Polymorphic Random Amplified Polymorphic DNA Research Articles

Comparison of AFLPs, RAPD Markers, and Isozymes for Diversity Assessment of Garlic and Detection of Putative Duplicates in Germplasm Collections

Garlic (Allium sativum L.) is an asexually propagated crop that displays much morphological diversity. Studies which have assessed garlic diversity with isozymes and randomly amplified polymorphic DNA (RAPD) markers generally agreed with the morphological observations but sometimes failed to discriminate clones. To discriminate among closely related garlic clones in more detail, we introduced amplified fragment-length polymorphism (AFLPs) to evaluate the genetic diversity and phenetic relatedness of 45 garlic clones and three A. longicuspis clones and we compared AFLP results with RAPD markers and isozymes. Three AFLP primer combinations generated a total of 183 polymorphic fragments. Although similarities between the clusters were low (≥0.30), some clones within the clusters were very similar (>0.95) with AFLP analysis. Sixteen clones represented only six different banding patterns, within which they shared 100% polymorphic AFLPs and RAPD markers, and likely are duplicates. In agreement with the results of other investigators, A. longicuspis and A. sativum clones were clustered together with no clear separation, suggesting these species are not genetically or specifically distinct. The topology of AFLP, RAPD, and isozyme dendrograms were similar, but RAPD and isozyme dendrograms reflected less and much less polymorphism, respectively. Comparison of unweighted pair group method with arithmetic averaging (UPGMA) dendrograms of AFLP, RAPD, and isozyme cluster analyses using the Mantel test indicated a correlation of 0.96, 0.55, and 0.57 between AFLP and RAPD, AFLP and isozyme, and RAPD and isozyme, respectively. Polymorphic AFLPs are abundant in garlic and demonstrated genetic diversity among closely related clones which could not be differentiated with RAPD markers and isozymes. Therefore, AFLP is an additional tool for fingerprinting and detailed assessment of genetic relationships in garlic.

Genetic diversity among elite Bulgarian barley varieties evaluated by RFLP and RAPD markers

Genetic variation among five elite winter barley cultivars (H. vulgare L.) currently grown in Bulgaria was assessed at the molecular level using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) markers. The present study sampled RFLPs in four well characterized multigene families in barley: the seed storage protein loci; the 18S, 5.8S and 26S ribosomal DNA loci; the loci coding for 5S ribosomal RNA and the loci coding subunit α of ATP-A complex in the mitochondrial genome. RFLPs were detected in three out of five investigated chromosomal loci in the barley cultivars studied. RAPD assay using arbitrary 10-base primers was applied to generate amplified length polymorphic markers in barley. Overall a total of 15 polymorphic phenotypes were found among the studied barley cultivars by using 11 out of 25 tested primers. All RAPDs were considered as dominant genetic markers except for two, where PCR and Southern blot analysis indicated the presence of codominant amplification products. Five RAPD polymorphisms in F1 and F2 progenies of the cross between Alpha and Obzor were inherited in Mendelian fashion. The determined values for the genetic variation proved a high genetic similarity among the tested cultivars. Genetic similarity (GS) calculated from RFLP and RAPD data ranged from 0.888 to 0.997 with a mean GS – 0.933.

RAPD polymorphisms in spring wheat cultivars and lines with different level of Fusarium resistance.

Random amplified polymorphic DNA (RAPD) markers have been used to characterize the genetic diversity among 35 spring wheat cultivars and lines with different levels of Fusarium resistance. The objectives of this study were to determine RAPD-based genetic similarity between accessions and to derive associations between Fusarium head blight (FHB) and RAPD markers. Two bulked DNA from either highly resistant lines or susceptible lines were used to screen polymorphic primers. Out of 160 screened primers, 17 primers generated reproducible and polymorphic fragments. Genetic similarity calculated from the RAPD data ranged from 0.64 to 0.98. A dendrogram was prepared on the basis of a similarity matrix using the UPGMA algorithm, which corresponded well with the results of principal component analysis and separated the 35 genotypes into two groups. Association analysis between RAPD markers and the FHB index detected three RAPD markers, H19(1000), F2(500) and B1(2400), significantly associated with FHB-resistant genotypes. These results suggest that a collection of unrelated genotypes can be used to identify markers linked to an agronomically important quantitative trait like FHB. These markers will be useful for marker-assistant breeding and can be used as candidate markers for further gene mapping and cloning.

