Assessment of genetic diversity in Calamus thwaitesii BECC. (Arecaceae) using RAPD markers

Calamus thwaitesii Becc. is a potentially useful rattan found in the Western Ghats of India and Sri Lanka. The wild stock of this rattan species is greatly diminished due to overexploitation for the furniture industry and increasingly rare. Genetic diversity was estimated in 80 samples representing eight populations from the Western Ghats and Sri Lanka using Random Amplified Polymorphic DNA (RAPD) markers. RAPDs generated a total of 120 markers with 10 decamer primers, of which 85% were found to be polymorphic. The percentage of polymorphic loci varied from 40.00 to 60.83 and genetic distance between populations ranged from 0.0332 to 0.2777. Among the analysed populations, Goa was found to be genetically superior followed by Achenkovil, Sinharaja and Talakkaveri. Majority of the genetic diversity was distributed within populations (70.79%) and only (29.21%) among populations. Genetic relationships estimated by the unweighted pair-group method with arithmetic averaging (UPGMA) cluster analysis and principal co-ordinate analysis failed to separate Indian and Sri Lankan populations geographically into two distinct groups.     

Genetic Diversity and Genetic Structure of an Endangered Species, Trillium tschonoskii

The genetic diversity and genetic structure of Trillium tschonoskii (Maxim) were investigated using amplified fragment length polymorphism markers. Eight primer combinations were carried out on 105 different individuals sampled from seven populations. Of the 619 discernible DNA fragments generated, 169 (27.3%) were polymorphic. The percentage of polymorphic bands within populations ranged from 4.52 to 10.50. Genetic diversity (H_E) within populations ranged from 0.0130 to 0.0379, averaging 0.0536 at the species level. Genetic differentiation among populations was detected based on Nei's genetic diversity analysis (53.03%) and analysis of molecular variance (AMOVA) (52.43%). AMOVA indicated significant genetic differentiation among populations (52.43% of the variance) and within populations (47.57% of the variance) (p < 0.0002). Gene flow was low (0.4429) among populations. Species breeding system and limited gene flow among populations are plausible reasons for the high genetic differentiation observed for this species. We propose an appropriate strategy for conserving the genetic resources of T. tschonoskii in China.     

An AFLP analysis of genetic diversity and structure of Caragana microphylla populations in Inner Mongolia steppe, China

Genetic diversity and structure of five natural populations of Caragana microphylla from the Inner Mongolia steppe were estimated using AFLP markers. Five pairs of primers generated a total of 312 bands among 90 individuals, with percentage of polymorphic bands (PPB) being 63% at the population level and 76% at the species level, respectively. The genetic diversity within populations was correlated significantly with the soil N:P ratio. AMOVA analysis revealed high genetic variations within populations (95.5%). The estimated number of migrants per generation (Nm) was 10.72, indicating a high level of gene flow among populations. There was no significant correlation (r = 0.36) between genetic distance and geographical distance. These results were discussed in terms of eco-geographical variations among populations, together with the life history traits and breeding system of the species. The knowledge obtained may have important implications for better conservation and wise use of the vegetation dominate by C. microphylla.     

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     

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.     

Genetic Variation Within and Among Populations of Rhodiola alsia (Crassulaceae) Native to the Tibetan Plateau as Detected by ISSR Markers

Genetic variation of 10 Rhodiola alsia (Crassulaceae) populations from the Qinghai–Tibet Plateau of China was investigated using intersimple sequence repeat (ISSR) markers. R. alsia is an endemic species of the Qinghai–Tibet Plateau. Of the 100 primers screened, 13 were highly polymorphic. Using these primers, 140 discernible DNA fragments were generated with 112 (80%) being polymorphic, indicating pronounced genetic variation at the species level. Also there were high levels of polymorphism at the population level with the percentage of polymorphic bands (PPB) ranging from 63.4 to 88.6%. Analysis of molecular variance (AMOVA) showed that the genetic variation was mainly found among populations (70.3%) and variance within populations was 29.7%. The main factors responsible for the high level of differentiation among populations are probably the isolation from other populations and clonal propagation of this species. Occasional sexual reproduction might occur in order to maintain high levels of variation within populations. Environmental conditions could also influence population genetic structure as they occur in severe habitats. The strong genetic differentiation among populations in our study indicates that the conservation of genetic variability in R. alsia requires maintenance of as many populations as possible.     

