Genetic Relationships Among Wild and Cultivated Accessions of Curry Leaf Plant (Murraya koenigii (L.) Spreng.), as Revealed by DNA Fingerprinting Methods

Murraya koenigii (L.) Spreng. (Rutaceae), is an aromatic plant and much valued for its flavor, nutritive and medicinal properties. In this study, three DNA fingerprinting methods viz., random amplification of polymorphic DNA (RAPD), directed amplification of minisatellite DNA (DAMD), and inter-simple sequence repeat (ISSR), were used to unravel the genetic variability and relationships across 92 wild and cultivated M. koenigii accessions. A total of 310, 102, and 184, DNA fragments were amplified using 20 RAPD, 5 DAMD, and 13 ISSR primers, revealing 95.80, 96.07, and 96.73% polymorphism, respectively, across all accessions. The average polymorphic information content value obtained with RAPD, DAMD, and ISSR markers was 0.244, 0.250, and 0.281, respectively. The UPGMA tree, based on Jaccard’s similarity coefficient generated from the cumulative (RAPD, DAMD, and ISSR) band data showed two distinct clusters, clearly separating wild and cultivated accessions in the dendrogram. Percentage polymorphism, gene diversity (H), and Shannon information index (I) estimates were higher in cultivated accessions compared to wild accessions. The overall high level of polymorphism and varied range of genetic distances revealed a wide genetic base in M. koenigii accessions. The study suggests that RAPD, DAMD, and ISSR markers are highly useful to unravel the genetic variability in wild and cultivated accessions of M. koenigii.     

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     

SPAR methods revealed high genetic diversity within populations and high gene flow of Vanda coerulea Griff ex Lindl (Blue Vanda), an endangered orchid species

Molecular genetic fingerprints of seven populations of Vanda coerulea comprising of thirty-two genotypes from Northeast India were developed using PCR based markers. Genetic variability in the wild genotypes of V. coerulea was analyzed using two different single primer amplification reactions (SPAR) methods, viz., random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR). A total of 32 genotypes were used to investigate the existing natural genetic diversity at intra-specific level. Two hundred and twenty six (226) amplification products were scored by RAPD and ISSR, both of which collectively showed 58.88% polymorphism with a mean intra-population genetic diversity (Hpop) of 0.119. However, their level of diversity at inter- and intra-population levels was significant, with the percentage of polymorphic loci (Pp) ranging from 17.70% to 45.13%, Shannon's information index (I) from 0.105 to 0.268 and Nei's gene diversity (h) from 0.072 to 0.185 with mean Nei's gene diversity 0.174 and the overall estimate of gene flow being (Nm) 1.165. Analysis of molecular variance (AMOVA) showed 96.07% of variation at intra-population level, whereas 3.93% variation was recorded at inter-population level. Only one major cluster was detected by cluster analysis using the unweighted pair-group method with arithmetic average (UPGMA). Present investigation suggests the efficiency of SPAR methods to estimate the genetic diversity of V. coerulea and can be seen as a starting point for future research on the population and evolutionary genetics of this species.     

Comparison of RAPD and ISSR markers for genetic diversity analysis among different endangered Mangifera indica genotypes of Indian Gir forest region

The genetic variability and relationships among 20 Mangifera indica genotypes representing 15 endangered and 5 cultivars, obtained from Indian Gir forest region, were analyzed using 10 random amplified polymorphic DNA (RAPD) and 21 inter simple sequence repeat (ISSR) markers. RAPD markers were more efficient than the ISSR assay with regards to polymorphism detection. Also, the average numbers of polymorphic loci per primer, average polymorphic information content (PIC) and primer index (PI) values were more for RAPD than for ISSR. But, total number of genotype specific marker loci, Nei’s genetic diversity (h), Shannon’s information index (I), total heterozygosity (Ht), average heterozygosity (Hs) and mean coefficient of gene differentiation (Gst) were more for ISSR as compared to RAPD markers. The regression test between the two Nei’s genetic diversity indexes showed low regression between RAPD and ISSR based similarities but maximum for RAPD and RAPD + ISSR based similarities. The pattern of clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrogram were compared. Thus, both the markers were equally important for genetic diversity analysis in M. indica.     

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.     

