Population genetic diversity and divergence of the halobiotic herb Limonium sinense estimated by AFLP and ISSR, and implications for conservation

Limonium sinense is a halobiotic herb endemic to China that has been traditionally used for hundreds of years for its good restorative function. Genetic variation and population structure of this species were investigated by using amplified fragment length polymorphisms (AFLPs) and inter simple sequence repeats (ISSRs). A high level of genetic diversity was detected [AFLP: H _E = 0.284, percentage of polymorphic loci (PPL) = 92.68 %; ISSR: H _E = 0.257, PPL = 85.71 %] at the species level with POPGENE. Based on analysis of molecular variation (AMOVA), the among-population component accounted for 29.03 % (AFLP) and 28.81 % (ISSR) of the genetic variation, indicating that most of the genetic variation was between individuals within populations. The Shannon diversity index (I) was higher for AFLP (0.432) than for ISSR (0.395). Five main clusters were shown in the unweighted pair-group method with arithmetic mean (UPGMA) dendrogram created using TFPGA, consistent with the result of principal coordinate analysis using NTSYS. In situ conservation is advocated first. Keeping a stable environment for this halobiotic herb is necessary. For ex situ conservation, it is important to establish a germplasm bank. AFLP and ISSR markers were proved to be efficient tools in assessing the genetic variation among populations of L. sinense. The patterns of variation appeared to be consistent for these two marker systems, and they can be used for management of genetic structure, protection of the halobiotic plant, and conservation of germplasm.     

DNA typing and genetic relations among populations of Kelussia odoratissima using ISSR and SRAP markers

Kelussia odoratissima is well known for its medicinal importance. It has been announced as an endangered species. Thus, examining the genetic variation and conservation of this plant is necessary. In the present study, inter simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) molecular markers were employed for the first time to access the genetic diversity and relationships of 77 wild individual plants of K. odoratissima collected from seven populations in Central Zagros region of Iran. A total of 146 bands were amplified by 12 ISSR primers, of which 129 (87.80 %) were polymorphic, while 69 polymorphic bands (83.30 %) were observed among 86 bands amplified by 11 SRAP primers. Polymorphic information content (PIC = 0.32), resolving power (Rp = 7.80), and marker informativeness (MI = 3.48) generated by ISSR primers were higher than that of SRAP analysis (PIC = 0.30, Rp = 5.61, and MI = 1.88). The study indicated that ISSR were more effective than SRAP markers for assessing the degree of genetic variation of K. odoratissima. In both UPGMA dendrograms of ISSR and SRAP, in most cases, individuals from each population were clustered in various groups without clear separation, which demonstrates the high variability of this germplasm in Iran. UPGMA cluster analysis revealed inconsistencies in the clustering patterns, as the Mantel’s test between the dendrograms for ISSR and SRAP data indicated a poor fit for the ISSR and SRAP data types (r = 0.10). Besides, principal coordinate analysis results showed that the first three principal coordinates account for 65.57 % of the total variation and studied seven populations were separated from each other and placed into five groups. These results have an important implication for K. odoratissima germplasm characterization, improvement, and conservation.     

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.     

Determination of genetic diversity among Saccharina germplasm using ISSR and RAPD markers

