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.     

Free-living amoebae isolated from water-hyacinth root (Eichhornia crassipes)

Free-living amoebae are widely distributed in aquatic environments and their hygienic, medical and ecological relationships to man are increasingly important. The purpose of this study was to isolate free-living amoebae from water-hyacinth root (Eichhornia crassipes) and the water of an urban lake in Mexico City. Five grams of wet root were seeded on non-nutritive agar with Enterobacter aerogenes (NNE). Water samples were concentrated by centrifugation at 1200g for 15 min and the pellet was seeded on NNE. Of the 16 isolated genera, 10 were detected in both habitats. The most frequent were Vannella in root and Acanthamoeba and Naegleria in water. The total number of isolates and genera isolated from root was higher than that isolated from water. The differences between root and water are probably due to the morphological characteristics of water-hyacinth root, which provides a large habitat and refuge area for many organisms.     

Analysis of genetic variation in grass carp (Ctenopharyngodon idellus) from native and colonized regions using ISSR markers

The grass carp (Ctenopharyngodon idella), a freshwater species native to China, have been introduced in more than 100 countries. In this paper, inter simple sequence repeat (ISSR) markers were used to evaluate genetic variation within and among grass carp populations sampled from its native (China) and colonized habitats (USA, Hungary, and Japan). Twenty-two ISSR primers were used to generate 207 bands, of which 82 (39.61 %) were polymorphic. The highest genetic diversity was observed in the Yangtze River population, whereas the lowest diversity was found in the Tone River population (Japan). Compared with colonized populations, the native populations possess approximately twice as much genetic diversity. The AMOVA analysis showed a relatively high level of genetic variation within populations. Significant genetic differences were revealed both among the native populations and between pooled native and colonized populations.     

赤霉素对入侵植物凤眼莲营养生长和克隆繁殖的调控

[目的]明确植物激素赤霉素对入侵植物凤眼莲营养生长和无性克隆繁殖的影响,探索凤眼莲快速生长繁殖的内在调控机理,为其科学防控及合理利用提供参考.[方法]在温室条件下用改良霍格兰营养液培养凤眼莲幼苗,并定期对其叶茎表面喷洒50μmol·L-1赤霉酸(GA3),培养4周后检测其营养生长和无性繁殖指标,包括株高、根长、膨大茎周...     

Clonal and spatial genetic structure in natural populations of Luohanguo (Siraitia grosvenorii), an economic species endemic to South China,as revealed by RAPD markers

Clonal growth is generally expected to have significant effects on the spatial genetic structure within populations. In this study, random amplified polymorphic DNA (RAPD) markers were used to reveal clonal and spatial genetic structure of four natural populations of Luohanguo (Siraitia grosvenorii), an economic vine species endemic to South China. A total of 351 ramets were assigned to 76 distinct multi-locus genotypes (i.e. genets), with the G/N varying from 0.121 to 0.350. No widespread genet was found across different populations. The clonal diversity (D) and evenness (E) ranged from 0.333 to 0.828 and from 0 to 0.741, respectively. While most genets consisted of fewer than five ramets, we observed some dominant genets that had much more (up to 69) ramets and spread over large areas. Spatial autocorrelation analyses revealed a spatial genetic structure (i.e. significant positive autocorrelation within 20 m and negative autocorrelation beyond 40 m) in one population, but not in other three populations with smaller population size. This study highlights the importance of clonal growth in shaping the spatial genetic structure in Luohanguo, which may have complex effects on the dynamics and evolution of its declining populations.     

我国特有植物青檀遗传结构的ISSR分析

青檀是我国特有的第三纪残遗植物,是宣纸纤维来源的重要原材料,现已被列入国家Ⅲ级保护植物。采用简单序列重复区间扩增多态性(ISSR)标记技术,对采自27个地理种群的628个青檀个体的遗传多样性和遗传结构进行了检测。8对引物共得到66个清晰的扩增位点,其中多态性位点63个,多态性位点百分率为95.45%。分析结果表明,青檀在物种水平上具有很高的遗传多样性(PPB=95.45%、Ao=1.9545、Ae=1.5729、He=0.3335、I=0.4980、Hb=0.3437)。在种群水平上的遗传多样性(PPB=69.98%、Ao=1.6998、Ae=1.4449、He=0.2561、I=0.3793、Hb=0.2656)和种群间遗传分化水平(Gst=0.23,Ф_ST=0.25)均处于中等水平。华北地区(30°-42°N)和华南地区(22°-30° N)青檀的遗传多样性和遗传结构的差异并不显著。古老的起源历史、较广的分布范围、异交的繁育系统、风媒种子、长命的生活史以及华南和华北地形和植被的差异可能是导致青檀目前遗传格局的主要原因。结合叶绿体序列(cpDNA)序列的遗传结构特征对青檀的保护策略进行了探讨。     

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.     