RAPD markers assisted varietal identification and genetic purity test in pepper, Capsicum annuum

The potential of randomly amplified polymorphic DNA (RAPD) markers in varietal identification and genetic purity test of hybrid varieties was evaluated. Five Jalepeno hybrid varieties and their corresponding parents were screened for polymorphic RAPD markers with 12 arbitrary 10-mer primers. Among a total of 177 bands observed, 14 bands contributed by nine primers were polymorphic in the five pepper varieties. Furthermore, four RAPD markers were found to be cultivar-specific markers for three hybrid varieties. Out of the 12 primers, six primers generated 11 useful RAPD markers to determine seed purity of all tested hybrid varieties. In conclusion, RAPD markers maybe useful for cultivar identification and hybrid purity test in pepper, Capsicum annuum, especially for routine seed quality control programme.

Genetic analysis of two common carp broodstocks by RAPD and microsatellite markers

The whole broodstock of two Hungarian common carp farms—80 and 196 individuals—was analyzed by using random amplified polymorphic DNA (RAPD) assay and microsatellite analysis. Ten polymorphic RAPD markers and four microsatellites were selected to genotype both of the stocks. As expected, microsatellite analysis revealed more detailed information on genetic diversities than RAPD assay. Results obtained with both types of DNA markers showed lack of major differences between the genetic structure of the two stocks: heterozygosity values and allele frequencies were very similar. Dendrograms created from both sets of data did not show grouping of individuals according to stocks. Genotypes from the two stocks were also compared to those from a limited number of samples collected from other hatcheries and two rivers. Allele frequencies in the groups were similar, with the exception of wild carps. An interesting observation was that three private microsatellite alleles were found in the eight wild carp individuals, compared to the seven detected in the rest of the samples tested (372 individuals).

RAPD Marker Diversity among Cultivated and Wild Soybean Accessions from Four Chinese Provinces

Wild soybean (Glycine soja Siebold & Zucc.) is the ancestor of cultivated soybean [Glycine max (L.) Merr.] and is widely distributed in China, Japan, Korea, Taiwan, and eastern Russia. In North America, modern soybean cultivars are derived from a very limited germplasm base. Use of more soybean introductions and G. soja lines in breeding programs to expand the genetic base of and incorporate specific traits into commercial soybean cultivars could be beneficial. This study was conducted to evaluate the genetic variation between and within annual Glycine spedes and to determine geographical patterns of variation within and between the annual Glycine species. Forty G. max and 40 G. soja accessions from four Chinese provinces, Heilongjiang, Shandong, Jiangsu, and Shanxi, were surveyed with random amplified polymorphic DNA (RAPDs). The results indicated that the genetic distance (GD) within the G. soja group was larger than that within the G. max group, but smaller than that between the G. max and G. soja groups. Twenty-three more polymorphic RAPD fragments were detected within the G. soja group than within the G. max group. Nine fragments were present only in G. soja lines. On the basis of AMOVA analysis,18% of the total variation could be accounted for by species, 15% by populations within species, and 67% by individuals within populations. Cluster and principal component analyses completely separated the G. max and G. soja groups. The groups formed by cluster analyses generally reflected the geographical regions of origin. Glycine max and G. soja lines for the same province were not more genetically related than were accessions of different species from different provinces, but the GDs between the G. soja lines from Heilongjiang and the G. max accessions from all other provinces were less than for the G. soja accessions from any other provinces. The results of this study could be used for exploiting the genetic diversity in the two species in breeding programs and in sampling and managing germplasm collections.

