes, depending on the marker used. Sequence analysis indicated a low level of variation (0.31%), with single-base-pair substitutions predominating.     

PCR-based molecular markers for assessment of somaclonal variation in <Emphasis Type="Italic">Pinus pinea</Emphasis> clones micropropagated <Emphasis Type="Italic">in vitro</Emphasis>

Four different markers [random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP), and selective amplified microsatellite polymorphism length (SAMPL)] were applied for evaluating somaclonal variation of micropropagated genotypes of stone pine (Pinus pinea L.). The total number of primers tested was 130, with 223 combinations assayed. A high number of them amplified successfully (178), representing 79.82 % of the total, and the average number of amplified fragments ranged from 2.47 (ISSR) to 65.76 (SAMPL). Based on internal controls, no problem of reproducibility was detected. Almost no somaclonal variation was detected within the clones. Of the tested markers, ISSR, AFLP, and SAMPL showed monomorphic amplification profiles, with only RAPD markers showing some interclonal variation.     

African sequence variation accounts for most of the sequence polymorphism in non-African Drosophila melanogaster

We compared the sequence polymorphism of 12 genomic fragments in six geographically dispersed African populations to one European Drosophila melanogaster population. On the basis of one African and one European population half of these fragments have strongly reduced levels of variability outside of Africa. Despite this striking difference in European variation, we detected no significant difference in African variation between the two fragment classes. The joint analysis of all African populations indicated that all high-frequency European alleles are of African origin. We observed a negative Tajima's D in all African populations, with three populations deviating significantly from neutral equilibrium. Low, but statistically significant, population differentiation was observed among the African populations. Our results imply that the population structure and demographic past of African D. melanogaster populations need to be considered for the inference of footprints of selection in non-African populations.     

Efficiency of ISSR and RAPD markers in genetic divergence analysis and conservation management of Justicia adhatoda L., a medicinal plant

Genetic variation within and among population is the basis for survival of the population both in short and long term. Thus, studying the plant genetic diversity is essential for any conservation program. Indigenous medicinal plants like Justicia adhatoda L. which are facing high rate of depletion from the wild population need immediate attention. DNA-based dominant molecular marker techniques, random amplification of polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) were used to unravel the genetic variability and relationships across thirty-two wild accessions of J. adhatoda L., a valuable medicinal shrub widespread throughout the tropical regions of Southeast Asia. Amplification of genomic DNA using 38 primers (18 RAPD and 20 ISSR) yielded 434 products, of which 404 products were polymorphic revealing 93.11 % polymorphism. The average polymorphic information content value obtained with RAPD and ISSR markers was 0.25 and 0.24, respectively. Marker index (RAPD = 3.94; ISSR = 3.53) and resolving power (RAPD = 4.24; ISSR = 3.94) indicate that the RAPD markers were relatively more efficient than the ISSR assay revealing the genetic diversity of J. adhatoda. The Shannon diversity index obtained with RAPD and ISSR markers was 0.40 and 0.38, respectively. The similarity coefficient ranged from 0.26 to 0.89, 0.33 to 0.93 and 0.31 to 0.90 with RAPD, ISSR and combined UPGMA dendrogram, respectively. PCA derived on the basis of pooled data of both the markers illustrated that the first three principal coordinate components accounted 79.27 % of the genetic similarity variance. The mantel test between two Jaccard’s similarity matrices gave r = 0.901, showing the fit correlation between ISSR- and RAPD-based similarities. Based on the results, ex-situ methods may be the most suitable and efficient measure for long-term conservation.     

Detecting the footprint of positive selection in a european population of Drosophila melanogaster: multilocus pattern of variation and distance to coding regions

The effects on nucleotide variation of adaptations to temperate habitats and of the possible bottleneck associated with the origin of European populations of Drosophila melanogaster should be detectable in DNA sequences given the short time elapsed relative to the species population size. We surveyed nucleotide variation in 109 fragments distributed across the X chromosome in a European population of D. melanogaster to detect the footprint of positive selection. Fragments were located primarily in large noncoding regions. Multilocus tests based on Tajima's D statistic revealed a significant departure from neutral expectations in a stationary panmictic population, with an important contribution from both positive and negative D values. A positive relationship between Tajima's D values and distance to coding region was detected, with a comparative excess of significantly negative D values in the subset of fragments closer to coding regions. Also, there was a significant heterogeneity in the polymorphism to divergence ratio, with 12 fragments contributing 42% to the test statistic. Moreover, these fragments were comparatively closer to coding regions. These findings would imply positive selection events, and thus selective sweeps, during the species expansion to Europe.     

