Polymorphic Random Amplified Polymorphic DNA Bands Research Articles

Assessment of Somaclonal Variations in Embryo-derived Axillary Shoots of Chickpea using Molecular Markers

Background: Somaclonal variation is considered as a source of genetic variation for crop improvement. It has been investigated using cytological, biochemical and molecular techniques.Methods: Genetic stability in the embryo-derived axillary shoots of 4 chickpea genotypes was assessed using eight inter-simple sequence repeat (ISSR) and 19 random amplified polymorphic DNA (RAPD) primers. Result: RAPD primers produced 94 and ISSR primers produced 38 distinct and scorable alleles, with an average of 4.9 alleles for RAPD and 4.75 for ISSR primers. The polymorphic information content (PIC) ranged from 0.36 to 0.90 for RAPD and from 0.50 to 0.87 for ISSR. ISSR recognized a 90%, but RAPD recognized 82% similarity value. No absolute similarity value was between the mother plant and the regenerated shoots for the overall genotypes. At a 90% similarity value, 15 out of the 20 regenerated shoots from ‘Giza 88’ group with their mother plant using ISSR markers; however, 11 regenerated shoots grouped with their mother plant in one central cluster for ‘Giza 4’ using RAPD markers. The observed variations in the total number of polymorphic RAPD and ISSR bands and the number of bands specific to the mother and regenerated shoots, detected intra-clonal variation and genetic instability seem to be genotype-dependent.

Genetic variation of maiden lily (Lilium rubellum Baker) within and among three natural populations in Fukushima Prefecture in Japan

Lily ( genus Lilium ) is one of the most important flowers for bulb and cut flower production in the world. Lilium rubellum Baker is a native species of Japan that exhibits early flowering, is pink coloured, and possesses a pleasant fragrance, which makes it an important genetic resource for breeding lilies. In this work, the genetic diversity of L. rubellum was studied among three natural populations located in Fukushima Prefecture, Japan, which have been designated as the Mt. Azuma, Nango and Atsushio-kano populations. A total of 31 accessions collected in 1997 were analysed using Random Amplified Polymorphic DNA (RAPD) markers. Eighteen decamer primers produced 98% polymorphic RAPD bands; furthermore, 11 of the 18 primers produced 10 or more polymorphic bands with a mean per primer Polymorphic Information Content of 0.382. Among the three populations, the mean Shannon index, Nei´s gene diversity and percentage of polymorphic loci were 0.2749, 0.4099 and 76.7%, respectively. These data revealed that there is a high genetic diversity within all the populations. Analysis of the three genetic diversity indices within populations showed that the Nango population had the highest genetic diversity, whereas the Atsushio-kano population had the lowest. An unweighted pair group method with arithmetic mean dendrogram based on Jaccard´s similarity coefficient was constructed, and the three populations of L. rubellum collected in the Fukushima prefecture were clearly differentiated at 0.61 similarity index. The Nango and Mt. Azuma populations were genetically closer than the Atsushio-kano population. The analysis of molecular variance showed a 29.53% variance among the populations. This study revealed that there is high genetic diversity within populations and moderate genetic diversity among the three natural populations of L. rubellum Baker in the Fukushima Prefecture of Japan.

Utilizing RAPD Analysis as Molecular Markers for comparison between Various Callus and Mother Plant of Jatropha curcas L.

Calli were induced from different explants of Jatropha curcas L. seedling on MS basal agar medium in presence of different plant growth regulators. at different levels for 4 weeks Random Amplified Polymorphic DNA (RAPD) markers were used for the assessment of genetic variation of callus grown in different hormonal combinations in comparison with the mother plant. A total of 8 arbitrary sequence primers were evaluated. Of 8 primers used for the RAPD analysis, primers showed consistent band patterns. In total, scorable bands were observed with the primers. The total number of amplicons produced per primer varied from 5 for OPA-07 and OPB-12 to as many as 8 bands for OPB-10, OPC- 09 and OPR-17. The average number of bands per primer was 6.625. Out of 53 bands, 36 were polymorphic (67.92%). The average number of polymorphic RAPD bands was 4.5 per primer. the highest similarity (0.71) with mother plant was recorded in leaf-derived callus (S4) grown on MS solid medium with 2.5 μM NAA plus 2.5 μM IBA, while endosperm callus maintained on MS with hormonal combination 12.5 μM IBA + BAP- 25.0 μM was found to show least similarity (0.54) with mother plant.In this concern, results have been suggested to be useful fast methods for comparing of geneticchanges and variation in plants.