Analysis of Genetic Diversity in Cicer arietinum L Using Random Amplified Polymorphic DNA Markers

PCR-based random amplified polymorphic DNA (RAPD) markers were employed to assess genetic diversity in 23 chickpea genotypes. Forty of the 100 random primers screened revealed polymorphism among the genotypes. Most of the primers revealed single polymorphic band, and only 14.1 2% of the products were polymorphic. Estimates of genetic similarity based on Jaccard’s coefficient ranged from 0.92 to 0.99, indicating narrow genetic variability among the genotypes based on RAPD markers.The 23 chickpea genotypes formed two major clusters in the dendrogram.The low RAPD polymorphism among chickpea genotypes suggests that more number of polymorphic primers need to be analysed to determine genetic relationships. It was observed that RAPD analysis employing 30 polymorphic primers could provide better estimates of genetic relationships in chickpea.

Evaluation of genetic diversity in 19 Glycine max (L.) Merr. accessions included in the Czech National Collection of soybean genotypes

The random amplified polymorphic DNA (RAPD) technique was used to evaluate both genetic diversity among 19 soybean accessions included in the Czech National Collection of Soybean Genotypes and their potential as a new source of genetic variations for soybean breeding programs. Only 22 of all the 40 random primers used in RAPD reactions showed polymorphism acceptable for an effective characterisation of these accessions. Altogether 122 highly reproducible RAPD fragments were generated, 55 of them were polymorphic (46%). However, because of the previously observed low degree of RAPD polymorphism in the case of Glycine max, fragments with low level of informativeness were evaluated, too. Presented results enable the selection of genetically distinct individuals. Such information may be useful to breeders willing to use genetically diverse introductions in soybean improvement process. 

RAPD Polymorphism as an Indicator of Population Structure, Breeding System, and Speciation in Fossombronia

Abstract The breeding system of Fossombronia foveolata and genetic differentiation in the F. foveolata species complex were studied using Random Amplified Polymorphic DNA (RAPD) markers. Polymorphism values for cloned siblings were not significantly different from those obtained from amplification of identical DNA samples. These results support the hypothesis that sporophytes display high levels of homozygosity. Nevertheless, low levels of genetic differentiation were detected between F. foveolata populations from spatially separated sites in southern Illinois. Monomorphism among siblings, low levels of polymorphism among southern Illinois populations, sporophyte production in clonal cultures maintained in an environmental chamber, growth of cultured clonal mats to sizes comparable to field populations, and short sperm and spore dispersal distances suggest that F. foveolata populations are probably established from single founder events and are maintained through inbreeding and vegetative reproduction. De...

Genetic variability evaluation in a Moroccan collection of barley, Hordeum vulgare L., by means of storage proteins and RAPDS

The genetic variation existing in a set of barley (Hordeum vulgare L.) landrace samples recently collected in Morocco was estimated. Two kinds of genetic markers, seed storage proteins (hordeins) and random amplified polymorphic DNA (RAPD), were used. Only six out of 31 landraces were subjected to RAPD analysis. Both kinds of markers, RAPD and storage proteins, yielded similar results, showing that the level of variation observed in Moroccan barley was high: all landraces showed variability; 808 different storage protein patterns (multilocus associations) were observed among 1897 individuals (2.32 seeds per association, on average) with an average of 43 multilocus associations per accession. In general, genetic variation within accessions was higher than between accessions. The 100 polymorphic RAPD bands generated by 21 effective primers were able to generate enough patterns to differentiate between uniform cultivars and even between individuals in variable accessions. One of the aims of this work was to compare the effectiveness of RAPD versus storage protein techniques in assessing the variability of genetic resource collections. On average hordeins were more polymorphic than RAPDs: they showed more alternatives per band on gels and a higher percentage of polymorphic bands, although RAPDs supply a higher number of bands. Although RAPD is an easy and standard technique, storage protein analysis is technically easier, cheaper and needs less sophisticated equipment. Thus, when resources are a limiting factor and considering the cost of consumables and work time, seed storage proteins must be the technique of choice for a first estimation of genetic variation in plant genetic resource collections.