Estimation of genetic variation in Cynodon dactylon accessions using the ISSR technique

Genetic variation in 55 accessions of Cynodon dactylon was estimated using inter-simple sequence repeat (ISSR) markers. The plant materials used in this study originated from 17 countries. A total of 236 ISSR fragments were generated with 14 primers. Fragment sizes ranged from 200 to 3000 bp. All scorable bands were polymorphic in nature and none of the primers used produced monomorphic bands, indicating a high level of genetic variation in this grass. The accessions were found to be clustered into eight major groups through the unweighted pair-group method with arithmetic averages. Genetic similarity coefficients (GSC) among the 55 accessions ranged from 0.52 to 0.95. The results clearly indicate that a high level of variation exists in Cynodon accessions. This study shows that the ISSR technique is a reliable tool for differentiating Cynodon accessions and for determining the genetic relationships among them.     

Population genetic differentiation of Schisandra chinensis and Schisandra sphenanthera as revealed by ISSR analysis

Schisandra chinensis (Turcz.) Baill. and Schisandra sphenanthera Rehd. et Wils. are well-known Chinese medicinal plants. The population genetic variation of the two species was studied using inter simple sequence repeat (ISSR) molecular markers. High levels of genetic diversity are revealed in both S. chinensis (P = 88.36%, h = 0.2894, I = 0.4396) and S. sphenanthera (P = 84.09%, H = 0.2782, I = 0.4280). However, the population genetic differentiation is significantly different between the two species. The S. sphenanthera harbors as high as 27% of the genetic variation among populations but 73% within populations, whereas in S. chinensis 17% of the genetic variation occurs among populations and 83% within populations. Both significant (P < 0.05) heterozygosity excess and shifted mode of allele frequency distribution are detected in four out of six populations of S. chinensis and one out of five populations of S. sphenanthera, suggesting the occurrence of recent population bottlenecks in the two species. The different patterns of genetic variation in S. chinensis and S. sphenanthera are discussed in relation to their differences in pollination mechanism, geographic distribution and historical events, and the level of gene flow and genetic drift.     

Population genetics studies of half-smooth tongue sole Cynoglossus semilaevis using ISSR markers

Inter-simple sequence repeat (ISSR) analysis was performed in order to evaluate the genetic diversity of wild and hatchery samples of half-smooth tongue sole Cynoglossus semilaevis. A group of 200 genotypes belonging to four wild samples, Laizhou (LZ), Weihai (WH), Qingdao (QD), Rizhao (RZ) and one hatchery sample, Mingbo (MB) were screened using 15 different ISSR primers. A total of 137 loci were produced in the five studied samples. 41.80%, 45.26%, 44.27%, 42.86% and 41.59% of these loci were polymorphic over all the genotypes tested in LZ, WH, QD, RZ and MB samples, respectively. The number of polymorphic loci detected by single primer combination ranged from 2 to 7. The average heterozygosity of LZ, WH, QD, RZ and MB samples were 0.0710, 0.0814, 0.0793, 0.0727 and 0.0696, respectively. The WH sample showed a higher genetic diversity including total number of ISSR bands (P < 0.05), total number of polymorphic bands (P < 0.05), average heterozygosity (P < 0.05) and total number of genotypes (P < 0.05) than all the other samples. Among the five studied samples, the hatchery sample (MB) showed the lowest genetic viability.     

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.     

