Unweighed Pair Group Method Research Articles

Genetic analysis of floating Enteromorpha prolifera in the Yellow Sea with AFLP marker

Extremely large accumulation of green algae Enteromorpha prolifera floated along China’ coastal region of the Yellow Sea ever since the summer of 2008. Amplified Fragment Length Polymorphism (AFLP) analysis was applied to assess the genetic diversity and relationships among E. prolifera samples collected from 9 affected areas of the Yellow Sea. Two hundred reproducible fragments were generated with 8 AFLP primer combinations, of which 194 (97%) were polymorphic. The average Nei’s genetic diversity, the coefficiency of genetic differentiation (Gst), and the average gene flow estimated from Gst in the 9 populations were 0.4018, 0.6404 and 0.2807 respectively. Cluster analysis based on the unweighed pair group method with arithmetic averages (UPGMA) showed that the genetic relationships within one population or among different populations were all related to their collecting locations and sampling time. Large genetic differentiation was detected among the populations. The E. prolifera originated from different areas and were undergoing a course of mixing.

Genetic diversity in Cinnamomum zeylanicum Blume. (Lauraceae) using random amplified polymorphic DNA (RAPD) markers

Cinnamomum zeylanicum Blume. is an important taxa cultivated in coastal plains for its stem bark, leaf, fruits and roots as spice and for its medicinal properties.Cinnamomum is an indicator plant of semi evergreen forest. In this study, RAPD-PCR analysis involving 11 decamer random primers was used to assess the genetic variation within C. zeylanicum in Western Ghats of southern India. Some primers showed appreciable intra-species variation or molecular polymorphism at amplicon levels. Despite morphological similarity, a great deal of genetic polymorphism was observed among the accessions. In this study, unweighed pair group method with arithmetic averages (UPGMA) analysis showed up to 89% genetic variation among these accessions, which is further supported by principle co-ordinate analysis (PCA). Key words: Genetic diversity, Cinnamomum zeylanicum, RAPD markers, DNA polymorphism.

Investigation of simple sequence repeats (SSR) marker-assisted genetic diversity among upland Bt- and non Bt-cotton varieties

Genetically modified with insect resistance gene (Cry1AC) and hybrid Bt-cotton genotypes were developed to redress cotton leaf curl virus epidemic. In this study, genetic diversity of 6 Bt- and 14 nonBt upland cotton genotypes was detected using simple sequence repeats (SSR) or micro-satellite markers. Out of 31 primer pairs, only 7 (22.6%) yielded polymorphic amplicons of 80 to 340 bp. The average loci per primer were 3.16 and 78.6% of them were informative. The unweighed pair-group method with arithmetic averages (UPGMA) dendrogram placed Bt- and non-Bt cotton varieties into four major groups. With the exception of 4 accessions (being similar), genetic dissimilarity coefficient among all other genotypes ranged from 0.50 to 0.98 suggesting a wide genetic heterogeneity among the selected collection. The lowest similarity was found between BH-160 and CIM-482, CIM-496 and MNH554, BH-160 and CIM-473, SARMAST and BH-160 (non-Bt cultivars), respectively as were some Btcultivars. In contrast to previous reports, these results show that some Bt- and non-Bt-cotton cultivars form a genetically diverse population. They could be used in future breeding programmes to develop new elite cotton cultivars.

Confirmation of sunflower F 1 hybrids using SDS-PAGE analysis

Among bio-chemical techniques, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) is most widely used due to its validity and simplicity for describing genetic structures of group of plants. In the present study, SDS-PAGE was used to confirm sunflower hybrids. Four male sterile lines, four restorer lines and their sixteen F1 hybrids were analyzed. Based on results of electrophoretic band spectra, Jaccard’s similarity index (S) was calculated for all possible pairs. The similarity matrix, thus, generated was converted to a dissimilarity matrix and used to construct dendrogram, using unweighed pair-group method with arithmetic means. Euclidean distance of 1.0 was observed among 5 comparisons, whereas maximum distance (3.87) was observed for 1 comparison only. In most of the cases, female parents, along with their respective crosses, were found in one cluster, indicating that SDS-PAGE can be used reliably for the identification of hybrids in sunflower.