RAPD markers reveal genetic variation between Cichorium spinosum L. and Taraxacum sp.; a substantial medicinal plants of Greece

Fifteen Random Amplified Polymorphic DNA (RAPD) markers were used to measure genetic diversity and genetic relationships between five endemic genera of Mediterranean basin (Greece). Three species of Cichorium spinosum were collected; two from island Crete (Greece) and one from island Kythnos, and other two species of Taraxacom sp. (Asteraceae); are from Orhomenos and Athens. Two hundred-forty amplified products and 163 RAPD bands were scored with an average of 67.91% of them revealing polymorphism across accessions. In this research OPD-05 primer with 24 bands showed the highest number of bands, while the OPM-18 and OPB-16, both of them with 8 bands showed the least number of bands. Also OPV-06 primer with 18 polimorphic bands showed the highest number of bands. The least number of polymorphic bandswere found in OPX-18. UBC-292, OPAN-01, OPB-16, OPM-18, OPD-05 primers. Subsequently, OPD-05 primer with 29.16% showed the least percentage of polymorphism degree, whereas OPM-18 and OPB-16 primers with 87.50% showed the highest percentage of polymorphism. UPGMA clustering based on data from polymorphic RAPD bands revealed two distinct group which joined to form one major cluster at 32% level of similarity. Also Cichorium spinosum, Crete and Cichorium spinosum, Kythnos, varieties with 100% similarity are synonyms. The similarity indices of the RAPD dendrogram ranged between 30% and 100% averagely high enough to suggest useful variability for genetic diversity and plant breeding.

English

Knowledge of genetic distances between genotypes is important for efficient organization and conservation of plant genetic resources for crop improvement programs. In this study genetic distances between genotype pairs (complements of Jaccard's similarity coefficient) were estimated from Random Amplified Polymorphic DNA (RAPD) data collected from 48 Zimbabwean sorghum landraces. These varieties showed variation in their seed proanthocyanidin (PAs) levels with 16 and 29 of them having detectable and non-detectable PA levels respectively. RAPDs revealed considerable genetic variation between the varieties used and 2.7 polymorphisms per primer were obtained.  Ninety nine polymorphic RAPD bands were used to calculate genetic distances and the mean genetic distance between the genotypes was 0.494 (± 0.113) with a range of 0.051 to 0.761. A multidimensional scaling (MDS) plot of the distance matrix revealed two distinct clusters of cultivated and wild sorghums. No clustering of genotypes according to their seed proanthocyanidin levels was revealed by MDS analysis; also the mean genetic distances of genotypes in the low, medium and high PA categories were not different from each other and none of them was significantly different from the mean genetic distances between all the groups. The RAPD markers used in the present study could not distinguish between sorghums with different PA levels in their seeds; however, the protocol established could be useful in further analysis of this trait in near isogenic lines.   Key words:  Genetic distances, multidimensional scaling, proanthocyanidins, Random Amplified Polymorphic DNA (RAPD), Sorghum bicolor, Zimbabwe.