Various species of genus Saccharina are economically important brown macroalgae cultivated in China. The genetic background of the conserved Saccharina germplasm was not clear. In this report, DNA-based molecular markers such as inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) were used to assess the genetic diversity and phylogenetic relationships among 48 Saccharina germplasms. A total of 50 ISSR and 50 RAPD primers were tested, of which only 33 polymorphic primers (17 ISSR and 16 RAPD) had an amplified clear and reproducible profile, and could be used. Seventeen ISSR primers yielded a total of 262 bands, of which 256 were polymorphic, and 15.06 polymorphic bands per primer were amplified from 48 kelp gametophytes. Sixteen RAPD primers produced 355 bands, of which 352 were polymorphic, and 22 polymorphic bands per primer were observed across 48 individuals. The simple matching coefficient of ISSR, RAPD and pooled ISSR and RAPD dendrograms ranged from 0.568 to 0.885, 0.670 to 0.873, and 0.667 to 0.862, revealing high genetic diversity. Based on the unweighted pair group method with the arithmetic averaging algorithm (UPGMA) cluster analysis and the principal components analysis (PCA) of ISSR data, the 48 gametophytes were divided into three main groups. The Mantel test revealed a similar polymorphism distribution pattern between ISSR and RAPD markers, the correlation coefficient r was 0.62, and the results indicated that both ISSR and RAPD markers were effective to assess the selected gametophytes, while matrix correlation of the ISSR marker system (r = 0.78) was better than that of the RAPD marker system (r = 0.64). Genetic analysis data from this study were helpful in understanding the genetic relationships among the selected 17 kelp varieties (or lines) and provided guidance for molecular-assisted selection for parental gametophytes of hybrid kelp breeding.     

Identification and genetic relationships among nine Albizzia species based on morphological and molecular markers

Abstract

Identifying germplasm is an important component for efficient and effective management of plant genetic resources. This investigation was undertaken for the identification and analysis of genetic variation within 9 species of Albizzia through 33 morphological parameters, and 15 Random Amplified Polymorphic DNA (RAPD) and 17 Inter Simple Sequence Repeat (ISSR) primers. The use of selected RAPD and ISSR primers generated a total of 163 and 201 amplified DNA fragments, respectively. High frequencies of polymorphism, 95.05% for RAPD and 96.02% for ISSR, were detected. Statistical approaches were employed to construct genetic relationships by RAPD, ISSR and morphological analysis. Cluster analysis by the unweighted pair-group method (UPGMA) of Nei's similarity generated dendograms with similar topology that gave a better reflection of the diversity and affinities between species. These molecular results were comparable to main morphological characteristics. The correlation matrices generated by RAPD and ISSR markers were highly correlated (r = 0.843 at p = 1.0), thereby indicating congruence between these two marker systems. Both morphometric data and molecular markers have the potential to analyse genetic variation among the nine species of Albizzia, thus providing a major input for management strategy of plant genetic resources.     

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 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.     

利用RAPD和ISSR分子标记分析怀地黄种质遗传多样性

用RAPD与ISSR技术对怀地黄的8个品种和2个脱毒品系进行了种质遗传多样性分析。分别从80条RAPD引物和44条ISSR引物中筛选出适合怀地黄种质分析的17条RAPD引物和10条ISSR引物,用于RAPD和ISSR分析。17条RAPD引物共扩增出177条带, 多态性位点数为109; 多态性位点比率为61.58%;平均多样性指数(I)为0.3135;每个位点的有效等位基因数（Ne）是1.3641; 10条ISSR引物共扩增出110条带. 多态性位点数为79; 多态性位点比率为71.58%;平均多样性指数(I)为0.3577;每个位点的有效等位基因数（Ne）是1.4037。 基于扩增条带数据库建立了各自的Jaccard遗传相关系数矩阵,构建了相似的分子树状图,将10个供试材料分为2类:一类群含组培85.5、大田85.5、组培9302、大田9302、金状元和金白6个材料;另一类群含北京1号、大红袍、地黄9104和野生地黄4个材料。两种分子标记的分析结果呈极显著正相关(r＝0.649)。结果表明,RAPD与ISSR标记适合于怀地黄种质遗传多样性分析,ISSR标记技术是一种多态性和重复性优于RAPD技术的实用技术。     

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 Characterization of <Emphasis Type="Italic">Barilius barna</Emphasis> (Hamilton, 1822) in the Teesta River of Sub-Himalayan West Bengal,India, Through RAPD and ISSR Fingerprinting