Genetic differentiation analysis of African cassava (Manihot esculenta) landraces and elite germplasm using amplified fragment length polymorphism and simple sequence repeat markers

Molecular‐marker‐aided evaluation of germplasm plays an important role in defining the genetic diversity of plant genotypes for genetic and population improvement studies. A collection of African cassava landraces and elite cultivars was analysed for genetic diversity using 20 amplified fragment length polymorphic (AFLP) DNA primer combinations and 50 simple sequence repeat (SSR) markers. Within‐population diversity estimates obtained with both markers were correlated, showing little variation in their fixation index. The amount of within‐population variation was higher for landraces as illustrated by both markers, allowing discrimination among accessions along their geographical origins, with some overlap indicating the pattern of germplasm movement between countries. Elite cultivars were grouped in most cases in agreement with their pedigree and showed a narrow genetic variation. Both SSR and AFLP markers showed some similarity in results for the landraces, although SSR provided better genetic differentiation estimates. Genetic differentiation (Fst) in the landrace population was 0.746 for SSR and 0.656 for AFLP. The molecular variance among cultivars in both populations accounted for up to 83% of the overall variation, while 17% was found within populations. Gene diversity (H_e) estimated within each population varied with an average value of 0.607 for the landraces and 0.594 for the elite lines. Analyses of SSR data using ordination techniques identified additional cluster groups not detected by AFLP and also captured maximum variation within and between both populations. Our results indicate the importance of SSR and AFLP as efficient markers for the analysis of genetic diversity and population structure in cassava. Genetic differentiation analysis of the evaluated populations provides high prospects for identifying diverse parental combinations for the development of segregating populations for genetic studies and the introgression of desirable genes from diverse sources into the existing genetic base.     

Genetic relationships among South-East Turkey wild barley populations and sampling strategies of Hordeum spontaneum

To assess the genetic diversity and the genetic structure of Turkish wild barley (Hordeum spontaneum Tell.) populations, 76 genotypes from ten ecologically and geographically different locations were analyzed by means of amplified fragment length polymorphism (AFLP) markers. Five primer combinations produced 187 scorable bands, of which 117 (62.6%) were polymorphic. Several population-specific and genotype-specific bands were identified, which differentiate populations or genotypes. Genetic distance, determined by Nei’s distance coefficient, varied from 0.07 to 0.21 with an average of 0.13. In the UPGMA dendrogram based on Nei genetic distances, the Hordeum spontaneum populations were separated into two major clusters. Genetic diversity was larger among (68%) than within (32%) populations. Eight AFLP bands were strongly correlated to the altitude of the collecting site, while no clear trend was detected between geographical origin and genetic diversity. Our results strongly suggest the need for a change in Hordeum spontaneum sampling strategy: more populations, rather then more individuals within population, should be sampled to appraise and safeguard genetic diversity in the wild barley gene pool.     

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.     

A Study of Conservation Genetics in Cupressus chengiana,an Endangered Endemic of China,Using ISSR Markers

ISSR markers were used to analyze the genetic diversity and genetic structure of eight natural populations of Cupressus chengiana in China. ISSR analysis using 10 primers was carried out on 92 different samples. At the species level, 136 polymorphic loci were detected. The percentage of polymorphic bands (PPB) was 99%. Genetic diversity (H _e) was 0.3120, effective number of alleles (A _e) was 1.5236, and Shannon’s information index (I) was 0.4740. At the population level, PPB = 48%, A _e=1.2774, H _e=0.1631, and I=0.2452. Genetic differentiation (G _st) detected by Nei’s genetic diversity analysis suggested 48% occurred among populations. The partitioning of molecular variance by AMOVA analysis indicated significant genetic differentiation within populations (54%) and among populations (46%; P < 0.0003). The average number of individuals exchanged between populations per generation (N _m ) was 0.5436. Samples from the same population clustered in the same population-specific cluster, and two groups of Sichuan and Gansu populations were distinguishable. A significantly positive correlation between genetic and geographic distance was detected (r=0.6701). Human impacts were considered one of the main factors to cause the rarity of C. chengiana, and conservation strategies are suggested based on the genetic characters and field investigation, e.g., protection of wild populations, reestablishment of germplasm bank, and reintroduction of more genetic diversity.     

Genetic diversity of forest arabica coffee (Coffea arabica L.) in Ethiopia as revealed by random amplified polymorphic DNA (RAPD) analysis

Genetic diversity within the forest Coffea arabica L. gene pool in Ethiopia has not been extensively examined with molecular markers. In the present study, a total of 75 polymorphic RAPD bands generated by twelve random primers were used to assess genetic diversity among 144 genotypes representing 16 C. arabica populations. The number of polymorphic bands detected with each primer ranged from 2 to 9 with a mean of 6.25 bands per primer. Banding patterns ranged in percentage polymorphism from 37% to 73% with an overall mean of 56% for the populations analyzed. The amount of genetic variation among populations estimated by Shannon-Weaver diversity index was (H = 0.30). The within population and between populations differentiation values were 0.65 and 0.35, respectively. Genetic differentiations within and between zones of sample collection sites were 0.80 and 0.20, respectively. Within population average similarities estimated by simple matching coefficients ranged from 0.72 to 0.85, with an overall average of 0.78. In the cluster analysis that used individual samples as operational taxonomic units, most of the representatives of the same population failed to cluster before they joined members of other populations. Nevertheless, most of the populations were clustered on the basis of their geographic closeness and an east west differentiation was observed at approximately 75% similarity. The results obtained provide information on how to select sites for in situ conservation of C. arabica germplasm.     