Analysis of genetic variability and population structure of the endemic medicinal Limonium sinense using molecular markers

Limonium sinense is an endemic medicinal herb used to treat fever, hemorrhage and other disorders. In the present study, population genetic diversity was elucidated using random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) primers. Percentage of polymorphic bands, Nei's gene diversity and Shannon's information index revealed a high level of genetic diversity at species level. The analysis of molecular variance revealed that 69.88% (RAPD), 71.19% (ISSR) and 70.97% (AFLP) of variability were partitioned among individuals within populations, which indicated the coherent trend by Gst (0.3849/0.3577/0.3670). Gene flow number (Nm) was 0.581/0.618/0.612, which indicated that there was a limited gene exchange between populations. The UPGMA clustering results showed that the genetic distance had no significant correlation with geographic distance. These results indicate that these markers were reliable tools for the differentiation and determination of the genetic diversity among the populations of L. sinense and the conservation of existing natural population is necessary.     

Genetic diversity and population structure of Butea monosperma (Lam.) Taub.- a potential medicinal legume tree

Three molecular marker systems, Random Amplified Polymorphic DNA (RAPD), Inter-Simple Sequence Repeats (ISSR) and Sequence-Related Amplified Polymorphism (SRAP) were employed to investigate the genetic structure and diversity among the 14 natural populations of Butea monosperma collected from different geographical regions of India. Detected by 17 RAPD, 15 ISSR and 11 SRAP primer combinations, the proportions of polymorphic bands were 84.2 %, 77.2 % and 91.9 %, respectively, and the mean Nei’s genetic distances among the populations were 0.13, 0.10 and 0.13, respectively. Partitioning of genetic variability by Analysis of molecular variance (AMOVA) revealed that the high genetic diversity was distributed within the populations. AMOVA also revealed that the coefficient of gene differentiation among populations based on F_ST was very high irrespective of markers used. The overall gene flow among populations (Nm) was very low. Cophenetic correlation coefficients of Nei’s distance values and clustering pattern by Mental test were statistically significant for all three marker systems used but poor fit for ISSR data than for RAPD, SRAP and combined data set of all three markers. For all markers, a high similarity in dendrogram topologies was obtained, although some differences were observed with ISSR. The dendrogram obtained by RAPD, SRAP and combined data set of all three markers reflect relationship of most of the populations according to their geographic distribution.     

The extent of clonality and genetic diversity in the rare Caldesia grandis (Alismataceae): Comparative results for RAPD and ISSR markers

Genetic variation and clonal diversity of three natural populations of the rare, highly clonal marsh herb Caldesia grandis Samuelsson were investigated using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Both of the markers worked effectively in clone identification of C. grandis. RAPD markers detected more diversity than ISSR markers in the three populations examined. Of the 60 RAPD primers screened, seven produced highly reproducible bands. Using these primers, a total of 61 DNA fragments were generated with 52 (85.25%) being polymorphic indicating considerable genetic variation at the species level. Analysis of molecular variance (AMOVA) showed that a large proportion of genetic variation (81.5%) resided within populations, while only a small proportion (18.5%) resided among populations. With the use of 52 polymorphic RAPD markers, we were able to identify 127 genets among 342 samples from three populations. The proportion of distinguishable genets (PD: mean 0.37), Simpson's diversity index (D: mean 0.91), and evenness (E: mean 0.78) exhibited high levels of clonal diversity compared to other clonal plants. These results imply that sexual reproduction has played an important role at some time during the history of these populations. Nevertheless, the high level of diversity could have been also partially generated from somatic mutations, although this is unlikely to account for the high diversity generally found among C. grandis genets.     