Efficiency of random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers for genotype fingerprinting and genetic diversity studies in canola (Brassica napus)

Genetic diversity evaluations among 10 canola ( Brassica napus ) genotypes were determined using RAPD and ISSR markers. The RAPD and ISSR primers with the highest degree of polymorphism were selected. A total of 67 bands of polymorphic RAPD bands were detected out of 77 bands, with an average of 13.4 polymorphic fragments per primer. The number of amplified fragments with RAPD primers ranged from 8 to 21, with the size of amplicons ranging from 162 to 3154 bp. The polymorphism ranged from 68.4 to 100.0, with an average of 87%. The five ISSR primers produced 94 bands across 10 genotypes, of which 76 were polymorphic, with an average of 15.2 polymorphic fragments per primer. The number of amplified bands varied from 4 to 32, with size of amplicons ranging from 127 to 3011 bp. The percentage of polymorphism using ISSR primers ranged from 25 to 100.0 with an average of 78.8%. Dice similarity coefficient was calculated for all pairwise comparisons and was used to construct a UPGMA dendrogram. Clustering of genotypes within the groups was not similar when RAPD and ISSR derived dendrograms were compared, whereas, the pattern of clustering of the genotypes remained akin in ISSR and the combined data of RAPD and ISSR. The similarity coefficient ranged from 0.44 to 0.78, 0.41 to 0.72 and 0.47 to 0.73 with RAPD, ISSR and combined dendrogram, respectively. These results provided valuable information for fingerprinting that can be used to create wider genetic base for future canola breeding program.

Essential oil composition and preliminary molecular study of four HungarianThymusspecies

Chemical and genetic differences of twenty taxa belonging to four Thymus species were studied in order to determine whether molecular characters and essential oil components could be used as taxonomic markers and to examine the correlation between them. Plant samples, representing different taxa and geographic regions, were collected from experimentally grown populations. Essential oil samples were analysed by GC/MS and cluster analysis of volatile composition resulted in segregation of thymol chemotypes from sesquiterpenic ones. Thymol was characteristic for all the populations of Thymus glabrescens and T. pannonicus as well as for certain taxa belonging to T. praecox and T. pulegioides. Sesquiterpenes occurred in only two taxa of T. glabrescens, in each sample of T. praecox and in three taxa of T. pulegioides. Plant samples were analysed by random amplified polymorphic DNA (RAPD). The obtained dendrogram revealed high gene diversity. The 13 primers resulted 114 polymorphic RAPD bands, and the average percentage of polymorphism was 80.8%. The RAPD dendogram showed separation neither at interspecific nor at interpopulational levels. Therefore, further specific molecular studies involving more taxa are suggested. Partial correlation have been found between molecular and chemical assessments.

Sequence characterized amplified region (SCAR) markers-based rapid molecular typing and identification of Cunninghamia lanceolata

Chinese fir ( Cunninghamia lanceolata (Lamb.) Hook.) is the main timber and afforestation tree species in South China. C. lanceolata clones, with similar morphological characteristics, make their exact identification very difficult. A simple and effective method is required to distinguish these clones in the clonal selection and breeding of C. lanceolata . However, C. lanceolata clones have so far been identified only through traditional phenotypic analysis. In the present study, 20 C. lanceolata clones from different geographic provenances were collected and analyzed using randomly amplified polymorphic DNA (RAPD) technique. Nine polymorphic RAPD bands were cloned and sequenced. Six stable informative dominant sequence characterized amplified region (SCAR) markers were developed by designing six pairs of specific SCAR primers from six sequenced polymorphic RAPD bands, respectively. On the basis of the differences in SCAR phenotypes detected among the 20 C. lanceolata clones, 10 individual clones were identified from 20 studied clones at first, and the other 10 clones were divided into 4 distinguishable groups. Some C. lanceolata clones from the same geographic provenances were also distinguished from one another. The experiment was the first report that SCAR markers-based molecular method was used in the research on molecular identification of C. lanceolata . By developing and jointly using specific useful SCAR makers, rapid molecular typing of C. lanceolata clones was accomplished, which could be used as a tool for the identification of C. lanceolata clones. Key words: Chinese fir, Cunninghamia lanceolata , tree breeding, SCAR markers, molecular typing, identification.

