Genotyping of Ganoderma species by improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis

Species of Ganoderma are used in traditional medicines. An improved random amplified polymorphic DNA (RAPD) analysis, where the RAMP time is prolonged, has been used to characterize the genetic variation in some well known species of Ganoderma. The DNA materials were collected from ten Ganoderma strains, amplified with randomly selected 24 RAPD primers and evaluated by agarose gel electrophoresis. A cluster dendrogram was constructed for genetic analysis on the basis of amplification results. The improved RAPD amplified DNA with consistent and clear banding patterns. A total of 316 bands were found with 93% polymorphism. There was a significant genetic distance between the different strains of Ganoderma, with an index of similarity coefficient in the range of 0.52–0.74. The inter-simple sequence repeat (ISSR) analysis of the Ganoderma DNA samples showed similar trend results to the RAPD analysis with 0.49–0.81 similarity coefficients. This study reports the high level of genetic differences between different species or strains of a single species of Ganoderma and confirms the significance of the improved RAPD method in genetic characterization of organisms. Therefore, the improved RAPD combined with ISSR techniques might be used for the genetic characterization of organisms.     

Pollen fertility of synthetic intra- and interspecific F1 hybrids in Canary Island Tolpis (Asteraceae)

Pollen fertility was determined for synthetic F₁ hybrids between members of the genus Tolpis endemic to the Canary Islands. Mean fertility varies from 1 to nearly 100%. High hybrid sterility is unusual for island lineages. Surprisingly, the fertility of hybrids between morphologically distinct species was generally higher than some crosses within the morphologically variable group that includes the Tolpis laciniata and T. lagopoda complexes. Within this group, fertility was lower particularly in hybrids involving one recognized segregate species (T. webbii), but none of the four potentially new species had reduced hybrid fertility. Despite the overall fertility differences between groupings within the T. laciniata and T. lagopoda complexes there is variation in fertility within groups; sterility factors have a complex inter-populational distribution. The cause of hybrid sterility is unknown, but preliminary data suggest that both chromosomal and genetic factors are involved.     

Genetic relationships among species of the genus Diplotaxis (Brassicaceae) using inter-simple sequence repeat markers

Inter-simple sequence repeat (ISSR) amplification was evaluated for its applicability as a genetic marker system to establish relationships among ten Diplotaxis species. ISSR amplification generated multiple banding profiles with the 12 primers from all DNA samples, with an average of 41.2 fragments per primer. This average was clearly higher for the 5′ triple-anchored primers than for other primers. The banding profiles were highly repeatable across separate PCR runs. DNA mixing procedures were found to be appropriate strategies to generate banding patterns representative of each species studied. Similarity values were calculated considering 494 ISSR bands, and a dendrogram was constructed based on the similarity matrix. The ten Diplotaxis species were clustered into two major groups. The first group consists of five species, Diplotaxis tenuifolia and Diplotaxis cretacea, and Diplotaxis muralis with their putative parents (D. tenuifolia and Diplotaxis viminea). In the second group three species are clustered that are closely related (Diplotaxis virgata, Diplotaxis catholica and Diplotaxis siettiana), in addition to Diplotaxis harra, and Diplotaxis erucoides, which has lowest similarity values with the rest of the species studied. The two groups defined in the present work may be concordant with the idea suggested by several authors of a biphyletic origin for Diplotaxis. The genetic relationships among the ten Diplotaxis species estimated by the polymorphism of ISSR markers are in agreement with those previously inferred by other morphological, biochemical and molecular data, indicating the reliability of the ISSR approach for this purpose. Received: 3 January 2000 / Accepted: 31 March 2000     

Use of inter-simple sequence repeat (ISSR) markers for discrimination between and within species of blackflies (Diptera: Simuliidae)

The present study is the first report of fingerprinting in blackflies (Diptera: Simuliidae), using inter-simple sequence repeat (ISSR) markers. Among five primers tested, three tetranucleotide repeat primers ((GACA)₄, (ACTG)₄, (ACAG)₄) generated a high proportion of polymorphic bands. Seven species representing various genera, subgenera or species groups were compared. No similar profiles were found. Intraspecific and interspecific banding patterns were analysed for two species in the Prosimulium hirtipes (Fries, 1824) species group and four species in the Simulium variegatum (Meigen, 1818) species group. The UPGMA cluster analysis based on Jaccard’s coefficient demonstrated the intraspecific and interspecific diversity and the resolving power of the ISSR markers to differentiate blackfly species and populations. In Simulium maximum (Knoz, 1961), geographically defined populations were successfully discriminated.     

