Association of loganin contents with the genetic characterization of natural populations of Palicourea rigida Kunth determined by AFLP molecular markers

Extracts of the medicinal plant Palicourea rigida Kunth, popularly known as douradinha, are widely used for treating urinary tract disorders. Unfortunately, nowadays this is one of the species endemic to Brazilian Cerrado that is at greatest risk of extinction.The aim of the this work was to use AFLP molecular markers to determine the genetic structure and diversity of eight natural populations of P. rigida and to associate their genetic characteristics with loganin production in order to obtain provide relevant information to promote programs for the conservation of this valuable medicinal plant.A total of 120 polymorphic bands were scored and higher proportion of genetic diversity was found in inter-populations (64%) rather than in intra-populations (36%). F_st value was found to be significantly greater than zero (0.3601), demonstrating the complex genetic structure of P. rigida populations. Accessions collected from Cristalina, GO, showed higher percentage of polymorphic loci (65.5%) and the highest genetic diversity. Analysis of Molecular variance (AMOVA) demonstrated 63.9% of intra-population genetic variation. The lowest genetic variability was detected among accessions from the population found in Sacramento, MG. No spatial standard was observed for P. rigida population, suggesting a partially isolated island model. It was observed a minor but significant positive correlation (r = 0.22) between chemical and genetic matrices. The association between chemical and genetic data indicated that environmental factors promoted the loganin production in populations growing in Luziânia, GO, and therefore accessions from those populations should be considered as prime material for initiating the conservation process of P. rigida.     

Genetic Characterization of the Gypsy Moth from China (Lepidoptera,Lymantriidae) Using Inter Simple Sequence Repeats Markers

This study provides the first genetic characterization of the gypsy moth from China ( Lymantria dispar ), one of the most recognized pests of forests and ornamental trees in the world. We assessed genetic diversity and structure in eight geographic populations of gypsy moths from China using five polymorphic Inter simple sequence repeat markers, which produced reproducible banding patterns. We observed 102 polymorphic loci across the 176 individuals sampled. Overall genetic diversity (Nei’s, H) was 0.2357, while the mean genetic diversity within geographic populations was 0.1845 ± 0.0150. The observed genetic distance among the eight populations ranged from 0.0432 to 0.1034. Clustering analysis (using an unweighted pair-group method with arithmetic mean and multidimensional scaling), revealed strong concordance between the strength of genetic relationships among populations and their geographic proximity. Analysis of molecular variance demonstrated that 25.43% of the total variability (F _ST = 0.2543, P < 0.001) was attributable to variation among geographic populations. The results of our analyses investigating the degree of polymorphism, genetic diversity (Nei’s and Shannon) and genetic structure, suggest that individuals from Hebei may be better able to adapt to different environments and to disperse to new habitats. This study provides crucial genetic information needed to assess the distribution and population dynamics of this important pest species of global concern.     

High genetic diversity detected in the endemic Primula apennina Widmer (Primulaceae) using ISSR fingerprinting

Primula apennina Widmer is endemic to the North Apennines (Italy). ISSR were used to detect the genetic diversity within and among six populations representative of the species distribution range. High levels of genetic diversity were revealed both at population percentage of polymorphic band (PPB = 75.92%, H _S = 0.204, H _pop = 0.319) and at species level (PPB = 96.95%, H _T = 0.242, H _sp = 0.381). Nei gene diversity statistics (15.7%), Shannon diversity index (16.3%) and AMOVA (14%) detected a moderate level of interpopulation diversity. Principal coordinate and Bayesian analyses clustered the populations in three major groups along a geographic gradient. The correlation between genetic and geographic distances was positive (Mantel test, r = 0.232). All together, these analyses revealed a weak but significant spatial genetic structure in P. apennina, with gene flow acting as a homogenizing force that prevents a stronger differentiation of populations. Conservation measures are suggested based on the observed pattern of genetic variability.     