Diversity analysis of Aegilops species from Morocco using RAPD markers

The diversity of 51 representative populations of the 5 Aegilops species from Moroccan collection was analyzed using 22 RAPD primers. We investigated the associations among these 5 Aegilops species (A. geniculata Roth (UUMM), A. triuncialis L. (UUCC), A. ventricosa Tausch (DDNN), A. peregrina (Hackel) Maire et Weiller (UUMM) and A. neglecta Req. ex Bert. subsp. recta (Zhuk.) K. Hammer (UUMMNN)); some diploid species considered as their ancestors; accessions of some neighboring countries and also accessions of Triticums. A total of 650 polymorphic RAPD fragments were amplified. A dendrogram was constructed using the un-weighed pair group method arithmetic average (UPGMA) and Jaccard`s similarity coefficient. The UPGMA clustering showed a regrouping to the level species with high level of the structuration of the diversity at A. geniculata. We confirm as reported by other authors, the proximity of N genome to U genome and C genome to M genome and also the difference between the genomes M and N. Thus, the phylogeny between the species and the different genomes were retracted.

Description and analysis of genetic diversity among squash accessions

In this work, the part of the squash core collection, maintained in the Greek Gene Bank, was assessed using the morphological and molecular data. Sixteen incompletely classified accessions of the squash were characterized along with an evaluation of their resistance against two isolates of Fusarium oxysporum. A molecular analysis using Random Amplified Polymorphic DNA (RAPD) markers was also performed, revealing high level of polymorphism. To study the genetic diversity among the squash accessions, a clustering procedure using Unweighed Pair Group Method and Arithmetic Average (UPGMA) algorithm was also adopted. Two independent dendrograms, one for the morphophysiological and one for molecular data were obtained, classifying the accessions into two and three main clusters, respectively. Despite the different number of the clusters there were many similarities between these two dendrograms, and a third dendrogram resulting from their combination was also produced, based on Gower's distance and UPGMA clustering algorithm. In order to determine the optimal number of clusters, the upper tail approach was applied. The more reliable clustering of the accessions was accomplished using RAPD markers as well as the combination of the two different data sets, classifying the accessions into three significantly different groups. These groups corresponded to the three different cultivated species of C. maxima Duch., C. moschata Duch., and C. pepo L. The same results were also obtained using Principal Component Analysis.

AFLP analysis of genetic variation of Hyphantria cunea (Drury) populations in Beijing and a nearby site

Hyphantria cunea (Drury) is a severe invasive pest in Beijing. It is important to understand the reason for its successful invasion and outbreak. Accordingly, this study was tailored to assess molecular variation and genetic relationships among three H. cunea populations in Beijing and one population from Sanhe in Hebei Province as control (totally 100 individuals). A silver-labeled amplified fragment length polymorphisms (AFLP) DNA profiling method was used to detect the genetic diversity and relationship among the populations. Five EcoRI and MseI primer combinations produced a total of 171 (91.2% polymorphic) informative fragments. The average Nei’s genetic diversity in four H. cunea populations analyzed by Popgene3.2 software was 0.2287, and the level of genetic diversity ranked in the order BDR (reared population in Daxing, Beijing) > BDN (natural population in Daxing, Beijing) > HSN (natural population in Sanhe, Hebei Province) > BFN (natural population in Fengtai, Beijing). The value of genetic differentiation among populations was 0.3321, and the gene flow was 1.0057. The genetic distance between these populations ranged from 0.0735 to 0.3309, 0.0531 on average. The result of UPGMA (unweighed pair group method with arithmetic averages) clustering showed that populations BFN and HSN were closely related, while BFN and BDN had the largest genetic distance. These results indicate that H. cunea populations in Beijing might originate from several areas.

