Inter-simple Sequence Repeat Polymerase Chain Reaction Research Articles

Genetic stability analysis of diapause‐induced multivoltine silkworm Bombyx mori germplasm using inter simple sequence repeat markers

AbstractConservation of silkworm [Bombyx mori L. (Lepidoptera: Bombycidae)] germplasm aids in prevention of genetic erosion and race extinction. Multivoltine silkworm germplasm maintenance involves rearing five generations per year and this requires expenditure, manpower, and infrastructure, in addition to frequent exposure of the silkworms to diseases, pests, and predators. To minimize these problems, an alternative protocol was developed by way of induction of egg diapause in the multivoltine silkworms through rearing of the fourth and fifth instars at low temperature (18–20 °C) under regulated photoperiod (L6:D18). This method allowed induction of egg diapause in the selected races at levels ranging from 26 to 94% and the diapause‐induced eggs were maintained under cold preservation conditions. The stability of genotypic characters was confirmed in the diapause‐induced silkworm batches after the seventh generation, by comparison with control batches of silkworm using inter simple sequence repeat‐polymerase chain reaction (ISSR‐PCR). Analysis of Nei’s genetic distance for the different races indicated no significant variation between control and diapause‐induced races. This reveals that all the selected races maintained genetic stability even after the seventh generation at the phenotypic and molecular level. Hence, it can be concluded that induction of egg diapause is an appropriate alternative method to preserve multivoltine races for longer periods of time.

Molecular characterization of Leishamania isolates from China by inter-simple sequence repeat polymerase chain reaction

Leishmania has distinct epidemiological and biological characteristics and causes a variety of clinical symptoms. To understand the genetic diversity and the phylogenetic relationships among Leishmania isolates from China, 29 Leishmania isolates from different geographic origins, vectors, and hosts were analyzed using 21 inter-simple sequence repeat polymerase chain reaction (ISSR-PCR) primers. A total of 864 polymorphic bands were obtained. According to the results of the neighbor-joining phylogenetic tree and principal component analysis, the 29 isolates studied clustered into six groups. Isolates of Leishmania donovani complex from China share the highest similarity with the reference strain of L. donovani (DD8). This study helps to elucidate the genetic relationship among Leishmania isolates from China and similarities between Chinese isolates and World Health Organization reference strains. Furthermore, ISSR-PCR could also be a quick, simple, and reliable method for Leishmania species identification.

Molecular Evidence Suggests That Populations of the Asian Citrus Psyllid Parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) from Texas, Florida, and Mexico Represent a Single Species

Abstract We genetically characterized Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) populations from Texas, Florida, and Mexico and the sister taxon Tamarixia triozae (Burks) by two molecular methods. T. radiata is an ectoparasitoid of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), nymphs. The populations were submitted to intersimple sequence repeat-polymerase chain reaction DNA fingerprinting with two primers. No fixed banding pattern differences were uncovered among the populations of T. radiata with either primer, whereas different patterns were observed in T. triozae, suggesting that there is no genetic differentiation among the populations. Support for these results was obtained by sequence analyses of the internal transcribed spacer region one and the mitochondrial cytochrome oxidase subunit one (COI) gene. In both genes, the intrapopulational variation range (percentage divergence, %D) fell within the interpopulational variation range. The %D at the COI gene between T. radiata and T. triozae was 9.0–10.3%. However, haplotype structure was uncovered among the populations. No haplotypes were shared between Florida and Texas/Mexico, whereas sharing was observed between Texas and Mexico. Two population-specific nucleotides were identified that allowed the discrimination of the Florida and Texas/ Mexico individuals. A neighbor-joining and a parsimonious tree clustered the populations into two distinct clades. The Florida population clustered into one clade, whereas the Texas/Mexico populations clustered into another clade. The COI phylogeographic analysis suggests that the population of T. radiata in Texas did not come from Florida. The data also suggest that the population in Texas came from Mexico, or vice versa, because the Mexico population showed less haplotype diversity.