Population genetic structure and conservation of the Azorean tree Prunus azorica (Rosaceae)

Prunus azorica is an endangered tree endemic to the Azores Archipelago, considered as a top priority species for conservation. Although propagation measures have been studied in detail, and a broad phylogeographic study on P. lusitanica was recently published, a detailed population genetics study devoted to Azorean taxon was lacking. To determine extant patterns of population genetic structure in P. azorica, we analysed eight populations from the five Azorean islands where the species presently occurs and the only extant individual from Flores Island. We also included samples of P. lusitanica subsp. hixa from the Canary Islands and Madeira, and of P. lusitanica subsp. lusitanica from mainland Portugal. Genotyping was undertaken for eight nuclear microsatellite polymorphic loci specifically isolated for P. azorica. Accessions of the different geographic regions were used to sequence ITS and trnL DNA regions. Regarding SSRs, the number of alleles ranged from 5 to 37 (mean = 12.6) per locus and from 2 to 64 per population (mean = 24). Our analysis showed a clear separation between samples from the Azores and those from other regions. Overall, São Miguel populations seemed to encompass the majority of the variability found within the archipelago. Regarding the Azorean populations only, the highest percentage of genetic variation was found within populations (92 %). Still, about 7 % of the variation was found among populations within islands. Expected heterozygosity ranged from values near 0 in the most depauperate populations up to 0.18. With a few exceptions, the level of differentiation between Azorean populations was generally low and gene flow was clearly above 1. Analysis of ITS sequences also detected differences between the Azores and the remaining regions but the trnL region did not reveal any variation. The genetic identity of P. azorica was recognised and thus should be preserved; however, the present results suggest that the Azorean taxon should be reinstated at the subspecies level.     

Analysis of the genetic structure of Rhodiola rosea (Crassulaceae) using inter-simple sequence repeat (ISSR) polymorphisms

The genetic diversity and differentiation of eleven R. rosea populations from different parts of its wide area of occurrence were studied by ISSR markers. Using eight primers, 252 DNA fragments were generated, and 243 of those DNA fragments were found to be polymorphic, indicating a high genetic variability at the species level (P = 96.4%, h = 0.176, SI = 0.291). Relatively low levels of diversity were determined at the population level (P 30.6-59.1%, h 0.088-0.165, SI 0.137-0.257). AMOVA analysis revealed that the majority of the genetic variation was within populations (65.42%), and the variance among populations was 34.58%. Cluster analysis revealed two groups of R. rosea populations; these groups likely represent distinct evolutionary lines in the species, which are different in genetic structure, evolutionary history and chorological migration routes.     

Cross-species amplification of microsatellites reveals incongruence in the molecular variation and taxonomic limits of the Pilosocereus aurisetus group (Cactaceae)

The Pilosocereus aurisetus group contains eight cactus species restricted to xeric habitats in eastern and central Brazil that have an archipelago-like distribution. In this study, 5–11 microsatellite markers previously designed for Pilosocereus machrisii were evaluated for cross-amplification and polymorphisms in ten populations from six species of the P. aurisetus group. The genotypic information was subsequently used to investigate the genetic relationships between the individuals, populations, and species analyzed. Only the Pmac101 locus failed to amplify in all of the six analyzed species, resulting in an 88?% success rate. The number of alleles per polymorphic locus ranged from 2 to 12, and the most successfully amplified loci showed at least one population with a larger number of alleles than were reported in the source species. The population relationships revealed clear genetic clustering in a neighbor-joining tree that was partially incongruent with the taxonomic limits between the P. aurisetus and P. machrisii species, a fact which parallels the problematic taxonomy of the P. aurisetus group. A Bayesian clustering analysis of the individual genotypes confirmed the observed taxonomic incongruence. These microsatellite markers provide a valuable resource for facilitating large-scale genetic studies on population structures, systematics and evolutionary history in this group.     

Genetic diversity in natural populations of Jacaranda decurrens Cham. determined using RAPD and AFLP markers

Jacaranda decurrens (Bignoniaceae) is an endemic species of the Cerrado with validated antitumoral activity. The genetic diversity of six populations of J. decurrens located in the State of São Paulo was determined in this study by using molecular markers for randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Following optimization of the amplification reaction, 10 selected primers generated 78 reproducible RAPD fragments that were mostly (69.2%) polymorphic. Two hundred and five reproducible AFLP fragments were generated by using four selected primer combinations; 46.3% of these fragments were polymorphic, indicating a considerable level of genetic diversity. Analysis of molecular variance (AMOVA) using these two groups of markers indicated that variability was strongly structured amongst populations. The unweighted pair group method with arithmatic mean (UPGMA) and Pearson''s correlation coefficient (RAPD -0.16, p = 0.2082; AFLP 0.37, p = 0.1006) between genetic matrices and geographic distances suggested that the population structure followed an island model in which a single population of infinite size gave rise to the current populations of J. decurrens, independently of their spatial position. The results of this study indicate that RAPD and AFLP markers were similarly efficient in measuring the genetic variability amongst natural populations of J. decurrens. These data may be useful for developing strategies for the preservation of this medicinal species in the Cerrado.     