Genetic variation of Carthamus tinctorius L. and related species revealed by SRAP analysis

Genetic diversity of 23 populations of Carthamus tinctorius L. and two populations of Carthamus lanatus L. in China was investigated using Sequence-related Amplified Polymorphism (SRAP). All populations could be uniquely distinguished by 30 primer combinations with 483 bands and 274 polymorphic bands which generated 57% of polymorphic ratio. Unweighed pair-group method of with arithmetical averages (UPGMA) cluster analysis enabled construction of a dendrogram for estimating genetic distances among different populations. The extreme variation was observed when No. 4 cultivated and No. 13 wild population of C. lanatus were grouped at GS = 0.58, and separated from 23 populations of C. tinctorius at GS = 0.10. The result suggested that the cultivated and wild populations of C. lanatus had close relationship with each other and far relationship with C. tinctorius . Dendrogram also revealed a large genetic variation in 23 C. tinctorius populations; different primer combinations allowed them distinctly distinguished one from others with relatively low genetic similarity. Furthermore, five typical representative fragments in C. lanatus were obtained by four most informative primer combinations, which provided a possibility to distinguish C. lanatus from the C. tinctorius evidently.

Genetic relatedness of Trichoderma isolates antagonistic against Fusarium oxysporum f.sp. dianthi inflicting carnation wilt

Twenty-eight isolates of Trichoderma belonging to four different species were screened in vitro for their antagonistic ability against Fusarium oxysporum f.sp. dianthi causing carnation wilt. Three different levels of antagonism observed in dual plate assay were further confirmed by cell-free culture filtrate experiments. Isolates showing class I level of antagonism produced maximum lytic enzymes, chitinases and beta-1,3-glucanases. Genetic variability of 25 selected isolates was assessed by random amplified polymorphic DNA technique and the amplified products were correlated for their level of antagonism. Unweighed pair-group method with arithmetical averages cluster analysis revealed prominent inter-and intraspecific genetic variation among the isolates. Based on their genetic relationship, the isolates were mainly distributed into 3 major groups representing T. atroviride, T. pseudokoningii and T. harzianum, with 20-35% interspecific dissimilarity. However, the polymorphism shown by the isolates did not correlate to their level of antagonism.

Genetic relatedness among cultivated and wild mulberry (Moraceae: Morus) as revealed by inter-simple sequence repeat analysis in China

The genetic diversity of 27 mulberry (Morus spp.) genotypes mainly from China was investigated using inter-simple sequence repeat (ISSR) markers to assist in addressing breeding objectives and conserving existing genetic resources. Of the 22 primers screened, 15 produced highly reproducible ISSR bands. Using these 15 primers, 138 discernible DNA fragments were generated with 126 (91.3%) being polymorphic, indicating considerable genetic variation among the mulberry genotypes studied. Genetic similarity ranged from 0.6014 between Yu 2 and Yu 711 to 0.9493 between Cuizhisang and Dejiang 10. The phenetic dendrogram based on ISSR data generated by the unweighed pair group method with arithmetical averages (UPGMA) method grouped the 27 accessions into two major clusters: cluster I, cultivated mulberry species (M. multicaulis Perr., M. alba Linn., M. atropurpurea oxb., M. bombycis Kiodz., M. australis Poir., M. rotundiloba Kiodz., M. alba var. pendula Dipp., M. alba var. macrophylla Loud., and M. alba var. venose Delile.); and cluster II, wild mulberry species (M. cathayana Hemsl., M. laevigata Wall., M. wittiorum Hand-Mazz., M. nigra Linn., and M. mongolica Schneid.). Our molecular analyses agree with the existing morphological classification of Morus and clarify the genetic relationships among mulberry species. Key words: Morus L., genetic diversity, inter-simple sequence repeat, relatedness