Molecular characterization of the wild relatives of wheat using CAAT-box derived polymorphism

A molecular assessment of genetic diversity was performed on a set of 228 selected accessions belonging to the different Aegilops and Triticum species using CAAT-box derived polymorphism (CBDP) markers. Fifteen CBDP primers generated 141 polymorphic fragments with an average of 9.40 per primer. The average of polymorphic information content and resolving power revealed a high efficiency of CBDP markers in analyzing genetic diversity among different wheat genotypes. The diversity indexes including polymorphic loci percent, number of observed and effective alleles, Shannon’s index and gene diversity among different populations were 76.24%, 1.67, 1.49, 0.42 and 0.28, respectively. These essentially coincided with the analysis of molecular variance results, indicating that 86, 68, and 59.46% of genetic variation were found within two Aegilops and Triticum genera and their populations, respectively. Genetic relationships inferred from cluster analysis was matched with STRUCTURE analysis, disclosing the accessions were grouped based on their genomic constitution. Furthermore, these results were confirmed by principal coordinate analysis (PCoA). Taken together, our results suggest that CBDP markers will be useful for genetic diversity assessment in the domesticated and wild relatives of wheat.     

Genetic diversity of heavy metal-tolerant populations in Silene paradoxa L. (Caryophyllaceae): a chloroplast microsatellite analysis

Eight populations of Silene paradoxa L. (Caryophyllaceae) growing in copper mine deposits, in serpentine outcrops or in uncontaminated soil in central Italy were studied. Genetic diversity was estimated using five polymorphic chloroplast microsatellite loci (cpSSR), identifying 27 different chloroplast haplotypes. The effective number of alleles, the haplotypic diversity and a stepwise mutational model-based parameter (DSH2) were computed. The effective number of alleles observed within populations from copper mine deposits was 20% that of the serpentine neighbouring populations, suggesting the occurrence of a founder effect. Moreover, 13 of the 27 different haplotypes scored were exclusive to only one population, indicating genetic isolation for all tolerant populations. Even the copper-tolerant populations appeared to have evolved independently. Finally, analysis of molecular variance (AMOVA) of the cpSSR markers gave statistical significance to the grouping of populations according to their geographical location. This study demonstrates that cpSSR markers could be a useful complementary tool to isoenzymes or random amplified polymorphic DNA markers for elucidating the pattern of genetic differentiation in heavy metal-tolerant populations.     

A study of conservation genetics in Cupressus chengiana, an endangered endemic of China, using ISSR markers

ISSR markers were used to analyze the genetic diversity and genetic structure of eight natural populations of Cupressus chengiana in China. ISSR analysis using 10 primers was carried out on 92 different samples. At the species level, 136 polymorphic loci were detected. The percentage of polymorphic bands (PPB) was 99%. Genetic diversity (He) was 0.3120, effective number of alleles (Ae) was 1.5236, and Shannon's information index (I) was 0.4740. At the population level, PPB = 48%, Ae = 1.2774, He = 0.1631, and I = 0.2452. Genetic differentiation (Gst) detected by Nei's genetic diversity analysis suggested 48% occurred among populations. The partitioning of molecular variance by AMOVA analysis indicated significant genetic differentiation within populations (54%) and among populations (46%; P < 0.0003). The average number of individuals exchanged between populations per generation (Nm) was 0.5436. Samples from the same population clustered in the same population-specific cluster, and two groups of Sichuan and Gansu populations were distinguishable. A significantly positive correlation between genetic and geographic distance was detected (r = 0.6701). Human impacts were considered one of the main factors to cause the rarity of C. chengiana, and conservation strategies are suggested based on the genetic characters and field investigation, e.g., protection of wild populations, reestablishment of germplasm bank, and reintroduction of more genetic diversity.     

Genetic structure of two Thalassia testudinum populations from the south Texas Gulf coast

The south Texas Gulf coast is a unique ecosystem that contains a number of different bay systems. We used random amplification of polymorphic DNA (RAPD) markers to assess genetic diversity, differentiation and genetic distance between populations from two different bays that differed significantly in terms of flowering rate and disturbance. We found that while each bay contained a number of unique RAPD profiles, the average genetic diversity in each population was low. Genetic distance between the two populations was also low (F_st = 0.084) and the majority (92%) of the genetic variation was attributed to differences between individuals within populations. The population from the Laguna Madre location, however, was polymorphic for a larger number of markers, had a higher average genetic diversity and a larger number of unique RAPD profiles. The higher level of flowering at this location most likely accounts for the higher diversity.     