Cultivar Identification in Olive Based on RAPD Markers

One hundred and thirteen olive (Olea europaea L.) accessions were characterized using randomly amplified polymorphic DNA (RAPD) markers. Forty-five polymorphic RAPD markers were obtained enabling us to distinguish 102 different RAPD profiles. The approximate estimation of the probability of obtaining the same RAPD profile for two different trees was between 6.75 × 10-5 and 4.82 × 10-14. A dendrogram was constructed using Ward's minimum variance algorithm based on chi-square distances. This led to a more clear-cut classification of profiles than the classical approach of unweighted pair group method with arithmetic average. Twenty-four clusters of RAPD profiles were shown in Ward's dendrogram. Reliability of the dendrogram structure was checked using variance analysis. RAPD data exhibited an acceptable resolving power for cultivar identification. A combination of three primers was proposed for rapid molecular identification of cultivars in collections and in nurseries.

HIERARCHICAL SPATIAL STRUCTURE AND DISCRIMINANT ANALYSIS OF GENETIC DIVERSITY IN THE RED ALGA MAZZAELLA LAMINARIOIDES (GIGARTINALES, RHODOPHYTA)

Our study of the genetic structure of Mazzaella laminarioides (Bory) Fredericq (Gigartinales) in the central Chilean region documented a high level of genetic diversity based on random amplified polymorphic DNA (RAPD) markers and indicated the occurrence of significant genetic structure at different spatial scales. A total of 288 haploid gametophytes was analyzed with 17 polymorphic RAPD bands, which produced 202 distinct multilocus genotypes. Within stands, mean gene diversity ranged from 0.210 to 0.249 and no significant linkage disequilibrium could be detected among pairs of alleles, revealing that recombination (sexual reproduction) regularly shuffles the genes at that scale. Analysis of molecular variance within stands (less than 30 m) showed that the structure was very low, only marginally significant, and did not increase with increasing hierarchical levels at this lowest spatial scale. In contrast, at a larger spatial scale (among stands, from 5 to 60 km), increasing geographical distance seemed to account for increasing isolation between populations even if natural barriers, such as sandy beaches or river estuaries, may play a role in such isolation. Moreover, the strong genetic differentiation occurring between locations separated by 60 km allowed the assignment of individuals to their original population through a canonical discriminant analysis. This approach further allowed the identification of potential recent migrants from one population to the other. Thus, in species like M. laminarioides for which the dominance of RAPD markers can be avoided by selecting haploid individuals, RAPD analysis appeared to be specially appropriate for the study of genetic differentiation.

GENETIC DIVERSITY AMONG CHICKEN BREEDS ESTIMATED THROUGH RANDOMLY AMPLIFIED POLYMORPHIC DNA

The randomly amplified polymorphic DNA (RAPD) markers were used to detect polymorphism among five breeds of chicken i.e. White Leghorn and Rhodes Island Red (selected for part period egg production and egg mass respectively), Red Cornish and White Plymouth Rock (selected for early body weights) and Kadaknath (native breed). Twelve of the fifty random primers screened yielded distinct polymorphic RAPD profiles. Of the total 96 fragments amplified, about 25 % showed polymorphism. Using the RAPD data matrix, the within population and between population genetic similarity was estimated. The selected improved breeds showed higher within population genetic similarity in comparison to the native breed. The two meat type breeds showed a high level of genetic similarity between themselves. The White Leghorn breed showed a low genetic similarity with other breeds. The native breed showed highest similarity with Rhodes Island Red. The dendogram was constructed to show phylogenetic relationship among these breeds. As expected, the genetic distances were lowest within similar type breeds and were highest between dissimilar type breeds. The results indicated the effectiveness of RAPD in detecting polymorphism between chicken populations and their applicability in population studies and establishing genetic relationships among the chicken populations.