Sequence characterized amplified region (SCAR) markers-based rapid molecular typing and identification of Cunninghamia lanceolata

Chinese fir ( Cunninghamia lanceolata (Lamb.) Hook.) is the main timber and afforestation tree species in South China. C. lanceolata clones, with similar morphological characteristics, make their exact identification very difficult. A simple and effective method is required to distinguish these clones in the clonal selection and breeding of C. lanceolata . However, C. lanceolata clones have so far been identified only through traditional phenotypic analysis. In the present study, 20 C. lanceolata clones from different geographic provenances were collected and analyzed using randomly amplified polymorphic DNA (RAPD) technique. Nine polymorphic RAPD bands were cloned and sequenced. Six stable informative dominant sequence characterized amplified region (SCAR) markers were developed by designing six pairs of specific SCAR primers from six sequenced polymorphic RAPD bands, respectively. On the basis of the differences in SCAR phenotypes detected among the 20 C. lanceolata clones, 10 individual clones were identified from 20 studied clones at first, and the other 10 clones were divided into 4 distinguishable groups. Some C. lanceolata clones from the same geographic provenances were also distinguished from one another. The experiment was the first report that SCAR markers-based molecular method was used in the research on molecular identification of C. lanceolata . By developing and jointly using specific useful SCAR makers, rapid molecular typing of C. lanceolata clones was accomplished, which could be used as a tool for the identification of C. lanceolata clones. Key words: Chinese fir, Cunninghamia lanceolata , tree breeding, SCAR markers, molecular typing, identification.

SCAR Makers and Multiplex PCR-Based Rapid Molecular Typing of Lentinula edodes Strains

Lentinula edodes is the second most important cultivated mushroom worldwide, the most commercial strains have been identified only through traditional phenotypic analysis. In this study, a simple rapid PCR-based molecular method was developed for distinguishing commercial strains of L. edodes by developing specific sequence characterized amplified region (SCAR) markers and establishing multiplex PCR assays with the SCAR primers. Derived from the randomly amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) techniques, 10 informative SCAR markers were generated from 10 polymorphic RAPD and SRAP bands. The differences in SCAR phenotypes among different strains made these SCAR markers potentially useful to characterize 6 strains and identify them from other studied strains. Moreover, different SCAR phenotypes also made the other 17 studied strains to be divided into four distinguishable groups. The multiplex PCR assays were further established for the joint use of some SCAR markers efficiently. Compared with some identification methods reported previously, the special feature of this new molecular method is technically rapid and convenient in the practical use and suitable for analyzing large numbers of samples. Thus, the simple rapid PCR-based molecular method can be used as a helpful assistant tool for the lentinula industry. To our knowledge, this study is the first to describe a development of a new SCAR maker-based multiplex PCR assay for rapid molecular typing of edible mushroom.

Genetic and Withaferin A Analysis of Iranian Natural Populations ofWithania SomniferaandW. Coagulansby RAPD and HPTLC

For successful conservation and breeding of a medicinal species, it is important to evaluate its genetic diversity as well as its content of phytochemical compounds. The aim of the present study was to investigate the genetic variation of Iranian natural populations of W. somnifera and W. coagulans, using the RAPD (random amplified polymorphic DNA) markers, and their withaferin A content. Using 16 RAPD primers, a total of 282 RAPD bands were achieved. The highest and lowest percentages of polymorphism were observed with primers OPAD-15 (100.0%) and OPC-06 (75.0%), respectively. Cluster analysis of the genotypes was performed based on data from polymorphic RAPD bands, using Dice's similarity coefficient and the UPGMA clustering method. Variations in the RAPD results were found to reflect geographical distribution and genetic factors of the plant populations. The HPTLC analysis of the studied samples revealed the presence of withaferin A in W. coagulans and W. somnifera. Moreover, the concentration of withaferin A had a range from 2.2 to 32.5 microg/g DW and was higher in the aerial part than in the root in all used samples. The results of the present study show that there is a high level of variation in the Iranian natural population of Withania, which is significant for conservation and breeding programs to improve production of withaferin A.