Genetic diversity of wild populations of Rheum tanguticum endemic to China as revealed by ISSR analysis

Rheum tanguticum is an important but endangered traditional Chinese medicine endemic to China. The wild resources have been declining. Establishing the genetic diversity of the species would assist in its conservation and breeding program. Inter-simple sequence repeats (ISSR) markers were used to assess the genetic diversity and population genetic structure in 13 wild populations of R. tanguticum from Qinghai Province. Thirteen selected primers produced 329 discernible bands, with 326 (92.94%) being polymorphic, indicating high genetic diversity at the species level. The Nei's gene diversity (He) was estimated to be 0.1724 within populations (range 0.1026–0.2104), and 0.2689 at the species level. Analysis of molecular variance (AMOVA) showed that the genetic variation was found mainly within populations (71.02%), but variance among populations was only 28.98%. In addition, Nei's differentiation coefficients (G_st) was found to be high (0.3585), confirming the relatively high level of genetic differentiation among populations. Mantel test revealed a significant correlation between genetic and geographic distances (r = 0.573, P = 0.002), and the unweighted pair-group method using arithmetic average (UPGMA) clustering and Principal coordinates analysis (PCoA) demonstrated similar results. Meanwhile, the genetic diversity of R. tanguticum positively correlated with altitude and annual mean precipitation, but negatively correlated with latitude and annual mean temperature. This result might be an explanation that the natural distribution of R. tanguticum is limited to alpine cold areas. We propose conservation strategy and breeding program for this plant.     

High genetic differentiation and low genetic diversity in Incarvillea younghusbandii, an endemic plant of Qinghai-Tibetan Plateau,revealed by AFLP markers

Incarvillea younghusbandii Sprague (Bignoniaceae) is a perennial herbaceous plant endemic to Qinghai-Tibetan Plateau. As a species of medical and horticulture importance, I. younghusbandii is threatened by over exploitation and habitat fragmentation. In this study, we analyze the genetic diversity and population structure of I. younghusbandii using amplified fragment length polymorphism (AFLP) markers. Our data reveal very low levels of genetic diversity in seven natural populations across Tibet. Specifically, at population level, the average Nei's genetic diversity index (H_E) and Shannon's diversity index (I) were 0.063 and 0.096, respectively. In contrast, high genetic differentiation among populations (Gst = 0.6238, Φ_ST = 0.614) is detected. The results of Neighbor-joining cluster, PCO, and STRUCTURE assignment reveal consistent pattern, suggesting seven well-defined genetic groups that are concordant with their geographical origins. The possible mechanisms and implications of these findings for conservation are discussed.     

Genetic relatedness of Portuguese almond cultivars assessed by RAPD and ISSR markers

Randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to analyse the genetic diversity of Portuguese Prunus dulcis cultivars and their relationship to important foreign cultivars. Of the primers tested, 6 (out of 60) RAPD and 5 (out of 18) ISSR primers were selected for their reproducibility and high polymorphism. Out of 124 polymerase chain reaction fragments that were scored, 120 (96.8%) were polymorphic. All the plants could be discriminated and constitute a very heterogeneous group. Five unidentified almond plants found in the region of Foz Côa (north Portugal) and wild almond (P. webbii) from Italy and Spain were also included. Four main groups of plants could be distinguished: P. dulcis cultivars; one Foz Côa plant; P. webbii; and P. persica (outgroup). The segregating Foz Côa plant may represent a feral individual or a hybrid between P. dulcis and P. webbii.Abbreviations dNTP Deoxynucleotide triphosphate - CTAB Cetyltrimethylammonium bromide - ISSR Inter-simple sequence repeats - PCR Polymerase chain reaction - RAPD Randomly amplified polymorphic DNA - RASTM Regional Agricultural Services of Trás-os-Montes - TE Tris-EDTA buffer - UPGMA Unweighted pair group method with arithmetical averagesCommunicated by P. Puigdoménech     

Conservation genetics of Heritiera littoralis (Sterculiaceae), a threatened mangrove in China,based on AFLP and ISSR markers

AFLP and ISSR markers were used to determine the genetic variations in eight mangrove and non-mangrove populations of Heritiera littoralis (Sterculiaceae), a threatened species in China. Our results showed a moderate to high level of genetic variation in this species (P = 63.69%, H_T = 0.20 for AFLP; P = 76.07%, H_T = 0.22 for ISSR), and a relatively high level of genetic differentiation among populations (G_ST = 0.24 for AFLP and 0.27 for ISSR). Life history traits, long-distance dispersal of floating seeds, and local environments may play important roles in shaping the genetic diversity and genetic structure of this species. Investigation of the plant’s reproductive capacity showed that the natural seed production of H. littoralis was low, as was the germination rate and the transformation rate from juvenile to adult. H. littoralis is seriously threatened and is in urgent need of conservation in China.     