The extent of clonality and genetic diversity in lingonberry (Vaccinium vitis‐idaea L.) revealed by RAPDs and leaf‐shape analysis

Numerous plant species reproduce mainly by clonal growth, implying that levels of genetic variation may be comparatively low. In this study we describe the genetic and genotypic diversity within and between four Swedish populations of the clonal shrub lingonberry, Vaccinium vitis-idaea L. Two approaches were used to assess the amount and partitioning of variation: automated image analysis of leaf shape and random amplified polymorphic DNA (RAPD) analysis. Morphometric analyses, using moment invariants and elliptic Fourier coefficients, revealed that most of the variation could be attributed to within-population variation. With the use of 43 polymorphic RAPD markers, we were able to identify 29 different genotypes (i.e. putative clones) among 129 plants from two populations. The genotypic diversity (D: mean 0.84) and evenness (E: mean 0.81) were higher than the average for clonal plant species. Within-population gene diversity was similar to values reported in nonclonal plants, suggesting that sexual reproduction has played a significant role in these populations despite low levels of seedling recruitment in present-day populations. An analysis of molecular variance revealed that most of the variation (89.2%) resided within populations. Comparisons between the different suites of characters indicated a congruent pattern of partitions of diversity, particularly when comparing moment invariants and RAPDs. When comparing the ability of the two descriptor suites to assign the plants to the RAPD-defined clones, elliptic Fourier coefficients yielded the best result; a classification test correctly reassigned 96.9% and 98.1% of the plant material in the two respective populations.

Estimating the genetic relationship of hybrid bromegrass to smooth bromegrass and meadow bromegrass using RAPD markers

AbstractThe two most widely grown bromegrass species in North America are smooth bromegrass, a hay type grass, and meadow bromegrass, a pasture type grass. Hybrid populations between these two species have been developed through hybridization and recurrent selection. The objective of this study was to determine the genetic relatedness of the hybrid bromegrass population S‐9073M to its parental populations using random amplified polymorphic DNA (RAPD) markers and to determine genetic variation within and between populations. Individuals from each of the three populations were genotyped at 43 polymorphic RAPD loci. One of the RAPD fragments was meadow bromegrass‐specific. Cluster analysis showed three groups representing the two parental populations and the hybrid population. An analysis of molecular variance (AMOVA), showed that the hybrid population had the highest within‐population variation, followed by meadow bromegrass and smooth bromegrass. The interpopulation genetic distance (phi‐statistic =Φst) was highest between meadow and smooth bromegrass and lowest between smooth and hybrid bromegrass. The hybrid population was genetically intermediate to smooth bromegrass and meadow bromegrass, but closer to smooth bromegrass, possibly reflecting the selection criteria used in the development of this hybrid.

Use of RAPD markers for the study of microbial community similarity from termite mounds and tropical soils

In this study, we test the use of the RAPD (Random Amplified Polymorphic DNA) molecular markers as a way to estimate the similarity of the microbial communities in various termite mounds and soils. In tropical ecosystems, termite activities induce changes in the chemical and physical properties of soil. The question then arises as to whether or not termites affect the presence of natural microbial communities. Successful 16S rDNA amplifications provided evidence of the occurrence of bacterial DNA in termite constructions including both soil feeder and fungus grower materials. A phenetic dendrogram using the similarity distance calculated from pairwise data including 88 polymorphic RAPD markers was reconstructed and bootstrap scores mapped. The microbial communities of the mounds of the four soil-feeding termites were clustered in the same clade, while those of the mounds of the fungus-growing species were distinct like those of control soils. Microbial changes in nests result from termite building behavior, depending on whether they include feces in their constructions for soil-feeders or use saliva as particle cement for fungus-growers. It is argued that RAPDs are useful markers to detect differences in microbial community structure not only between termitaries and control soils but also between mounds of soil-feeders.