Genetic variation in Ural populations of the rare plant species <Emphasis Type="Italic">Adenophora lilifolia</Emphasis> (L.) DC. on the basis of analysis of polymorphism of ISSR markers

The genetic variation in four populations of Adenophora lilifolia (L.) DC., a rare plant species of the Perm region, was analyzed using 56 ISSR markers. The characteristics of DNA polymorphism and population genetic diversity were determined. These data demonstrate a high level of DNA polymorphism (P ₉₅ = 82.14%). The studied A. lilifolia populations are weakly differentiated; the intrapopulation variation is the main contributor to the genetic variation.     

Genomic instability in phenotypically normal regenerants of medicinal plant Codonopsis lanceolata Benth. et Hook. f., as revealed by ISSR and RAPD markers

Codonopsis lanceolata Benth. et Hook. f., commonly known as bonnet bellflower, is a high-valued herb medicine and vegetable. In this study, a large number of plants were regenerated via organogenesis from immature seed-derived calli in C. lanceolata by a simple and efficient method. Compared with the mother donor plant, the regenerated plants did not exhibit visible phenotypic variations in six major morphological traits examined at the stage of one-season-maturity under field conditions. To gain insight into the genomic stability of these regenerated plants, 63 individuals were randomly tagged among a population of more than 2,000 regenerants, and were compared with the single mother donor plant by two molecular markers, the inter-simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD). Apparent genomic variation was detected in the 63 regenerants, whereas preexisting heterozygosiy in the donor plant was deemed minimal by testing 30 seedlings germinated from selfed seeds of the same donor plant. The percentages of polymorphic bands (PPB) in the ISSR and RAPD analysis were respectively 15.7 and 24.9% for the 63 regenerated plants. Cluster analysis indicates that the genetic similarity values calculated on the basis of RAPD and ISSR data among the 64 plants (63 regenerated and one donor) were respectively 0.894 and 0.933, which allow classification of the plants into distinct groups. Nineteen randomly isolated bands underlying the changed RAPD or ISSR patterns were sequenced, and three of them showed significant homology to known-function genes. Detailed pairwise sequence comparison at one locus between the donor plant and a regenerant revealed that insertion of two short (24 and 19 bp) stretches of nucleotides in the regenerated plant relative to the donor plant occurred in an apparently stochastic manner.Electronic Supplementary Material Supplementary material is available for this article at     

Assessment of genetic stability in tissue-cultured products and seedlings of Saussurea involucrata by RAPD and ISSR markers

To control the genetic quality during the whole process of tissue culture of the traditional Chinese medicinal plant, Saussurea involucrate Kar. et Kir., DNA polymorphisms and genetic variations were investigated using randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers. The genetic stability/variation in tissue-cultured products, including three calli, three adventitious shoots, regenerated plantlets and 2 year-old regenerated plantlets cultivated in the planting base in Tianshan Mountain, were assessed compared with 1 year-old and 2 year-old seedlings cultivated in the same planting base using aseptic seedlings as reference. Apparent genetic variation was detected in the 11 type of plant materials. The percentages of polymorphic bands in the RAPD and ISSR analysis were, respectively, 35% and 33%. Cluster analysis indicated that the genetic similarity values calculated on the basis of RAPD and ISSR data among the 11 type of plant materials were respectively ranged from 0.823 to 0.995 with a mean of 0.878 and 0.825 to 0.974 with a mean of 0.885, which classified the samples into three groups. The similarity coefficient also revealed that differences among three calli were not remarkable by both RAPD and ISSR analysis, and only chemical components and growth properties needed consideration in the screening of callus used for the next redifferentiation studies. But there are remarkable differences among three adventitious shoots analyzed by ISSR markers. Therefore, RAPD and ISSR markers are efficient tools in genetic variation assessment and quality control in plant tissue culture process.     

ISSR as New Markers for Identification of Homokaryotic Protoclones of Agaricus bisporus

To accelerate the breeding of Agaricus bisporus, quick and reliable methods to identify the infrequent homokaryons are necessary. A new marker, inter simple sequence repeat (ISSR) fingerprinting, is described for differentiation of homo- and hetero-karyotic protoclones. Nine slow growing protoclones, two strandy and seven appressed, were analyzed for the first time with ISSR amplifications. The patterns were highly polymorphic and very reproducible. Among 40 primers tested, 7 ISSR primers were selected for the analysis of genomic DNA and generated a total of 68 ISSR fragments. ISSR fingerprinting detected 44.12% polymorphic loci. All appressed homokaryons carried a subset of ISSR markers found in the heterokaryons, and clustered separately in dendrogram. These were not able to produce a fruiting body. A test of cross-fertility and the following fruiting trial proved that 7 of the 9 protoclones with different ISSR fingerprints were homokaryons. These results demonstrated that ISSR markers provide an efficient alternate for identification of homokaryons and suggest these markers be considered as new tools for the survey of Agaricus species.     