Genetic characterization of Barilius barna, an economically important freshwater fish in the Indian scenario, is unexplored in the sub-Himalayan Dooars region of West Bengal, India. This study is the first attempt to characterize the genetic architecture of Barilius barna from the Teesta river of this region. We have studied loci polymorphism, genetic diversity, Shannon’s information index and the measure of evenness in the two populations of this river through ten RAPD and seven ISSR primer-based PCR amplifications. The result showed 89.52 and 82.21% polymorphisms in RAPD and ISSR amplification respectively. The Nei’s genetic diversity and Shannon’s information index varied from 0.172 ± 0.189SD to 0.293 ± 0.164SD and 0.265 ± 0.268SD to 0.445 ± 0.220SD respectively, which indicated low level of genetic variation. AMOVA revealed significant level of variance within the population and gene flow between the populations. Low levels of genetic variation and moderate to high levels of genetic relatedness were found in the studied populations. Expectedly, the populations were genetically not very distant from each other, as evident from the Nei’s unbiased measure of genetic distance and identity. As the species is commercially important and the region is located in the sub-Himalayan region, the management and proper rehabilitation of this ichthyofauna in the wild is urgently required. Our results may serve as a guideline for adopting such management decisions.     

Genetic variability and structure of Quercus brantii assessed by ISSR,IRAP and SCoT markers

Persian oak (Quercus brantii Lindl.) is one of the most important woody species of the Zagros forests in Iran. Three molecular marker techniques: start codon targeted (SCoT), inter-simple sequence repeat (ISSR) and inter-retrotransposon amplified polymorphism (IRAP) markers were compared for fingerprinting of 125 individuals of this species collected from different geographical locations of north-west of Iran. A total of 233 bands were amplified by 18 ISSR primers, of which 224 (96.10%) were polymorphic, and 126 polymorphic bands (97.65%) were observed in 129 bands amplified by 10 IRAP primers. Besides, 118 bands were observed for all 10 SCoT primers, of which 113 were polymorphic (95.71%). Average polymorphism information content (PIC) for ISSR, IRAP and SCoT markers was 0.30, 0.32 and 0.38, respectively, and this revealed that SCoT markers were more informative than IRAP and ISSR for the assessment of diversity among individuals. Based on the three different molecular types, cluster analysis revealed that 125 individuals taken for the analysis can be divided into three distinct clusters. The Jaccard's genetic similarity based on the combined data ranged from 0.23 to 0.76. These results suggest that efficiency of SCoT, IRAP and ISSR markers was relatively the same in fingerprinting of individuals. All molecular marker types revealed a low genetic differentiation among populations, indicating the possibility of gene flow between the studied populations. These results have an important implication for Persian oak (Q. brantii) germplasm characterization, improvement, and conservation.     

Inter and intra-population variability of Pongamia pinnata: a bioenergy legume tree

Pongamia pinnata is an oil-producing tree species with multiple uses and considerable potential as a bioenergy crop. This investigation was carried out to assess the extent of genetic structure in a representative set of 226 individuals of Pongamia pinnata encompassing seven populations as a prelude to utilization of promising and genetically divergent material in breeding programmes. Molecular polymorphism was 67.18% with ten inter simple sequence repeats (ISSR) between the individuals indicating modest levels of genetic variation in the Pongamia pinnata germplasm collected. The within-population variation based on ISSR polymorphism was 32.34% and polymorphism at the species level was 94.3%. Genetic differentiation between populations (GST = 0.61) was positively correlated with geographical distance. The data obtained indicate an immediate need to widen the genetic base of Pongamia pinnata germplasm for proper characterization, and for extensive plantations of elite varieties to meet the demands for biodiesel.     