Genetic diversity within and among the wild populations of Murraya koenigii (L.) Spreng., as revealed by ISSR analysis

Murraya koenigii (L.) Spreng., commonly known as curry leaf plant, is found in the different hilly regions of India. In the present study, fifty-nine accessions representing eight wild populations of M. koenigii were analyzed using thirteen ISSR primers. A total of 152 bands were amplified, out of which, 136 were polymorphic corresponding to 89.47% polymorphism across the accessions. The pairwise population genetic distances were calculated for all the populations that varied from 0.05 to 0.13 between the populations of M. koenigii. AMOVA and Nei’s genetic diversity analysis revealed higher genetic variations within populations than among the populations. The clustering of populations in the dendrogram was not in congruence with geographical affiliations. The results indicate that the ISSR method is sufficiently informative and powerful to estimate the genetic diversity in M. koenigii populations. As M. koenigii is an important wild plant genetic resource, therefore, information on genetic variability might be a potential source as breeding material for development of commercially valuable traits in M. koenigii plants.     

Isozyme variation in Desmodium nudiflorum

Five populations of Desmodium nudiflorum were assayed for genetic variability in eight enzyme systems encoding thirteen genetic loci. The proportion of polymorphic loci for the species as a whole is 46.2% while the proportion of polymorphic loci within populations averages 13.5%. Average individual heterozygosity is 2.3%. D. nudiflorum shows significant differences in gene frequencies between populations, and genotypic frequencies within populations conform to Hardy-Weinberg expectations.     

Isolation and characterization of microsatellite marker loci in the Wagner’s mustached bat <Emphasis Type="BoldItalic">Pteronotus psilotis</Emphasis> (Chiroptera: Mormoopidae) and cross-amplification in other related species

Pteronotus psilotis, a mormoopid bat, is an insectivorous, gregarious and strict cave-dwelling species that is found areas between the sea level and an elevation of about 1000 masl. This species is present in diverse habitats ranging from rain forest to dry deciduous forest. Nine microsatellite loci were developed for Wagner’s mustached bat, Pteronotus psilotis using the next-generation sequencing approach, and their utility for population genetics studies was assessed. All loci were polymorphic (7–15 alleles) and characterized in 30 individuals from three P. psilotis populations, with the levels of observed and expected heterozygosity ranging from 0.280 to 0.867 and 0.584 to 0.842, respectively. One locus showed significant departures from Hardy–Weinberg expectations after Bonferroni correction. Cross-amplification in 11 other bat species was tested, for which eight microsatellites were successfully amplified, and of these seven were polymorphic. The development of these new microsatellite loci will contribute to investigations of genetic population structure, genetic diversity and gene flow in P. psilotis populations, as well as in other closely related bat species.     

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

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

Distribution of genetic variability in southern populations of Scots pine (Pinus sylvestris L.) from the Alps to the Apennines

Alpine and Northern Apennine populations of Pinus sylvestris collected from eight different Italian sites were analyzed by mitochondrial nadI intron and InterSimple Sequence Repeat (ISSR) markers, in order to describe the natural level of genetic variability and to clarify their genetic relationships. The small Northern Apennine populations are the southernmost populations of this conifer in Italy. All the analyzed populations were spontaneous and reforested areas were excluded. The analysis of the polymorphisms in the nad 1 intron sequence confirmed that the Italian P. sylvestris populations have the same mitotype (mitotype a) as the Central European ones. In the genomic ISSR analysis the proportion of shared alleles between the individuals showed the highest degree of differentiation between French and Italian populations and a divergence between the Alpine and Apennine populations. Alpine populations showed a higher genetic variability (GD 0.310±0.0252) than Apennine samples (GD 0.217±0.019). In addition, the individuals from the Apennines did not show a clear population structure, suggesting a common genetic constitution of Apennine P. sylvestris. It is likely that this constitution is the result of a progressive genetic isolation between the Alpine and the Northern Apennine populations from the early Holocene. The genetic constitution of the Northern Apennine populations suggests the opportunity of a management where in situ conservation of such small populations could be coupled to their use as sources of suitable local reforesting materials.     