Biodiversity of moderately halophilic bacteria in hypersaline habitats in Egypt

Screening bacteria from different saline environments in Alexandria. Egypt, lead to the isolation of 76 Gram-negative and 14 Gram-positive moderately halophilic bacteria. The isolates were characterized taxonomically for a total of 155 features. These results were analyzed by numerical techniques using simple matching coefficient (SSM) and the clustering was achieved by the unweighed pair-group method of association (UPGMA). At 75% similarity level the Gram-negative bacteria were clustered in 7 phena in addition to one single isolate, whereas 4 phena represented the Gram-positive. Based on phenotypic characteristics, it is suggested that the Gram-negative bacteria belong to the genera Pseudoalteromonas, Flavobacterium, Chromohalobacter, Halomonas and Salegentibacter, in addition to the non-identified single isolate. The Gram-positive bacteria are proposed to belong to the genera Halobacillus, Salinicoccus, Staphylococcus and Tetragenococcus. This study provides the first publication on the biodiversity of moderately halophilic bacteria in saline environments in Alexandria, Egypt.

Genetic variation in zinc-tolerant populations of Glyceria fluitans

Amplified fragment length polymorphism (AFLP) was used to conduct a study of the genetic diversity of zinc-tolerant populations of Glyceria fluitans from 10 sites from across Europe. Six different primer combinations were used on five to nine plants from each of the 10 sites to generate a total of 796 bands, of which 670 were polymorphic. These data were then used to calculate a dendrogram by agglomerative clustering using the unweighed pair group method with average linkage (UPGMA). The dendrogram contained two distinct clusters, with little overlap between populations. Genetic diversity between populations of G. fluitans did not always correlate with geographical distances, for example, plants from the Navan population from Ireland were more genetically similar to populations from Poland than other populations from Ireland. In other instances, geographical origin was significant, for example, all Polish populations were genetically similar to each other. Populations from two English sites only 160 km apart, showed such a high degree of genetic diversity that they were placed in different clusters in the dendrogram. They were more closely related to Irish and Polish populations than to each other. Plants from different zinc-contaminated sites were found not to cluster together. The conclusion was that the cluster groupings were not related to exposure to zinc at the sites of origin, and that the drive to generate distinct metal-tolerant populations may not occur in this species due to the existence of a constitutive tolerance to metals.

MOLECULAR IDENTIFICATION OF EGG PARASITOID, TELENOMUS SPP. (HYMENOPTERA: SCELIONIDAE) FROM SEVERAL LOCATIONS IN JAVA USING RAPD-PCR

Random amplified polymorphic DNA (RAPD) amplified by the polymerase chain reaction (PCR) was used to determine the differences of four Telenomus species and five populations of T. rowani from several locations in Java. Amplification of genomic DNA by using primer P2 (Amersham Pharmacia Biotech) indicated that each Telenomus species had a unique set of RAPD bands. Two bands which characterized the genus are estimated to be 300 and 430 bp. Each species had three specific bright bands except T. dignoides which only had two specific bright bands. However, no bands are unique to any of the five populations of T. rowani and all of the bands are less than 500 base-pair. Cluster analysis using UPGMA (Unweighed Pair Group Method With Arithmatic Mean) showed that the four Telenomus species consist of two groups, T. rowani and T. remus in one cluster and T. dignus with T. dignoides belonging to another cluster. Key words : PCR-RAPD / clustering / Telenomus spp.