Toxicity of Carbon Tetrachloride to Dunaliella salina, an Environmentally Tolerant Alga

Carbon tetrachloride (CCl4), a water disinfection by-product, at low environmentally relevant concentrations exerts adverse effects on mammals.The unicellular microalga Dunaliella salina possessing a remarkable degree of environmental adaptation was selected as test organism to investigate low-level exposure to CCl4. With incubation with less than 0.13% CCl4, algae responses were similar to control as evidenced by cell growth and levels of β-carotene, a marker of adaptation. The maximal concentration of CCl4 that D. salina could tolerate was 0.2%. Algae incubated with more than 0.32% CCl4 showed decreased growth and reduced β-carotene levels, which were nondetected after a few days. However, after 98 d, D. salina seemed to revive as evidenced by growth and returned to the biomass similar to control in another 25 d. Randomly amplified polymorphic DNA (RAPD) method was used to compare the genomic DNA difference between control and recovered cells. Polymorphic and repeatable RAPD bands indicated that chronic effects of CCl4 to D. salina led generation of altered genomic DNA, which may enable the microalga to adapt to survival in an apparently toxic substance environment.

Relationships among some cornelian cherry genotypes (Cornus mas L.) based on RAPD analysis

Turkey is an important producer of cornelian cherries (Cornus mas L.), especially in northern Anatolia. Seed propagation and long-term human selection has given rise to a great diversity of trees. Twenty-six cornelian cherry genotypes (CC1–CC26) from the Coruh Valley in northern Anatolia were evaluated for genetic relationships by using Randomly Amplified Polymorphic DNA (RAPD) markers, based on 56 decamer random primers, seven of which showed reliable polymorphisms. These seven primers generated 80 markers, with 77 (96.25%) displaying polymorphisms. Cluster analysis of the cornelian cherry genotypes was performed based on data from polymorphic RAPD bands, by using Jaccard’s similarity coefficient and the Unweighted pair-group method with arithmetic average (UPGMA) clustering method. A similarity matrix showed that the highest genetic similarity (0.913) was between CC15 and CC16 and the least (0.129) was between CC4 and CC16. The cophenetic correlation coefficient between the similarity matrix and the cophenetic matrix of the dendrogram was relatively high (r = 0.87), supporting the validity of the dendrogram. Based on these results, RAPD analysis can be used for the characterization and grouping of cornelian cherry genotypes. Genetically divergent genotypes identified in this study may be useful for future breeding programs. This is the first study demonstrating that RAPD analyses can be used to differentiate and classify cornelian cherry genotypes.

GENETIC STRUCTURE OF NATURAL POPULATIONS IN THE RED ALGAEGELIDIUM CANARIENSE(GELIDIALES, RHODOPHYTA) INVESTIGATED BY RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) MARKERS1

Random amplified polymorphic DNA (RAPD) marker variation was analyzed in female gametophytes in natural populations ofGelidium canariense(Grunow) Seoane‐Camba ex Haroun, Gil‐Rodríguez, Diaz de Castro et Prud'Homme van Reine from the Canary Islands to estimate the degree and distribution of genetic variability and differentiation. A total of 190 haploid individuals were analyzed with 60 polymorphic RAPDs bands which produced 190 distinct multilocus genotypes. A high level of polymorphism was detected in all populations analyzed. Within‐population gene diversity ranged from 0.156 to 0.264. The populations on the island of Gran Canaria showed higher genetic variation than the other populations analyzed. The partitioning of molecular variance by analysis of molecular variance showed that most genetic variation resides within populations (68.85%). These results suggest that sexual reproduction is the predominant mode of reproduction forG. canariensegametophytic populations, and the main determinant in reaching high levels of genetic diversity. The Neighbor‐Joining tree and FCA analysis displayed two subclusters that correspond to the populations from the western islands (Tenerife, La Palma, Gomera) and the eastern island (Gran Canaria). In addition, we have detected a significant relationship betweenFST/(1−FST) and geographical distance consistent with data on water circulation and age of islands. The results obtained agree with an isolation by distance model, with gene flow from eastern to the western islands, and a high level of genetic differentiation between populations (FST=0.311,P<0.001).