Molecular genetic diversity of Satureja bachtiarica

Fifty-seven genotypes from eight population of Satureja bachtiarica was evaluated using fifteen ISSR and eleven RAPD markers. DNA profiling using RAPD primers amplified 84 loci, among which 81 were polymorphic with an average of 7.36 polymorphic fragments per locus. Also, using RAPD markers maximum and minimum polymorphic bands observed for Semyrom (77.38 %) and Farsan (40.48 %) populations, respectively. Semyrom population recorded the highest unbiased expected heterozygosity (0.259) and Shannon’s Indices (0.38). While, the lowest values of unbiased expected heterozygosity (0.172) and Shannon’s Index (0.245) were recorded for Eghlid and Farsan populations, respectively. On the other hand, ISSR primers produced 136 bands, from which 134 were polymorphic with an average of 9.06 polymorphic fragments per primer (98.52 %). The ISSR markers evaluation revealed that maximum and minimum polymorphic bands observed for Semyrom (66.18 %) and Farsan (31.62 %), respectively. Shahrekorud population recorded the highest unbiased expected heterozygosity (0.211) and Shannon’s Indices (0.301). While, the lowest value of unbiased expected heterozygosity (0.175) observed for Farsan and Yazd populations and the lowest Shannon’s Index (0.191) recorded by Farsan population. The overall results of the study revealed that both ISSR and RAPD markers were effective for evaluation of genetic variation of S. bachtiarica.     

Genetic diversity of commercially grown Moringa oleifera Lam. cultivars from India by RAPD, ISSR and cytochrome P450-based markers

Moringa oleifera is a less used, drought-tolerant tropical plant, rich in nutritionally and nutraceutically important bioactive compounds. It is native to India and now under cultivation in many countries, but no data is available on genetic variability. Three DNA marker techniques, i.e., random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and cytochrome P₄₅₀ gene-based markers were used for the detection of genetic variability in eight Indian cultivars of M. oleifera, collected from various states of India. A total of 17 RAPD, 6 ISSR and 7 pairs of cytochrome P₄₅₀-based markers generated 48.68, 48.57 and 40.00 % polymorphisms, respectively. The marker index (MI) for each of these marker systems (3.25 for RAPD, 4.73 ISSR and 2.95 for Cyt P₄₅₀-based markers) suggest that ISSR markers are the most effective for assessment of genetic diversity. Based on the three types of marker data, the eight cultivars of M. oleifera were grouped into four sub-clusters in a dendrogram, but without any distinct geographical pattern. This suggests spread of planting material and high rates of gene flow through cross pollination. High bootstrap values (94.4 and 82.3) were obtained at major nodes of the dendrogram using the winboot software. The dendrogram and PCA plots generated from the binary data matrices of the three marker systems were found highly concordant to each other. This study reveals a huge genetic diversity among the cultivars and this can be utilised for conservation and cultivar development in breeding programmes to produce high yielding, nutritionally superior cultivars.     

Genetic diversity in Monilinia laxa populations in stone fruit species in Hungary

The objectives of this study were firstly, to determine the genetic diversity of Monilinia laxa isolates from Hungary, using the PCR-based inter-simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) technique; secondly, to prepare genetic diversity groups based on the dendrograms; and finally, to select some relevant isolates to study their fungicide sensitivity. 55 and 77 random amplified polymorphic ISSR and RAPD markers, of which 23 and 18 were polymorphic and 32 and 59 monomorphic, respectively, were used to assess the genetic diversity and to study the structure of M. laxa populations in Hungary. 27 isolates out of 57 ones were confirmed as M. laxa from several orchards (subpopulations) in three geographical regions, in various inoculum sources and in various hosts, were used. 10 fungicides and 12 isolates selected from genetic diversity groups based on the ISSR dendrograms were used to determine the fungicide sensitivity of the selected isolates. The analysis of population structure revealed that genetic diversity within locations, inoculum sources and host (H _S ) accounted for 99 % of the total genetic diversity (H _T ), while genetic diversity among locations, inoculum sources and host represented only 1 %. The relative magnitude of gene differentiation between subpopulations (G _ST ) and the estimate of the number of migrants per generation (Nm) averaged 0.005–0.009 and 53.9–99.2, respectively, for both ISSR and RAPD data set. The results obtained in dendrograms were in accordance with the gene diversity analysis. Grouping of isolates in the dendrograms was irrespective of whether they came from the same or different geographical locations. There was no relationship between clustering among isolates from inoculum sources and hosts. In the fungicide sensitivity tests, five isolates out of 12 were partly insensitive to boscalid+piraclostrobin, cyprodinil, fenhexamid or prochloraz. Obtained results in genetic diversity of M. laxa populations are discussed together with implications for the management of brown rot.     