Molecular characterization of Jatropha genetic resources through inter-simple sequence repeat (ISSR) markers

The genetic diversity among eight Jatropha species and three Jatropha curcas accessions were analyzed using ISSR-PCR. Nine ISSR primers generated reproducible amplification banding pattern of 61 polymorphic bands out of 64 scored accounting for 98.14% polymorphism across the species. The ISSR primers viz., I1, I2, I3, I4, I5, I6, I7 and I10 generated 100% polymorphic patterns. Jaccard’s coefficient of similarity varied from 0.346 to 0.807, indicative of high level of genetic variation among the genotypes studied. The UPGMA cluster analysis indicated three distinct clusters, one comprising all accessions of J. curcas L (TNMJ1, TNMJ 22 and TNMJ 23), while second included four species viz., J. tanjorensis J. L. Ellis et Saroja., J. gossypifolia L., J. podagrica Hook and J. maheshwarii Subrum and M.P. Nayer and the third cluster included another four species viz., J. villosa Wight J. multifida L., J. integerrima Jacq and J. glandulifera Roxb. The overall grouping pattern of clustering corresponds well with principal component analysis (PCA) confirming patterns of genetic diversity observed among the species. So far, there are no reports on the molecular diversity of the Jatropha species through ISSR marker. This study provides valid guidelines for collection, conservation and characterization of Jatropha genetic resources and also for further breeding programme towards biodiesel production.     

Genetic diversity and phylogenetic relationships of the genus Lycopersicon (Tourn.) Mill. as revealed by inter-simple sequence repeat (ISSR) analysis

Inter-simple sequence repeat (ISSR) analysis was for the first time used to study the genetic diversity and phylogenetic relationships in 54 wild accessions and cultivars of the genus Lycopersicon. Analysis involved 14 ISSR primers homologous to microsatellite repeats and containing additional selective anchor nucleotides. In total, 318 ISSR fragments were amplified for the wild and cultivated tomato genomes. The interspecific polymorphism revealed with the ISSR primers was 95.6%. Species-specific ISSR fragments were detected for each tomato species. The highest number (more than 20) of species-specific fragments were obtained for L. esculentum sensu lato, although the intraspecific variation of ISSR patterns was low. UPGMA cluster analysis was used to construct a dendrogram and to estimate the genetic distances between the species of the genus Lycopersicon; between populations of L. peruvianum, L. pimpinellifolium, and L. esculentum; and between tomato cultivars. The ISSR-based phylogeny was generally consistent with Lycopersicon taxonomy based on morphological and molecular evidence, suggesting the applicability of ISSR analysis for genotyping and phylogenetic studies in tomato.     

Population genetics of Ageratina adenophora using inter-simple sequence repeat (ISSR) molecular markers in China

Abstract

Understanding distribution and diversity of invasive weeds is essential for the development of efficient control measures against it. In the present study, inter-simple sequence repeat (ISSR) markers were used to assess the biogeographic relationships among populations of the invasive Crofton weed (Ageratina adenophora (Spreng.)) during 2004–2006 in China. A total of 100 ISSR primers with di-, tri-, tetra- and penta-nucleotide repeats were screened, from which 20 polymorphic and informative primers were selected. Amplification of the 20 primers generated a total of 479 polymorphic bands among the 64 weed populations, and a high level of genetic diversity (H _E = 0.1541 ± 0.0193) was detected in A. adenophora. Neighbor-joining (NJ) cluster analysis based on genetic distances among populations grouped the populations according to their geographical origin, i.e. (1) populations of southwestern Guizhou, (2) populations of Liangshan city in Sichuan, (3) populations of western Guizhou, (4) Guangxi populations plus Chongqing populations, (5) populations of southern Yunnan, and (6) populations of Yangtze River Valleys in Sichuan plus populations of western Yunnan. A significant positive correlation between geographical and genetic distance was detected by the Mantel test (r = 0.183, p = 0.0012). Based on the divergence relationships revealed by ISSR markers, it was assumed that A. adenophora mainly dispersed through wind and water in China.     