FORCING AND MOLECULAR CHARACTERIZATION OF CAMPANULA

Campanula portenschlagiana Roem and Schult germplasm was evaluated horticulturally and using a molecular marker. Flowering of C. portenschlagiana collected in the United States (CPC) was accelerated by 16-hour long day (LD) photoperiod at 7.5°C. However, flowering of C. portenschlagiana collected from Japan (CPJ) was not affected by LD at 7.5°C.). CPC and CPJ were grouped closely when polymorphic RAPD bands were analyzed. Seedlings of C. portenschlagiana from Germany (CPG) were grouped distantly from CPJ and CPC and were significantly different in growth habit. The results of greenhouse forcing and randomly amplified polymorphic DNA (RAPD) analysis, species and cultivars should be characterized and identified morphologically and using molecular markers.

CHARACTERIZATION OF ACER GRISEUM AND ITS PUTATIVE INTERSPECIFIC HYBRIDS

Acer griseum (Franchet) Pax, A. maximowiczianum Miquel, A. triflorum Komarov and putative interspecific hybrids between A. griseum and A. maximowiczianum were characterized using RAPD markers. Variations in randomly amplified polymorphic DNA (RAPD) banding patterns were observed among A. griseum seedlings. According to neighbor-joining analysis, several A. griseum trees growing in the US National Arboretum were very distinct from A. griseum accessions growing at the Arnold Arboretum and at the Morris Arboretum. Hybrids between A. griseum and A. maximowiczianum had morphological characteristics that were intermediate between both parents. However, these hybrids were grouped closer to A. maximowiczianum than to A. griseum according to phylogenetic distance analysis generated by polymorphic RAPD markers. Variations among A. griseum accessions could be the result of natural segregation within species or introgression between A. griseum and other species belonging to Section Trifoliata.

Use of random amplified polymorphic DNA (RAPD) markers to reveal genetic diversity within and between populations of cashew (Anacardium occidentale L.)

SummaryRAPD (random amplified polymorphic DNA) markers were used to determine genetic diversity within and between populations of cashew (Anacardium occidentale L.). Efficient and novel procedures for extracting PCR-amplifiable, high molecular weight DNA from young cashew leaves were developed and the PCR conditions for RAPD analysis of cashew DNA using random 10.bp primers were defined. Optimized PCR-reaction conditions were then used to analyse differences in RAPD profiles within and between a selection of cashew varieties obtained from diverse geographical locations around the world and within a pool of twenty elite Tanazanian cashew lines. RAPD polymorphisms were obtained among the Tanzanian lines and between and within the geographically diverse lines. The relatively uniform RAPD profiles for the selection of random primers tested suggested a high degree of DNA level similarity between the Tanzanian accessions. Accessions from India, Mozambique and Tanzania showed the closest relationship, with accessions from Brazil being the most distinct from the other provenances. A specific RAPD-PCR product was linked with cashew accessions from the Cook Islands.

Microsatellite and RAPD polymorphisms in Ontario corn hybrids are related to the commercial sources and maturity ratings

Random-amplified polymorphic DNA (RAPD) and microsatellite markers were used to estimate the genetic relationships among 37 Ontario corn hybrids. Almost all (95%) of the 160 RAPD fragments and all of the 79 microsatellite alleles were polymorphic across the 37 hybrids. Similarity values among the hybrids ranged from 31% to 86% when based on the RAPD data. The similarities based on microsatellite markers ranged from 12% to 77%. The genetic diversity revealed by microsatellite marker analysis was higher than that obtained from RAPD analysis. The similarity matrices for the microsatellite data and the RAPD data were moderately correlated (0.43). Cluster analyses based on either type of marker showed that most of the hybrids from the same company were closely related to each other. Both dendrograms clustered similar pairs or groups of hybrids. A principal component analysis, based on the combined RAPD and microsatellite data, yielded a good separation of the hybrids with Ontario Corn Heat Unit (OCHU) values 2800. Seventeen RAPD markers and 5 microsatellite markers were significantly associated with the OCHU ratings of the hybrids.