New Approaches to Analysis of Variations in Brain Ganglioside Composition Related to Idioadaptations and Aromorphosis of Vertebrates

For objective interpretation of results of comparative studies of lipids, performed for many years at Institute of Evolutionary Physiology and Biochemistry, it is essentially important to use modern methods of analysis, first of all, polythetic methods of cluster analysis. Using the unweighed pair group method of the mean values, it has been shown that the dendrograms constructed on the basis of analysis of characters of molecular organization of the carbohydrate part of the ganglioside molecule correspond on the whole to the tree of classical taxonomy of vertebrates. Carbohydrate components of glycoconjugates, including gangliosides, play an important role in intercellular interactions, in processes of cell adhesion, recognition, and apoptosis. It may be suggested that changes of the carbohydrate component of the ganglioside molecule in the course of evolution of vertebrates contribute to achievement of a qualitatively new level of biochemical organization of organs and tissues, providing a possibility of aromorphic modifications of organization of animals, which are essential for the appearance of new classes. On the contrary, analysis of the data on molecular organization of a fatty acid component of vertebrate brain gangliosides by the same method could not reveal any relation of the species position on the dendrogram to its taxonomic belonging. At the same time, it was found out that the content of various fatty acids (FA) of brain gangliosides of ectothermic vertebrates correlated statistically significantly with the environmental temperature, positively in the case of saturated acids and negatively in the case of unsaturated ones. This agrees with the previously obtained data on the significance of differences in FA composition of warm-water and cold-water stenothermic species of teleosts. Changes of the FA composition of brain gangliosides seem to contribute to processes of idioadaptations to environmental temperature in various groups of ectothermic vertebrates, these adaptations aimed at maintaining properties of cell membranes, which are optimal for their vital activity.

Genetic diversity in Fusarium oxysporum f. sp. melonis*

Fusarium oxysporum f. sp. melonis is an important vascular wilt pathogen of melon. Races 1, 2 and 1–2 of this fungus have been identified in Portugal by pathogenicity tests with appropriate hosts. The aim of this research was to examine the relationships between different races of F. o. melonis of Portuguese and French origin through analysis of random amplified polymorphic DNAs (RAPDs). DNA fingerprint profiles were developed for all the accessions. Each isolate showed 5–10 DNA bands with each of the 16 primers employed. A total of 126 bands was obtained. The size of amplified DNA fragments generated with these primers ranged from 0.5 to 3.2 kb. A phenogram based on the Jaccard coefficient of similarity was computed by the unweighed pair group method using arithmetic averages (UPGMA). It was found that Portuguese race 2 is very similar to French race 1, while French race 2 is the most dissimilar being clearly separated from all other races. The genetic diversity of these isolates is also being studied for vegetative compatibility by using the nit mutant system.

Genetic variation in the African rodent subfamily Otomyinae (Muridae): immuno-electrotransfer of liver proteins of someOtomys irroratus(Brants 1827) populations

Liver homogenates from 12 populations of the vlei rat Otomys irroratus (Brants 1827) were subjected to Sodium Dodecyl Sulfate Polyacrylamide electrophoresis on 7–17% gradient gels followed by Western blotting. Detection of antigenic proteins was carried out with an antiserum against liver proteins of one of these populations. Blots thus obtained were digitized into computer images and the gray level values of all electromorphs were recorded. A matrix of these values was then constructed and subjected to statistical analyses (discriminant function and cluster analysis by the Unweighed Pair Group Method with Averages). Boot-strapping parsimony analysis was performed on a multistate character matrix derived from the gray level data. Although immunoblotting seemed to be sufficiently sensitive to detect extensive individual variation, there was only partial agreement between the dendrograms thus generated and the trees previously obtained from chromosome studies (CONTRAFATTO et al. 1992b). Evolutionary implica...

A COMPARISON OF TEMPERATURE-INDUCED POLYPHENISM IN AFRICAN BICYCLUS BUTTERFLIES FROM A SEASONAL SAVANNAH-RAINFOREST ECOTONE.