Genetic relationship among species in the genus Sonneratia in China as revealed by inter-simple sequence repeat (ISSR) markers

Inter-simple sequence repeat (ISSR) analysis was used to establish the genetic relationship among six Sonneratia species in China. A total of 100 primers were screened, of which 11 polymorphic and informative patterns were selected to determine the genetic relationship. Four hundred and eighty five DNA bands were amplified, among which 481 bands (99.18%) were polymorphic. The average number of DNA bands amplified by each primer was 44. Similarity coefficients were calculated and a dendrogram was constructed by using UPGMA algorithm. The six Sonneratia species were divided into two major groups. Group I consisted of Sonneratia caseolaris, Sonneratia × gulngai, Sonneratia alba, and Group II included Sonneratia × hainanensis, Sonneratia ovata and Sonneratia apetala. In Group I, S. × gulngai was close to S. alba, and in Group II, S. × hainanensis was close to S. ovata. The genetic relationships estimated by the polymorphism of ISSR markers are basically in agreement with those previously inferred by morphological data. Thus, ISSR approach is a reliable marker system that can be used to study genetic relationship in the genus Sonneratia.     

甘肃省冬虫夏草遗传多样性的ISSR分析

对采自甘肃省3个冬虫夏草主产区域6个具有代表性地方的18个样本进行ISSR分析。15条ISSR引物共扩增得到条带清晰并呈多态性的95条谱带,每一引物扩增获得的ISSR条带数在3－9条之间,扩增片段集中在300－3,000bp。基于遗传相似性系数（GS）、不加权成对群算术平均法（UPGMA）构建的系统树分析,可以看出：分离自同一株虫草上不同部位的样本无显著遗传差异;同一地点样本间的遗传分化较小;不同地域的样本间存在着较大的遗传分化,遗传多样性较丰富。同时6个地方的冬虫夏草明显地分为3个区域,而在同一区域不同     

Phylogenetic relationships among strains of the entomopathogenic fungus, Nomuraea rileyi, as revealed by partial beta-tubulin sequences and inter-simple sequence repeat (ISSR) analysis

AIMS: To elucidate the phyletic relationships among three members of the entomogenous fungal genus, Nomuraea, with an emphasis on N. rileyi. METHODS AND RESULTS: Relationships were evaluated by analysis of the beta-tubulin gene and of inter-simple sequence repeats (ISSR). The amplification product of the partial beta-tubulin gene was larger for N. atypicola than for N. rileyi, and sequencing of this gene fragment confirmed that N. atypicola possesses approximately 25 more nucleotides than N. rileyi and N. anemonoides. Based on neighbor joining and bootstrap analysis of the partial beta-tubulin gene, N. atypicola failed to form a monophyletic grouping with the other two species of Nomuraea. In contrast, the single isolate of N. anemonoides clustered with the N. rileyi isolates, and both taxa grouped with Epichloe typhina (Hypocreales: Clavicipitaceae). Results from this study suggested that N. rileyi and N. anemonoides are closely related to the Clavicipitaceae. In contrast, evidence indicated that N. atypicola is not closely related to this family, and that this taxon is not a Nomuraea. Based on the 83 polymorphic loci of ISSR, it was observed that isolates of N. rileyi from diverse geographical origins were distinctly different from both N. atypicola and N. anemonoides. Considerable heterogeneity was observed in the 18 isolates of N. rileyi tested, and several clusters contained isolates from disparate geographical locations and hosts. However, three isolates from the Philippines (three host species) and three strains isolated from velvetbean caterpillar (Anticarsia gemmatalis) larvae in South America did cluster together. Two other strains from Brazil (isolated from Spodoptera spp.) were distinct from the velvetbean caterpillar isolates from South America. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The beta-tubulin gene was generally too conserved to resolve intraspecies variability. However, ISSR did identify polymorphisms among the isolates of N. rileyi tested. The results of this study indicate that ISSR may be used as robust molecular markers for studying the population genetics of this entomopathogenic fungus.     