Microsatellite Markers Reveal Genetic Variation within Sclerotinia sclerotiorum Populations in Irrigated Dry Bean Crops in Brazil

Microsatellites are powerful markers to infer population genetic parameters. We used 10 microsatellite loci to characterize the genetic diversity and structure of 79 samples of Sclerotinia sclerotiorum isolated from four Brazilian dry bean populations and observed that eight of them were polymorphic within populations. We identified 102 different haplotypes ranging from 6 to 18 per locus. Analyses based on genetic diversity and fixation indices indicated variability among and within populations of 28.79% (F_ST = 28793) and 71.21%, respectively. To examine genetic relatedness among S. sclerotiorum isolates, we used internal spacer (ITS1‐5.8S‐ITS2) restriction fragment length polymorphism (PCR‐RFLP) and sequencing analysis. PCR‐RFLP analysis of these regions failed to show any genetic differences among isolates. However, we detected variability within the sequence, which does not support the hypothesis of clonal populations within each population. High variability within and among populations may indicate the introduction of new genotypes in the areas analysed, in addition to the occurrence of clonal and sexual reproduction in the populations of S. sclerotiorum in the Brazilian Cerrado.     

Population genetic structure of the sidespot barb,Barbus neefi,from the north-eastern escarpment,South Africa

Allozyme analysis was used to determine patterns of genetic variation within and between populations of Barbus neefi. The products of 29 loci were analysed, with 17 loci being monomorphic in all populations. The genetic variability estimates compared well with values reported in the literature. The percentage of polymorphic loci (0.95 criterion) ranged from 0–20.69% and average expected heterozygosity from 0.004–0.069. Headstream populations generally revealed lower genetic variability than populations in low lying areas, which emphasizes the importance of conserving headstream populations. Fixed allele mobility differences were observed at the MPI-1 protein coding locus between the Selons River population and the other populations. The measures of genetic differentiation as assessed by F-statistics, the effective number of migrants per generation and Nei's unbiased genetic distance consistently separated the Selons River population from the rest. Nevertheless, these estimates all fell within the range considered for conspecific populations. There was also a clear genetic division between populations from north and south of the Olifants River. The three populations from the Dorps, Spekboom and Blyde Rivers (Limpopo River system) and the population from the Crocodile River (Incomati River system) showed negligible genetic differentiation. Human-assisted transfer of populations may be responsible for this close similarity.     

Genetic Diversity and Population Structure of Teucrium polium (Lamiaceae) in Tunisia

Random amplified polymorphic DNA markers were used to assess the genetic diversity within and among seven Tunisian diploid and polyploid populations of Teucrium polium L. from five bioclimatic areas. Out of the 141 bands generated from eight selected primers, 124 were polymorphic. The genetic diversity within a population (Shannon’s index) was high and varied according both the ploidal levels and bioclimatic zones. The genetic differentiation among populations assessed by G _ST and Φ_ST statistics was high, suggesting a low level of gene flow among them. The major proportion of the variation was attributable to individual differences within populations. The UPGMA analysis based on Nei and Li’s coefficient showed that individuals from each population clustered together. In a dendrogram using the Φ_ST distance matrix, population grouping is concordant with bioclimates and cytotypes. Conservation strategies should take into account the level of the genetic diversity of the populations according to their bioclimate and ploidal levels.     

Microsatellite Polymorphisms in Cassava Landraces from the Cerrado Biome, Mato Grosso do Sul, Brazil

Using nine microsatellite loci, we investigated genetic structure and diversity in 83 Brazilian cassava accessions, including several landraces, in the Cerrado biome in Mato Grosso do Sul, Brazil. All nine loci were polymorphic, averaging 6.00 alleles per locus. Treating each of seven municipalities as a cassava group or population, they averaged 3.5 alleles per locus, with 97% polymorphic loci, high values for observed heterozygosity (0.32) and gene diversity (0.56). Total genetic variability was high (0.668), and most of this genetic variability was concentrated within municipalities (0.577). Cluster and structure analyses divided accessions into two major clusters or populations (K = 2). Also, a significant genetic versus geographic correlation was found (r = 0.4567; P < 0.0260). Migratory routes in the Cerrado are considered main contributors to the region’s high cassava diversity and spatial genetic structure, amplifying interactions between traditional farmers and the evolutionary dynamics of this crop.     