Temperature-induced variation and norms of reaction have been analyzed for wing pattern elements of six species belonging to the African butterfly genus Bicyclus (Lepidoptera, Nymphalidae, Satyrinae). Five of these species are sympatric in Malawi and exhibit seasonal polyphenism in the savannah-rainforest ecotone. The sixth species originated from Cameroonian equatorial rainforest. The organisms were laboratory reared under four different temperature conditions ranging from 17-28°C. The variation in response to temperature is described by principal component analysis (PCA). Discrimination on the basis of plastic wing pattern characters was performed by discriminant function analysis (DFA) and unweighed pair-group method algorithm (UPGMA) clustering. A phylogenetic reconstruction based on adaptive plastic wing characters was compared with a cladogram built on "nonadaptive" characters. Results demonstrate that: (1) Phenotypic plasticity of wing pattern characters in response to temperature in laboratory-reared organisms is reminiscent of variation induced by seasonal change in the field. (2) Different wing pattern characters are under different control: "exposed" characters of butterflies at rest position are highly sensitive to temperature variation, whereas "hidden" characters, only visible during active behavior, are dominated by species differences. In general the sensitivity of the former can be attributed to their proposed function in deflecting predators. (3) The sexes differ especially in the size of those eyespots that are displayed during active behavior. (4) Species from seasonal and aseasonal environments react in a broadly similar manner to temperature variation. However, savannah species and species of aseasonal rainforest exhibit relatively shallow reaction norms, whereas reaction norms are steeper in species from the savannah-rainforest ecotone. Such a strong response was also apparent in so-called correlation networks between principal components for these species. (5) Phylogenetic distances are to some extent reflected in ordination in both PCA-space and DFA-space: closely related species of the safitza group remain close in both ordinations. The more distantly related species differ in ordination from a pattern as suggested by a phylogenetic reconstruction. It is argued that the wing pattern variation of these species reflects both adaptive processes and historical relationships.

Hepatitis C virus types 7, 8 and 9 should be classified as type 6 subtypes

Tokita et al. reported the identification of hepatitis C virus type 7, 8 and 9 in Southeast Asia, based on the unweighed pair-group method with the artihmetic mean, a method that may not be appropriate when the nucleotide substitution rate is not constant over time. To determine more accurately the relationship of these proposed types 7, 8, and 9 with other types, a more appropriate approach, the neighbor-joining method, was applied to re-evaluate the phylogenetic relationship. A total of 660 HCV sequences, including the reported sequences, were collected from DNA databases. For every region, the nucleotide substitution rate was estimated by the 6-parameter method, and phylogenetic trees were constructed using the neighbor-joining method. The results of this analysis indicate that the newly proposed HCV types 7, 8 and 9 should be classified as type 6 subtypes. HCV type 6 appears to be diversified

Identities and Relationships among Chinese Vegetable Brassicas as Determined by Random Amplified Polymorphic DNA Markers

Fifty-two germplasm accessions of Chinese vegetable brassicas were analyzed using 112 random amplified polymorphic DNA (RAPD) markers. The array of material examined spanned a wide range of morphological, geographic, and genetic diversity, and included 30 accessions of Brassica rapa L. (Chinese cabbage, pakchoi, turnip, and broccoletto), 18 accessions of B. juncea (L.) Czern. (leaf, stem, and root mustards), and four accessions of B. oleracea L. ssp. alboglabra (Chinese kale). The RAPD markers unambiguously identified all 52 accessions. Nei-Li similarities were computed and used in unweighed pair group method using arithmetic means (UPGMA) cluster analyses. Accessions and subspecies were clustered into groups corresponding to the three species, but some accessions of some subspecies were most closely related to accessions belonging to other subspecies. Values for Nei-Li similarities suggest that Chinese cabbage is more likely to have been produced by hybridization of turnip and pakchoi than as a selection from either turnip or pakchoi alone. RAPD markers are a fast, efficient method for diversity assessment in Chinese vegetable brassicas that complements techniques currently in use in genetic resources collections.

Variation in Spore Polypeptides from Four Species of Vairimorpha

Spore polypeptides from four species of Vairimorpha were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis for identification and classification. The spore profile for each Vairimorpha species is unique and reproducible. The unweighed pair-group method with arithmethic means is used to construct a phenogram to summarize the relationship of the Vairimorphe species.