樟科濒危植物思茅木姜子遗传多样性的ISSR分析

本文采用ISSR标记对中国特有且仅在云南南部狭域分布的樟科濒危植物思茅木姜子(Litseaszemaois)现存8个居群的遗传多样性进行了研究。从96条引物中筛选出了10条,对103个个体进行了扩增,共扩增出77条条带,其中多态性条带为67条。分析结果表明:(1)思茅木姜子的遗传多样性水平很高。在物种水平上,多态位点百分率PPB=87.01%,平均每个位点的有效等位基因数Ne=1.4006,Nei’s基因多样度指数H=0.2466,Shannon多样性信息指数Hsp=0.3826;在居群水平上,PPB=37.99%,Ne=1.2500,H=0.1418,Shannon多样性信息指数Hpop=0.2088。(2)居群间的遗传分化较低。基于Nei’s遗传多样性分析得出的居群间遗传分化系数Gst=0.3700;Shannon’s居群分化系数((Hsp–Hpop)/Hsp)为0.45。AMOVA分析显示:思茅木姜子的遗传变异主要存在于居群内,占总变异的72.99%,居群间的遗传变异占27.01%,表明思茅木姜子属于异交种。(3)两两居群间的Nei’s遗传一致度(I)的范围为0.8233–0.9761。经Mantel检测,居群间的遗传距离和地理距离之间不存在显著的正相关关系(r=0.0925,P=0.6931)。我们推断人类活动的干扰和生境的片断化是导致思茅木姜子濒危现状的主要因素。考虑到目前其遗传多样性水平虽然很高,但各居群个体数量很少,因此应该对思茅木姜子各居群的所有个体实施及时的就地保护;而遗传变异大部分存在于居群内的个体间,所以在迁地保护时应在各居群内大量采样。     

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.     

Genetic relationships among cherry species with transferability of simple sequence repeat loci

Sweet and sour cherries are two economically important species in the world. The capability to distinguish among cherry genotypes in breeding, cultivation and germplasm collection is extremely important for scientific as well as economic reasons. In the present research, sixteen simple sequences repeat (SSR) loci were used to estimate the relationships among sweet, sour, duke and wild cherries. All of the SSR markers showed high transferability across the studied species that allowed us to study genetic diversity in them. Totally 96 alleles were generated with SSR loci, of which 93 were found polymorphic with 97.57 % polymorphism. Values of genetic similarity between genotypes varied from 0.16 to 0.97 which indicated high level of genetic diversity. On the basis of their genetic similarities, SSR analysis allowed to group the genotypes into three main clusters according to their species. These results have an important implication for cherry germplasm characterization, improvement, and conservation.     

Genetic variation in five Mediterranean populations of Juniperus phoenicea as revealed by inter-simple sequence repeat (ISSR) markers

BACKGROUND AND AIMS: The assessment of the genetic variability and the identification of isolated populations within a given species represent important information to plan conservation strategies on a genetic basis. In this work, the genetic variability in five natural populations of Juniperus phoenicea, three from Sardinia, one from Cyprus and the last one in the Maritime Alps was analysed by means of ISSRs, on the hypothesis that the latter could have been a refugial one during the last glaciation. METHODS: ISSRs were chosen because of their ability to detect variation without any prior sequence information. The use of three primers yielded 45 reproducible, polymorphic bands, which were utilized to estimate the basic parameters of genetic variability and diversity. KEY RESULTS: All of the populations analysed harboured an adequate amount of genetic variability, with H(S) = 0.1299. The proportion of genetic diversity between populations has been estimated by G(ST) = 0.12. The three Sardinian populations are separated, as tested by AMOVA, from the Cyprus and the continental ones. CONCLUSIONS: The results indicate that geographical isolation has represented a major barrier to gene flow in Juniperus phoenicea. This work represents a first step towards a full genetic characterization of a conifer from the Mediterranean, a world biodiversity hotspot confronted with climate change, and thus contributes towards the planning of genetics-informed conservation strategies.     