A method to measure genetic distance between allogamous populations of alfalfa (Medicago sativa) using RAPD molecular markers

 Alfalfa (Medicago sativa L.) is a forage legume of world-wide importance whose both allogamous and autotetraploid nature maximizes the genetic diversity within natural and cultivated populations. This genetic diversity makes difficult the discrimination between two related populations. We analyzed this genetic diversity by screening DNA from individual plants of eight cultivated and natural populations of M. sativa and M.  falcata using the RAPD method. A high level of genetic variation was found within and between populations. Using five primers, 64 intense bands were scored as present or absent across all populations. Most of the loci were revealed to be highly polymorphic whereas very few population-specific polymorphisms were identified. From these observations, we adopted a method based on the Roger’s genetic distance between populations using the observed frequency of bands to discriminate populations pairwise. Except for one case, the between-population distances were all significantly different from zero. We have also determined the minimal number of bands and individuals required to test for the significance of between-population distances. Received: 7 July 1997 / Accepted: 28 October 1997     

Inter-simple sequence repeat (ISSR) variation in forest coffee trees (Coffea arabica L.) populations from Ethiopia

Genetic variation of forest coffee trees (Coffea arabica L.) from four regions of Ethiopia was investigated using inter-simple sequence repeat (ISSR) markers. A total of 160 individuals representing 16 populations were sampled. Eleven ISSR primers amplified a total of 123 fragments of which 31 fragments (25%) were polymorphic. Estimate of total gene diversity (H _T), and the coefficient of genetic differentiation (G _ST) were 0.37 and 0.81, respectively. This indicates that most of the variability is between populations than within populations. The partitioning of genetic variation into within and between populations based on Shannon’s information index also revealed more differentiation between populations (0.80) than within populations (0.20). In the phenogram most of the coffee tree samples were clustered on the basis of their regions of origin but failed to cluster according to their respective populations, which could be attributed to the presence of substantial gene flow between adjacent populations in each region assisted by man in the process of transplantation or by wild animals such as monkeys, which eat the berries and defecate the seeds elsewhere. On the other hand, the inter-regional clustering of some coffee tree samples from Bale and Jimma regions could be due to the transport of coffee seeds across regions and their subsequent planting. Although ISSR markers detected lower polymorphic loci than previously reported results with random amplified polymorphic DNA (RAPD) markers on the same materials, it can be used as an alternative method for molecular characterization of C. arabica populations. The results may provide information to select sites for in situ conservation.     

Intra-specific genetic diversity in wild olives (Olea europaea ssp cuspidata) in Hormozgan Province, Iran

Wild olive (O. europaea ssp cuspidata) plants grow in various regions of Iran and are expected to have considerable genetic diversity due to adaptation to the various environmental conditions. We examined the genetic diversity of four populations of wild olive growing in Hormozgan Province located in southern Iran by using 30 RAPDs and 10 ISSR markers. The mean value of polymorphism for RAPD loci was 73.71%, while the value for ISSR loci was 81.74%. The Keshar population had the highest value of intra-population polymorphism for both RAPD and ISSR loci (66.86 and 62.71%, respectively), while the Tudar population had the lowest values (20.35 and 28.81%, respectively). Similarly, the highest and lowest number of effective alleles, Shannon index and Nei's genetic diversity were also found for these two populations. The highest value of H(pop)/H(sp) within population genetic diversity for RAPD and ISSR loci was found for the Keshar population (H(pop) = 0.85 and H(sp) = 0.90). OPA04-750, OPA13-650 and OPA02-350 RAPD bands were specific for Tudar, Bondon and Keshar populations, respectively, while no specific ISSR bands were observed. Analysis of molecular variance as well as the pairwise F(ST) test showed significant differences for RAPD and ISSR markers among the populations. The NJ and UPGMA trees also separated the wild olive populations from each other, indicating their genetic distinctness. UPGMA clustering of the four wild olive populations placed the Tudar population far from the other populations; Keshar and Bokhoon population samples revealed more similarity and were grouped together. We conclude that there is high genetic diversity among O. europaea ssp cuspidata populations located in southern Iran. We also found RAPD and ISSR markers to be useful molecular tools to discriminate and evaluate genetic variations in wild olive trees.     