Planchonella, first record of gynomonoecy for the family Sapotaceae

Inter-simple sequence repeat (ISSR) markers were used to investigate the levels and pattern of genetic variation within and among populations of Pteroceltis tatarinowii Maxim., an endangered plant endemic to China. Of the 76 primers screened, 11 produced highly reproducible ISSR bands. A total of 118 bands were presented from the 11 selected primers across all individuals of five natural populations, corresponding to an average of 10.73 bands per primer. The size of the ISSR bands ranged from 200 to 2,000 bp. The percentage of polymorphic loci at the population level ranged from 77.97 to 86.44%, with an average value of 82.54%. Genetic differentiation among populations was revealed based on Nei’s genetic diversity analysis (19.41%) and the nonparametric analysis of molecular variance (20.62%). The Mantel test showed a significant positive correlation between geographic distance and genetic distance (r = 0.7758, P < 0.05), indicating a role of geographic isolation in shaping the present population genetic structure of P. tatarinowii. The size of the natural populations of P. tatarinowii was noted in field observations to be very small, chiefly owing to habitat destruction and overexploitation in the past decades. Therefore, effective measures for preserving genetic diversity of this species at the population level are needed and should include protecting its natural habitats and increasing the numbers of individuals. To meet the commercial demand for this species, P. tatarinowii plantations and cultivation facilities should be established as an alternative source of raw materials.     

Evaluation of genetic diversity of <Emphasis Type="Italic">Clinacanthus nutans</Emphasis> (Acanthaceaea) using RAPD,ISSR and RAMP markers

Three polymerase chain reaction (PCR) techniques were compared to analyse the genetic diversity of Clinacanthus nutans eight populations in the northern region of Peninsular Malaysia. The PCR techniques were random amplified polymorphic deoxyribonucleic acids (RAPD), inter-simple sequence repeats (ISSR) and random amplified microsatellite polymorphisms (RAMP). Leaf genomic DNA was PCR amplified using 17 RAPD, 8 ISSR and 136 RAMP primers . However, only 10 RAPD primers, 5 ISSR primers and 37 RAMP primers produced reproducible bands. The results were evaluated for polymorphic information content (PIC), marker index (MI) and resolving power (RP). The RAMP marker was the most useful marker compared to RAPD and ISSR markers because it showed the highest average value of PIC (0.25), MI (11.36) and RP (2.86). The genetic diversity showed a high percentage of polymorphism at the species level compared to the population level. Furthermore, analysis of molecular variance revealed that the genetic diversity was higher within populations, as compared to among populations of C. nutans. From the results, the RAMP technique was recommended for the analysis of genetic diversity of C. nutans.     

Diversity among wild accessions of Bacopa monnieri (L.) Wettst. and their morphogenetic potential

Biochemical and molecular diversity among 14 accessions of Bacopa monnieri (L.) Wettst. collected from various locations of India was investigated. A significant variation was recorded in bacoside A contents of these accessions. A scatter plot of principle component analysis based on bacoside A contents clubbed these populations into two major groups and accession BM14 was placed separately. Similarly, about 35 % variations were detected in these populations based on combined data of random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR). Individually, ISSR markers detected higher variation (44.9 %) as compared to RAPD markers (23 %). Clustering based on molecular marker data grouped these accessions into two major groups and also placed accession BM14 as an out group. The shoot organogenic potential of leaf explants taken from microshoots and rooting of microshoots also varied among accessions. Maximum shoot organogenic potential was observed in accession BM5 and maximum rooting potential was observed in accessions BM1, BM2, BM7, BM10 and BM14. Present study is an important step for developing long-term strategy for conservation of this important medicinal herb.     

Genetic Variation in Natural Populations of Stipa tenacissima from Algeria

Intermicrosatellite PCR [inter-simple sequence repeat (ISSR)-PCR] markers and cytogenetics criteria were used to assess the level of genetic diversity and genetic structure in 17 populations of Stipa tenacissima (Gramineae) from Algeria. All populations sampled in the steppe area were diploids (2n = 2x = 24), and those sampled in the dry area were hexaploids (2n = 6x = 72). The dendrogram based on ISSR-PCR showed homogeneity within populations and large variability among populations. All individuals of the same population were gathered and formed groups clearly separated in all populations. These groups were separated into two clusters related to biotope, one from the steppe area and the other from the dry area. AMOVA indicated low genetic diversity among populations (30% of variation) and high within populations (70%). This variation pattern would constitute an adaptive strategy to grow in various ecological conditions.     