Highly differentiated populations of the narrow endemic and endangered species Primula cicutariifolia in China,revealed by ISSR and SSR

The perennial herb Primula cicutariifolia Pax is an endangered and endemic species with narrow distribution in eastern Anhui and Zhejiang provinces of China. In this study, the levels of genetic variation and the pattern of genetic structure in five natural populations of P. cicutariifolia were assessed by inter-simple sequence repeats (ISSR) and simple sequence repeat (SSR) markers. Both markers revealed that there was remarkably low genetic variation within populations (e.g., H_e = 0.19 and 0.18, for ISSR and SSR respectively) and high differentiation among populations (G_ST = 0.714 and 0.611; Φ_ST = 0.698 and 0.599, for ISSR and SSR respectively). The level of population genetic diversity was correlated to population size only detected by ISSR markers. The genetic structure of P. cicutariifolia may be explained by limited gene flow that was caused by habitat fragmentation and limited seeds and pollen dispersal ability, self breeding system and biennial life form. To protect and avoid disappearance of P. cicutariifolia, much more attentions should be paid to protect all the remnant populations and their habitats, and three management units, i.e. Tianmushan, Damodao, and Panan units, were proposed.     

Genetic diversity of yacon (Smallanthus sonchifolius (Poepp. & Endl.) H. Robinson) and its wild relatives as revealed by ISSR markers

Set of 29 accessions of Smallanthus sonchifolius, an important tuber crop from South America, and its three wild relatives were analysed using the Inter Simple Sequence Repeats (ISSR) markers. Seven primers out of 30 primers screened gave clear and reproducible spectra. The range of amplified bands was from 2500 bp to 300 bp. These seven primers generated in total 77 bands, from which 75 (97.4%) were polymorphic. Nei's genetic distances between samples varied from 0.01 to 0.24. The Shannon's index (I) was estimated as 0.0392. The UPGMA dendrogram created using the Neighbour joining method and based on the Dice's dissimilarity coefficient separated clearly the wild accessions from all S. sonchifolius samples, which remained close to each other, confirming the clonal origin and thus a very low genetic variability within the genus.     

广东省五节芒遗传多样性的ISSR分析

采用ISSR分子标记技术,对广东省内分布的能源植物五节芒(Miscanthus floridulus)13个野生居群共215个个体的遗传多样性进行了研究.9个ISSR引物共扩增到了81个位点,其中76个是多态性位点.结果表明,广东五节芒的遗传多样性水平很高,物种水平上,多态位点百分率(PPB)为93.83％,Nei's...     

Assessment of genetic stability in tissue-cultured products and seedlings of Saussurea involucrata by RAPD and ISSR markers

To control the genetic quality during the whole process of tissue culture of the traditional Chinese medicinal plant, Saussurea involucrate Kar. et Kir., DNA polymorphisms and genetic variations were investigated using randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers. The genetic stability/variation in tissue-cultured products, including three calli, three adventitious shoots, regenerated plantlets and 2 year-old regenerated plantlets cultivated in the planting base in Tianshan Mountain, were assessed compared with 1 year-old and 2 year-old seedlings cultivated in the same planting base using aseptic seedlings as reference. Apparent genetic variation was detected in the 11 type of plant materials. The percentages of polymorphic bands in the RAPD and ISSR analysis were, respectively, 35% and 33%. Cluster analysis indicated that the genetic similarity values calculated on the basis of RAPD and ISSR data among the 11 type of plant materials were respectively ranged from 0.823 to 0.995 with a mean of 0.878 and 0.825 to 0.974 with a mean of 0.885, which classified the samples into three groups. The similarity coefficient also revealed that differences among three calli were not remarkable by both RAPD and ISSR analysis, and only chemical components and growth properties needed consideration in the screening of callus used for the next redifferentiation studies. But there are remarkable differences among three adventitious shoots analyzed by ISSR markers. Therefore, RAPD and ISSR markers are efficient tools in genetic variation assessment and quality control in plant tissue culture process.