Estimation of genetic variability and population structure in Sapindus trifoliatus L., using DNA fingerprinting methods

Genetic variability and population structure of Sapindus trifoliatus L. (Sapindaceae), collected from Gujarat, Karnataka and Uttar Pradesh states were estimated using three DNA fingerprinting methods viz., random amplified polymorphic DNA (RAPD), directed amplification of minisatellite DNA (DAMD) and inter-simple sequence repeats (ISSR). The cumulative data analysis carried out for all three markers showed 69.42 % polymorphism. The intra-population genetic diversity analysis revealed the highest values of Nei’s genetic diversity (0.16), Shannon information index (0.24) and polymorphic loci (43.99 %) among Bhavnagar (BH) population, whereas lowest values were found in Junagarh (JU) population. The maximum inter-population average genetic distance (0.20) was between Allahabad (AL) and JU populations. Analysis of molecular variance (AMOVA) showed highest percentage of variation among individuals of populations (56 %) followed by 25 % among populations and 19 % among regions. Principal coordinate analysis and UPGMA dendrogram revealed that genetic diversity was in congruence with the geographical diversity. The data strongly suggest that low genetic flow, geographic isolation and to some extent genetic drift are the major factors responsible for high genetic differentiation. Preservation of genetic diversity of S. trifoliatus is important, both to promote adaptability of the populations to changing environment as well as to preserve a large gene pool for future genetic improvement. The present study using RAPD, DAMD and ISSR profiles of S. trifoliatus provide the means of rapid characterization of accessions within the populations, and thus enable the selection of appropriate accessions for further utilization in conservation and prospection programs of this important plant genetic resource.     

Assessment of genetic variability through ISSR and RAPD markers in <Emphasis Type="Italic">Commiphora wightii</Emphasis> (Arn.) Bhandari

Commiphora wightii (Arn.) Bhandari is a commercially, medicinally and traditionally important tropical shrub widely used to treat various ailments and disorders. Demand of this plant is increasing in the pharmaceutical and perfumery industries due to the presence of guggulsterone E and Z, two important isomers conferring lipid- and cholesterol-lowering, and anti-cancerous properties. Ruthless and unscientific harvesting of oleo-gum resin by local populations from the wild, with negligible conservation efforts has made this species endangered and led to its inclusion in the Red Data Book of IUCN. It is imperative to have broad information regarding the extent of genetic variability available in the species to accelerate the breeding and conservation programs. Therefore, the present study was undertaken to analyze the extent of genetic variability existing among the C. wightii germplasm collected from Rajasthan and Haryana, the diversity rich Indian states, using ISSR and RAPD markers. A total of 100 (50 each) RAPD and ISSR markers were screened of which 37 RAPD and 43 ISSR primers were able to amplify DNA fragments. RAPD markers were more efficient, detecting 74.16 % polymorphism, compared to ISSR which detected 62.52 % polymorphism. Also, the values of average number of polymorphic bands per assay, polymorphism information content (PIC), diversity index (DI) and marker index (MI) were more for RAPD (7.76, 0.19, 0.38 and 2.53, respectively) than for ISSR (7.02, 0.13, 0.32 and 1.88) markers. The UPGMA dendrogram constructed using individual as well as combined data of the two marker systems separated the collected accessions into two major clusters containing 47 and 4 accessions, respectively, while one accession from Bikaner was not included in any cluster. Genetic similarity values obtained from Jaccard’s coefficient using combined data of both the marker systems were between 0.50 and 0.97. These results indicated the existence of wide genetic variability within this species and can be used for further research in the area of germplasm conservation, population genetics and plant breeding.     

Genetic diversity across natural populations of <Emphasis Type="Italic">Dendrobium officinale</Emphasis>, the endangered medicinal herb endemic to China,revealed by ISSR and RAPD markers

Dendrobium officinale is a rare and endangered herb with special habitats and endemic to China. Genetic diversity was examined within and among nine natural populations using inter-simple sequence repeat (ISSR) and random amplified polymorphic (RAPD) for conservation. Both molecular markers revealed a high percentage (>89%) of polymorphic bands and ISSR markers detected more diversity than RAPD markers. Analysis of molecular variance (AMOVA) revealed that 78.84% (ISSR) and 78.88% (RAPD) of variability was partitioned among individuals within populations. This genetic structure was probably due to severe genetic drift resulting from habitat fragmentation and human overexploitation since 1950s. Moreover, there is a lack of significant association between genetic and geographic distances (r = 0.276; p > 0.05) in the populations of D. officinale. From the conservation point of view, populations GL, GS and GSD with higher genetic diversity should be protected firstly to maintain the species potential for evolutionary change and population YG with lower diversity but representing a novel evolutionary unit should also be paid more attention to during D. officinale conservation practice. Published in Russian in Genetika, 2009, Vol. 45, No. 3, pp. 375–382. The article is published in the original.     