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.     

AFLP Analysis of Genetic Diversity of the Endangered Species Sinopodophyllum hexandrum in the Tibetan Region of Sichuan Province,China

Amplified fragment length polymorphism (AFLP) markers were used to estimate the genetic diversity of seven wild populations of Sinopodophyllum hexandrum (Royle) Ying from the Tibetan region of Sichuan Province, China. Six primer combinations generated a total of 428 discernible DNA fragments, of which 111 were polymorphic. The percentage of polymorphic bands (PPB) was 25.93 at the species level, and PPB within population ranged from 4.91 to 12.38%. Genetic diversity (H _E) within populations varied from 0.01 to 0.04, averaging 0.05 at the species level. As revealed by the results of AMOVA analysis, 58.8% of the genetic differentiation occurred between populations, and 41.2% within populations. The genetic differentiation was, perhaps, due to the limited gene flow (N _m=0.43) of the species. The correlation coefficient (r) between genetic and geographical distance using Mantel's test for all populations was 0.698 (P=0.014). The UPGMA cluster analysis revealed a similar result in that the genetic distances among the populations show, to a certain extent, a spatial pattern corresponding to their geographic locations. On the basis of the genetic and ecological information, we propose some appropriate strategies for conserving the endangered S. hexandrum in this region.     

Determination of genetic relationships among shape Phaseolus vulgaris populations in a conical cross from RAPD marker analyses

Random-amplified polymorphic DNA (RAPD) markers were used to determine genetic relationships among Phaseolus vulgaris breeding populations. Genetic distances were calculated from the distribution of 317 RAPD markers among 8 parents, 10 individuals from 8 cycle-one populations, 10 individuals from 6 cycle-two populations and 10 individuals from 2 cycle-three populations of a conical cross. Genetic distances between populations and parents were consistent with their degree of relationship in the crossing scheme indicating that a RAPD analysis is a sensitive and useful method for categorizing breeding materials according to their genetic similarities. Genetic variation among individuals within populations increased from cycle one to cycle three and variation among populations within the cycles decreased from cycle one to cycle three in the conical cross. The results showed that this crossing scheme can be used to collect the genetic diversity in eight parents into a single plant breeding population. Abbreviations: CBB, common bacterial blight; GD, genetic distance; RAPD, random-amplified polymorphic DNA     

Genetic diversity assessment in Portugal accessions of <Emphasis Type="Italic">Olea europaea</Emphasis> by RAPD markers

Eighty seven olive (Olea europaea ssp. sativa L.) cultivar accessions from Portugal were characterized by means of randomly amplified polymorphic DNA (RAPD) markers. Of the 11 arbitrary 10-mer primers tested a total of 92 polymorphic bands were obtained, representing 87.6 % of the total amplification products. Twenty nine different genotypes were clearly discriminated. Differences were not found among the amplification profiles from different individuals of the same cultivar. All the genotypes could be identified by the combination of three primers: OPR-1, OPK-14 and OPA-1, seven genotype-specific markers being detected. Genetic relationships were estimated by the unweighted pair-group method with arithmetic averaging (UPGMA). The genetic analysis of the results showed a gradual distance between the various cultivars, making it difficult to identify well-differentiated phylogenetic groups, although two clusters were distinguishable with 35 % similarity, in addition to three independent branches with lower similarity: Galega, Tentilheira and Redondal. The dendrogram reflect some relationships for most of the cultivars according to the use of the fruit and ecological adaptation.     

Genetic differentiation between sun and shade habitats in populations of Lindera benzoin L.

Differences in selection patterns among habitats can alter the distribution of genetic diversity even when this is estimated with neutral markers. For plants, light is an essential resource that can influence both abiotic and biotic components of habitat. We examined genetic differentiation between sun and shade habitats in Lindera benzoin L. (Spicebush), a perennial understory shrub. Genetic diversity of 127 plants from sun and shade habitats in two populations of L. benzoin was determined using 12 polymorphic microsatellite markers. We analyzed patterns of genetic diversity using analysis of molecular variance (AMOVA), and we assessed correlation between genetic and geographic distance using Mantel tests. We found (1) low levels of differentiation among populations (F _ST = 0.028), (2) little evidence of genetic structure within populations due to isolation-by-distance, and (3) some evidence of habitat-based genetic differentiation. Specifically, the AMOVA showed a small (0.5%) but significant portion of overall variation could be explained by differences between habitats. The overall low levels of differentiation we saw were likely a result of extensive gene flow in this dioecious, bird-dispersed species.