Isolation and characterization of dinucleotide repeat microsatellites in Drosophila ananassae

Drosophila ananassae is a cosmopolitan species with a geographic range throughout most of the tropical and subtropical regions of the world. Previous studies of DNA sequence polymorphism in three genes has shown evidence of selection affecting broad expanses of the genome in regions with low rates of recombination in geographically local populations in and around India. The studies suggest that extensive physical and genetic maps based on molecular markers, and detailed studies of population structure may provide insight into the degree to which natural selection affects DNA sequence polymorphism across broad regions of chromosomes. We have isolated 85 dinucleotide repeat microsatellite sequences and developed assay conditions for genotyping using PCR. The dinucleotide repeats we isolated are shorter, on average, than those isolated in many other Drosophila species. Levels of genetic variation are high, comparable to Drosophila melanogaster. The levels of variation indicate the effective population size of an Indonesian population of D. ananassae is 58,692 (infinite allele model) and 217,284 (stepwise mutation model), similar to estimates of effective population size for D. melanogaster calculated using dinucleotide repeat microsatellites. The data also show that the Indonesian population is in a rapid expansion phase. Cross-species amplification of the microsatellites in 11 species from the Ananassae, Elegans, Eugracilis and Ficusphila subgroups indicates that the loci may be useful for studies of the sister species, D. pallidosa, but will have limited use for more distantly related species.     

Population structure in African Drosophila melanogaster revealed by microsatellite analysis

Tropical sub-Saharan regions are considered to be the geographical origin of Drosophila melanogaster. Starting from there, the species colonized the rest of the world after the last glaciation about 10 000 years ago. Consistent with this demographic scenario, African populations have been shown to harbour higher levels of microsatellite and sequence variation than cosmopolitan populations. Nevertheless, limited information is available on the genetic structure of African populations. We used X chromosomal microsatellite variation to study the population structure of D. melanogaster populations using 13 sampling sites in North, West and East Africa. These populations were compared to six European and one North American population. Significant population structure was found among African D. melanogaster populations. Using a Bayesian method for inferring population structure we detected two distinct groups of populations among African D. melanogaster. Interestingly, the comparison to cosmopolitan D. melanogaster populations indicated that one of the divergent African groups is closely related to cosmopolitan flies. Low, but significant levels of differentiation were observed for sub-Saharan D. melanogaster populations from West and East Africa.     

Genetic analysis of Pinus strobus and Pinus monticola populations from Canada using ISSR and RAPD markers: development of genome-specific SCAR markers

Pinus is the largest genus of conifers, containing over 100 species and is also the most widespread genus in the Northern Hemisphere. Pinus monticola and P. strobus are two closely related and economically important species in Canada. Morphological and allometric characteristics have been used to assess genetic variation within these two species but these markers are not reliable due to ecological variations. The purpose of the present study was to determine the level of genetic diversity within and among Canadian populations from the two species using molecular markers and to identify and characterize genome-specific inter-simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers. The level of genetic variation among populations was much lower for P. monticola than P. strobus. For both species, the among population variation values were smaller than within population variation. The populations from P. monticola were more closely genetically related than populations from P. strobus based on ISSR and RAPD analyses. Six ISSR and four RAPD markers specific to either P. monticola or P. strobus were cloned and sequenced. Primer pairs flanking these specific sequences were designed and genome specific SCAR markers for P. monticola and P. strobus were developed and characterized.     

Studying plant genome variation using molecular markers

The authors' studies on the organization and variation of plant genome with the use of molecular markers are briefly reviewed with special emphasis on random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), sequence characterized amplified region (SCAR), and cleaved amplified polymorphic sequence (CAPS) markers detected with the use of polymerase chain reaction (PCR). These markers have been demonstrated to be promising for identifying cultivars and determining the purity of genetic strains of pea. Genetic relationships between strains, cultivars, and mutants of pea have been studied. The role of molecular markers in molecular genetic mapping and localizing the genes of commercially important characters of pea has been shown. The possibility of the use of molecular markers for studying somaclonal variation and detecting mutagenic factors in plants during long-term spaceflights is considered. The prospects of using DNA markers for understanding the organization and variability of higher plant genomes are discussed.     