Comparison of RAPD,ISSR, and AFLP Molecular Markers to Reveal and Classify Orchardgrass (Dactylis glomerata L.) Germplasm Variations

Three different DNA-based techniques, Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR) and Amplified Fragment Length Polymorphism (AFLP) markers, were used for fingerprinting Dactylis glomerata genotypes and for detecting genetic variation between the three different subspecies. In this study, RAPD assays produced 97 bands, of which 40 were polymorphic (41.2%). The ISSR primers amplified 91 bands, and 54 showed polymorphism (59.3%). Finally, the AFLP showed 100 bands, of which 92 were polymorphic (92%). The fragments were scored as present (1) or absent (0), and those readings were entered in a computer file as a binary matrix (one for each marker). Three cluster analyses were performed to express–in the form of dendrograms–the relationships among the genotypes and the genetic variability detected. All DNA-based techniques used were able to amplify all of the genotypes. There were highly significant correlation coefficients between cophenetic matrices based on the genetic distance for the RAPD, ISSR, AFLP, and combined RAPD-ISSR-AFLP data (0.68, 0.78, 0.70, and 0.70, respectively). Two hypotheses were formulated to explain these results; both of them are in agreement with the results obtained using these three types of molecular markers. We conclude that when we study genotypes close related, the analysis of variability could require more than one DNA-based technique; in fact, the genetic variation present in different sources could interfere or combine with the more or less polymorphic ability, as our results showed for RAPD, ISSR and AFLP markers. Our results indicate that AFLP seemed to be the best-suited molecular assay for fingerprinting and assessing genetic relationship among genotypes of Dactylis glomerata.     

DNA fingerprints of living fossil Ginkgo biloba by using ISSR and improved RAPD analysis

In this study, we have aimed to genetically characterize Ginkgo biloba. Nine G. biloba samples from different places of China were collected, and DNA was extracted from the leaves of these samples for inter-simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) analysis. ISSR analysis showed high genetic variation among the nine varieties of G. biloba; the polymorphism and similarity coefficients were 87% and 0.40–0.84, respectively. RAPD analysis also showed 93% polymorphism, and the similarity coefficients ranged from 0.44 to 0.87. Persistent genetic isolation that developed for millions of years might influence the genetic variability between the samples of G. biloba. This study generates a genetic map of G. biloba, and reports the highly variable intra-species genetic characteristics of this living fossil among different geographical locations of China. Our study also suggests that ISSR and the improved RAPD markers are useful molecular tools for the genetic characterization of plants.     

ISSR and RAPD based evaluation of genetic fidelity and active ingredient analysis of regenerated plants of Picrorhiza kurroa

Genetic stability and phytochemical analysis of in vitro established plants of Picrorhiza kurroa Royle ex Benth, have been carried out. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers were used to assess the genetic fidelity of tissue culture products including three adventitious shoots from three calli and 6 months old tissue culture raised plants growing in green house condition with mother plant. Apparent genetic variation was detected in the five types of plant materials. The percentage of polymorphic bands in the RAPD and ISSR analysis were 16.25 and 14.54 %, respectively. The genetic similarity was calculated on the basis of RAPD and ISSR data among the five types of plant materials and were ranged from 0.5 to 1.0 (mean 0.75) and 0.47 to 1.0 (mean 0.73), respectively. The similarity coefficient by both RAPD and ISSR analysis revealed that differences between tissue culture raised plants and mother plant was not remarkable, but notable differences were observed among three adventitious shoots regenerated from three calli. The phytochemical analysis of tissue culture raised products showed higher secondary metabolite (picrotin and picrotoxinin) content as compare to mother plant. The information gained on genetic stability/variability will be valuable for the large scale propagation and secondary metabolite production of P. kurroa.