珍稀濒危植物长叶红砂种群遗传多样性的ISSR分析

采用ISSR分子标记技术,对濒危小灌木长叶红砂(Reaumuria trigyna) 集中分布的5个种群的遗传多样性水平和遗传结构进行了研究。14条引物共检测到114个位点,其中99个为多态位点,多态位点比率为86.84％,长叶红砂种群具有较高的遗传多样性。物种水平上Shannon多样性指数(I)为0.468 8,Nei基因多样性指数(H)为0.308 4;种群水平上,多态位点比率P为77.89%,I为0.410 6,H为0.260 9,基因分化系数Gst为0.106 9,揭示了长叶红砂种群遗传变异多存在于种群内,种群间的遗传分化较小,占10.69%。 基因流(Nm)为4.178 7＞1,说明种群间的基因交流,防止了由于遗传漂变导致的遗传分化。聚类分析表明长叶红砂种群遗传距离与地理距离之间无显著的相关性。研究结果说明遗传多样性水平与物种本身特性和所处不同群落有关,濒危植物并不一定表现为遗传变异水平的降低。     

Genetic diversity in inter‐simple sequence repeat profiles across natural populations of Indian pomegranate (Punica granatum L.)

Pomegranate (Punica granatum L.), in the monogeneric family Punicaceae, is found in Iran, Afghanistan, India and Mediterranean countries. Iran is considered to be its primary centre of origin. In India, pomegranate occurs naturally only in the Western Himalayan regions of Jammu and Kashmir, Himachal Pradesh and Uttarakhand States. However, there is no information about genetic variation in wild pomegranate at population level. In this paper, we describe genetic diversity across natural populations of Indian pomegranate based on inter‐simple sequence repeat (ISSR) markers. Forty‐nine accessions representing eight populations from two regions were analysed using ISSR. Seventeen ISSR primers resulted in 268 polymorphic bands, with 87.01% polymorphism throughout the accessions. Pair‐wise population genetic distances ranged from 0.05 to 0.45, with a mean of 0.25 between populations. amova and Nei’s genetic diversity analyses revealed higher genetic variation within populations than among populations. A higher genetic differentiation (G_ST) was observed between the spatially distant populations, indicating a low level of genetic exchange (Nm) among these populations. However, clustering of populations was not in accordance with their geographical affiliations in the tree. The results indicate that the ISSR method is sufficiently informative and powerful to assess genetic variability in pomegranate, and that patterns of genetic variability observed among populations of wild pomegranate from the Western Himalaya differ. Estimation of genetic variation reported here provides a significant insight for in situ conservation and exploitation of genetic resources for this economically important species as potential breeding material.     

Genetic Variation in Chinese Pine (Pinus tabulaeformis), a Woody Species Endemic to China

Random amplified polymorphic DNA markers were used to investigate genetic variation of the Chinese pine (Pinus tabulaeformis Carr.), a species endemic to China and the most widely distributed pine species in North China. The results revealed that P. tabulaeformis populations had a relatively high level of genetic diversity (H _t = 0.3268), distributed mainly within (79.2%) rather than among (20.8%) populations. The populations of Lingkong Mountain and Wuling Mountain had a higher level of diversity (0.2687) than the other four populations (0.2537). No statistically significant relationships were found between genetic diversity and climatic factors by correlation analysis and between genetic distance and geographic distance by the Mantel test. These results suggest that the partitioning of genetic diversity in each population might have been influenced not only by water and temperature conditions but also by other factors such as human activities and the Holocene postglacial history of these populations.     

Genetic diversity of Sinojackia dolichocarpa (Styracaceae), a species endangered and endemic to China,detected by inter-simple sequence repeat (ISSR)

Sinojackia dolichocarpa, a species endangered and endemic to China, is distributed only in the regional area of Shimen and Sangzhi in Hunan Province. Inter-simple sequence repeat (ISSR) markers were used to investigate the genetic diversity within and among the four natural populations of S. dolichocarpa. Leaf samples were collected from 84 individuals. Thirteen ISSR primers selected from 80 primers gave rise to 137 discernible DNA bands of which 100 (72.99%) were polymorphic. On average each primer gave rise to 10.5 bands including 7.7 bands with polymorphic profile. At the species level, high genetic diversity was detected (PPB: 72.99%; H_E: 0.2255; Ho: 0.3453). However, relatively low genetic diversity existed within populations. Population in Maozhuhe (MZH) exhibits the greatest level of variability (PPB: 40.38%, H_E: 0.1566, Ho: 0.2330), whereas the population in Jingguanmen (JGM) finds its own variability at the lowest level (PPB: 30.66%; H_E: 0.1078; Ho: 0.1601). A high level of genetic differentiation among populations was revealed by Nei's gene diversity statistics (45.30%), Shannon's information measure (45.24%) and analysis of molecular variance (AMOVA) (52.88%). The main factors responsible for the high level of differentiation among populations are probably related to the selfing reproductive system and the isolation of populations. The strong genetic differentiation among populations indicates that the management for the conservation of genetic variability in S. dolichocarpa should aim to preserve every population.