The effect of genetic environment on locus-specific instability of the yellow gene in Uman' population of Drosophila melanogaster

The effects of genotype of the laboratory strains, C(1)DX, ywf/Y, 23.5 MRF/CyL4, and C(1)DX,yf; pi2, on locus-specific instability in the yellow gene of the strains y(2-717, y(2-715), and y(2-700 ) from Uman' population of Drosophila melanogaster was studied. Crosses of the males from Uman'-derived lines with the C(1)DX, ywf/Y females yielded a cascade of derivatives, mostly consisting of y+ and y2 alleles, while their crosses with the 23.5 MRF/CyL4 and C(1)DX,yf; pi2 females mostly resulted in the appearance of y+ and y(1) derivatives. The genomes of laboratory strains used in the study contained the full-sized hobo elements, which could differ from one another relative to the structure of variable region and affinity to different DNA sequences.     

Analysis of genetic diversity in earthworms using DNA markers

Earthworms are one of the most important and beneficial macrofauna, and are used extensively in organic farming. Earthworms mediate soil biological regulation systems, and produce biogenic structures. They help to maintain soil structure, water infiltration, and regulate the availability of nutrients assimilated by plants. The objectives of this study were to perform morphological and molecular characterizations of 24 earthworm individuals collected from geographically diverse locations to assess the level of genetic variation. For molecular analysis, the effectiveness of RAPD, ISSR, and Universal rice primers (URPs) markers was investigated to identify polymorphism among 24 isolates of earthworms. A total of 62 molecular markers were used for amplification of genomic DNA of earthworms. Of these, 10 RAPD, 10 ISSR, and 10 URPs markers were used for characterization, which showed 95.7%, 96.7% and 98.3% polymorphism, respectively. The dendrogram, generated from the DNA markers by the unweighted pair group method using arithmetic averages, grouped all the isolates into two main clusters. All Eisenia fetida isolates were clustered in group A, whereas group B included three isolates belonging to Eudrilus eugeniae. Molecular markers allowed a rapid assessment of genetic variation among these closely related isolates of earthworms. These results suggest that molecular markers are a good choice for diversity analysis of earthworm individuals.     

Detecting DNA polymorphism and genetic diversity in Lentil (Lens culinaris Medik.) germplasm: comparison of ISSR and DAMD marker

Genetic diversity and interrelationships among 31 lentil genotypes were evaluated using 10 Inter-Simple Sequence Repeat (ISSR) and 10 directed amplification of minisatellite DNA region (DAMD) primers. A total of 43 and 48 polymorphic bands were amplified by ISSR and DAMD markers, respectively. Average polymorphism information content (PIC) for ISSR and DAMD markers were 0.37 and 0.41, respectively. All 31 lentil genotypes could be distinguished by ISSR markers into three groups and by DAMD markers into two groups. Various molecular markers show a different efficiency for evaluating DNA polymorphism in lentil and indicate that the patterns of variation are clearly influenced by the genetic marker used. Comparatively, the genetic diversity of examined lentil genotypes by two different marker techniques (ISSR and DAMD) was high and indicated that ISSR and DAMD are effective and promising marker systems for fingerprinting in lentil and give useful information on its genetic relationships.     

Clonal diversity in the rare Calamagrostis porteri ssp. insperata (Poaceae): comparative results for allozymes and random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) markers

Four populations of the rare, highly clonal grass Calamagrostis porteri ssp. insperata were examined using allozymes and the two polymerase chain reaction (PCR)-based markers, random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) bands. Only one of the 15 allozyme loci was variable and two alleles were detected, both of which were found in two populations, while only one genotype was detected in the other two populations. ISSR and RAPD markers detected more genotypes within populations than did allozymes. ISSR markers detected more diversity than RAPD markers in three of the four populations examined. In one population, no RAPD diversity was found whereas eight different genotypes were found among the 10 plants with ISSR markers. This diversity is present despite rare flowering, no documented occurrence of seed set in natural populations and very low seed set with experimental pollinations, all of which suggest that sexual reproduction rarely occurs. The subspecies is self-compatible, but seed initiation is lower in selfed ovules; also, there is high embryo abortion regardless of pollen source. Variation detected by RAPD and ISSR primers may reflect higher levels of sexual reproduction in the past, very rare sexual reproduction in extant populations, somatic mutations, or a combination of the three. Although the PCR-based markers identify several multilocus genotypes within populations, it is not known whether these all represent distinct genets generated by sexual reproduction or result from somatic mutations in the old, perennial and highly clonal plants.