Sex identification and genetic variation of Saccharina (Phaeophyta) gametophytes as revealed by inter-simple sequence repeat (ISSR) markers

Inter-simple sequence repeat (ISSR) polymerase chain reaction (PCR) markers were utilized to investigate the genetic variation between male and female gametophyte populations of strains Rongfu and 901 of Saccharina. In total, 11 ISSR primers were able to generate 135 satisfactory and reproducible loci, of which 134 were polymorphic with 99.26 % polymorphism. The percentages of polymorphism of female gametophyte populations (60 and 62 % for their respective strains) were higher than those of the males (53 %), and the Nei’s genetic diversity and Shannon’s information index showed a similar tendency. The clustering of gametophytes of the same sex from each strain was well resolved by both an unweighted paired group method using the arithmetic average and a principal component analysis, suggesting that any male/female gametophyte pair could represent each strain. However, a single pair was not adequate for germplasm maintenance because the genetic variance among individuals within a population accounted for 57.45 % of the total (P?<?0.0001), as shown by the analysis of molecular variance. The gametophyte sex could be identified by amplification with primer UBC809 because of a differential band present in the females. According to the sequence of this band, a pair of ISSR-derived sequence-characterized amplified region (SCAR) primers was designed. With the primers, one female-specific fragment was detected using PCR and Southern blot hybridization. This converted SCAR marker was localized on one unique chromosome of the female gametophytes of these two strains by use of fluorescence in situ hybridization, confirming that it was a female chromosome-specific marker.     

Evaluation of genetic diversity amongst <Emphasis Type="Italic">Descurainia sophia</Emphasis> L. genotypes by inter-simple sequence repeat (ISSR) marker

Descurainia sophia is a valuable medicinal plant in family of Brassicaceae. To determine the range of diversity amongst D. sophia in Iran, 32 naturally distributed plants belonging to six natural populations of the Iranian plateau were investigated by inter-simple sequence repeat (ISSR) markers. The average percentage of polymorphism produced by 12 ISSR primers was 86 %. The PIC values for primers ranged from 0.22 to 0.40 and Rp values ranged between 6.5 and 19.9. The relative genetic diversity of the populations was not high (Gst =0.32). However, the value of gene flow revealed by the ISSR marker was high (Nm = 1.03). UPGMA clustering method based on Jaccard similarity coefficient grouped the genotypes into two major clusters. Graph results from Neighbor-Net Network generated after a 1000 bootstrap test using Jaccard coefficient, and STRUCTURE analysis confirmed the UPGMA clustering. The first three PCAs represented 57.31 % of the total variation. The high levels of genetic diversity were observed within populations, which is useful in breeding and conservation programs. ISSR is found to be an eligible marker to study genetic diversity of D. sophia.     

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.     

Negligence in the Atlantic forest,northern Brazil: a case study of an endangered orchid

Currently, many Brazilian orchids are threatened with extinction resulting from habitat loss and intense harvesting pressure stemming from their value as ornamental plants. Therefore, the genetic diversity in remaining populations is fundamental to the survival of these species in natural environments. In order to inform conservation strategies, this study evaluated the genetic diversity and structure of Cattleya granulosa populations. The sample consisted of 151 individuals from 12 populations in the Atlantic Forest, northeastern Brazil, evaluated using 91 ISSR markers. Genetic variability was assessed through molecular variance, diversity indexes, clusters of genotypes through Bayesian analysis, and tests for genetic bottlenecks. From all polymorphic loci, genetic diversity (H_E) varied between 0.210 and 0.321 and the Shannon index ranged from 0.323 and 0.472. Significant genetic differentiation between populations (Φ_ST = 0.391; P < 0.0001) resulted in the division of the populations into five groups based on the log-likelihood Bayesian analysis. We found significant positive correlation between geographical and genetic distances between populations (r = 0.794; P = 0.017), indicating isolation by distance. Patterns of allelic diversity within populations suggest the occurrence of bottlenecks in most C. granulosa populations (n = 8). Therefore, in order to maintain the genetic diversity of the species, the conservation of spatially distant groups is necessary.