Genetic diversity and relationships of sweetpotato and its wild relatives in Ipomoea series Batatas (Convolvulaceae) as revealed by inter-simple sequence repeat (ISSR) and restriction analysis of chloroplast DNA

Genetic diversity and relationships of 40 accessions of Ipomoea, representing ten species of series Batatas, were examined using ISSR markers and restriction-site variation in four non-coding regions of chloroplast DNA. A total of 2071 ISSR fragments were generated with 15 primers in these accessions and, on average, 52 bands per accession were amplified. Most of the primers contained dinucleotide repeats. The ISSR fragments were highly polymorphic (62.2%) among the 40 accessions studied. Restriction analysis of chloroplast (cp) DNA revealed 47 informative restriction-site and length mutations. Phylogenetic analyses of ISSR and cpDNA datasets generally revealed similar relationships at the interspecific level, but the high polymorphism of ISSRs resulted in a better separation of intraspecific accessions. However, the combined ISSR and cpDNA dataset appeared to be appropriate in resolving both intra- and interspecific relationships. Of the species examined, I. trifida was found to be the most closely related to cultivated sweetpotato, the hexaploid I. batatas, while I. ramosissima and I. umbraticola were the most distantly related to I. batatas within the series. Ipomoea triloba, hitherto considered to be one of the ancestors of sweetpotato, was only distantly related to sweetpotato based on ISSR similarity index. Received: 4 January 1999 / Accepted: 27 September 1999     

Genetic diversity and variance of Stentor coeruleus (Ciliophora: Heterotrichea) inferred from inter-simple sequence repeat (ISSR) fingerprinting

We used inter-simple sequence repeat fingerprinting to analyze the genetic structure of 16 populations of Stentor coeruleus from three lakes and three ponds in China. Using 14 polymorphic primers, a total of 99 discernible DNA fragments were detected, among which 76 (76.77%) were polymorphic, indicating median genetic diversity in these populations. Further, both Nei's gene diversity (h) and Shannon's information index (I) between the different populations revealed a median genetic diversity. At the same time, gene flow was interpreted to be low. The main factors responsible for the median level of diversity and low gene flow within populations are probably due to a low frequency of sexual recombinations. Analysis of molecular variance showed that there was high genetic differentiation among the five water bodies. Both cluster analysis and a nonmetric multidimensional scaling analysis suggested that genotypes isolated from the same locations displayed a higher genetic similarity than those from different ones, separating populations into subgroups according to their geographical locations. However, there is a weak positive correlation between the genetic distance and geographical distance.     

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.     

Estimation of genetic diversity in some Iranian wild Prunus subgenus Cerasus accessions using inter-simple sequence repeat (ISSR) markers

As Iran is one of the main origins of Prunus germplasm. In this study, ISSR markers were used for genetic diversity evaluation of 39 accessions of subgenus Cerasus belonging to six species i.e. Prunus avium L., Prunus cerasus L., Prunus mahaleb L., Prunus incana Pall., Prunus microcarpa Boiss., and Prunus brachypetala Boiss.. With 12 ISSR primers, 151 polymorphic bands were detected with polymorphism ratio range of 81.8%–100%. The lowest similarity (0.04) was found between P. avium and P. microcarpa genotypes and the mean of similarity between all genotypes was 0.28. Cluster analysis separated improved cultivars from wild accessions. Improved cherry cultivars and rootstocks were placed closer to the P. avium than the other species. The principal coordinate analysis (PCoA) supported the cluster analysis results. The wild accessions were separated according to their species and collection sites. ISSR markers are useful techniques for genetic diversity evaluation in Prunus subgenus Cerasus.     

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.