Segregation patterns of several morphological characters and RAPD markers in interspecific hybrids between Dianthus giganteus and D. carthusianorum

The segregation ratio of morphological characters (15 quantitative and 4 qualitative) and random amplified polymorphic DNA (RAPD) markers in 55 interspecific hybrids between Dianthus giganteus and D. carthusianorum and their parents were studied. Interspecific hybrids exhibited either intermediate characteristics between parents shorter, fewer characteristics than parents except for stem length and height of corolla where heterosis was found. Segregation of four qualitative characters such as profile of the lower part of the corolla, stigma color, the main secondary color of petal blade, and arrangement of individual flowers, were exhibited as either characteristics of female parent or male parent. Two hundred and sixteen polymorphic RAPD bands detected in 55 interspecific hybrids were divided into four types: AB type (both-parental type) which was presented in both female parent and male parent, Ab type (female parental type) which was presented in only female parent, aB type (male parental type) which was presented in only male parent, and ab type (non-parental type) which was presented in only hybrids except their parents. The number of AB, Ab, aB, and ab type bands were 33 (15.3%), 56 (26.0%), 50 (23.1%), and 77 (35.6%), respectively. The proportion of parental type bands to non-parental type bands was two to one and only 54.6% of four type bands coincided with expected ratio.

Conservation Genetics of Mexican Beech, Fagus grandifolia var. mexicana

Fagus grandifolia var. mexicana(Fagaceae) is a Mexican endemic tree, currently threatened with extinction. In order to assess the level and structure of genetic variation in four remaining populations, leaf samples were analysed using random amplified polymorphic DNA (RAPD) and cpDNA PCR-RFLP markers. A sample of the more widespread congener, F. grandifoliavar. grandifolia from the USA was also analysed for comparison. Thirty-three polymorphic RAPD bands were produced using 18 10-mer primers. AMOVA of RAPD data indicated significant (P < 0.002) population differentiation, with 15.6% of variation recorded between Mexican populations. PCR-RFLP analysis enabled three cpDNA haplotypes to be identified, denoted types A, B, and C. Types A and B were each restricted to an individual Mexican population, whereas Type C was fixed for two Mexican populations, and the population from the USA. Within-population genetic variation, quantified as percentage polymorphic bands, Shannon's Diversity Index and Nei's gene diversity measure, was found to be lower in Mexican populations than in that from the USA, and was positively related to population size. These results suggest that an unexpectedly high degree of genetic variation exists within Mexican beech, and this variation should be considered in developing the conservation strategy that is urgently required if extinction of this taxon is to be prevented.

RAPD Variations in Selected and Unselected Blue Grama Populations

Blue grama (Bouteloua gracilis H.B.K. Lag. ex Steud.) is one of the most widespread native grasses in western North America. Little is known about the genetic diversity of this species and the genetic shift in selected populations. Random amplified polymorphic DNA (RAPD) markers were used to assess the genetic variations of blue grama plants in 11 natural source populations (SPs) in southern Manitoba and two selected populations. Population ‘BMSC’ was a balanced multisite composite of 99 clones selected for higher seed yield from the 11 SPs. Population ‘MSC’ was a mass‐selected composite of 25 clones with higher seed yield from eight SPs. Twelve RAPD primers were used to assay 108 original propagules from the 11 SPs and 96 seedlings from each selected population. A total of 69 polymorphic RAPD bands were detected. No unique RAPD bands were found for any SPs and 97.8% of the total RAPD variation was detected within SPs. Variation in RAPD markers was not associated with geographical distances. Highly significant changes in RAPD band frequency from their SPs were detected in both selected populations, but only MSC displayed the fixation of polymorphic bands. The estimated genetic shifts were small, 0.6% for BMSC and 1.9% for MSC.

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.

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.

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.