Trichinellosis survey in the wild boar from the Toledo mountains in south-western Spain (2007–2008): molecular characterization of Trichinella isolates by ISSR-PCR

In Spain, trichinellosis represents a public health problem, with an average of five outbreaks per year, wild boar meat being the main source of infection. A trichinellosis survey (2007-2008 hunting campaign) was carried out on wild boars in the Toledo Mountains (south-western Spain, EU) in the context of a surveillance programme on wildlife diseases. A total of 2216 wild boars from different locations of the region were examined. The examination was carried out by veterinarians in the local abattoir (Matadero Municipal de Toledo). The positive samples were sent to the Department of Parasitology (Facultad de Farmacia, UCM) for experimental isolation and specific identification by inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR). Using this technique we identified 17 isolates as Trichinella spiralis with an electrophoretic profile indistinguishable from the T. spiralis reference strain (ISS48). We confirmed that ISSR-PCR is a robust technique for the molecular identification of Trichinella isolates. According to our results, the prevalence of T. spiralis in wild boars from the Toledo Mountains (>800 m above sea level) during the hunting season was approximately 0.77%. The prevalence of T. spiralis (100% of our observations) is a good example of the persistence of this species in sylvatic conditions (coming from the domestic cycle), if a good wild host is abundant. Our observations confirm the major prevalence of T. spiralis over T. britovi in this region, as well as the risk to human health represented by the consumption of uninspected wild boar meat.

Determination of the population genetic structure of the invasive weed Ageratina adenophora using ISSR-PCR markers

A genetic analysis of nuclear DNA was performed by inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) technique on 32 populations of Ageratina adenophora, an invasive triploid weed in China. Among the 100 ISSR markers detected, 12 showed genetic variation both within and among the populations. Among the 446 amplified bands, 93.5% were found polymorphic. Most individuals (99%) displayed a unique ISSR fingerprint pattern, which yielded a high level of polymorphism (Po = 93.5%) and genetic diversity (Nei’s HT = 0.2354). The estimates of population variation, based on ISSR-PCR, were high, as measured by the analysis of molecular variance (AMOVA, FST = 0.3140), the Wright’s F-statistics (GST = 0.3453), and the Shannon’s information index (Hsp = 0.3716). AMOVA revealed 68.6% genetic variation within the populations and 91.2% within the provinces. The Mantel test showed that genetic distance was significantly correlated with geographic distance.

Identification of Spanish Trichinella isolates by ISSR-PCR: Intra-specific variability of Trichinella britovi

In the present work, we investigated genetic variability of the Spanish Trichinella isolates by ISSR-PCR (inter-simple sequence repeat polymerase chain reaction), a technique that is being successfully used to study diversity among related populations. We recovered a total of 43 isolates from different host and geographic localization and identified them by molecular techniques (RAPD and multiplex-PCR) and by Western blot with monoclonal antibodies US5 and US9. Nineteen (44.2%) out of 43 were identified as Trichinella spiralis and 24 (55.8%) as Trichinella britovi. When these samples were analysed by the ISSR technique, all the T. spiralis isolates presented a pattern similar to the T. spiralis ISS116. By contrast, the ISSR-PCR analysis of the isolates identified as T. britovi, showed two different banding profiles compatible with the European T. britovi isolate pattern (ISS2), and the autochthonous Spanish T. britovi isolate (ISS11). Three of these 43 isolates were involved in human outbreaks; the three were identified as T. britovi and showed a pattern similar to the European isolate ISS2. As conclusion, we highlight that an intra-species variability within the Spanish T. britovi isolates analysed was observed, with a predominant group similar to T. britovi ISS2, while T. spiralis group isolates were more homogeneous. No correlations were found between the different ISSR-PCR T. britovi types and the host/geographical origin of the isolates.

Microsatellite markers for the red band needle blight pathogen, Dothistroma septosporum

Twelve microsatellite markers were developed for population analyses of the fungal pathogen, Dothistroma septosporum. Intersimple sequence repeat polymerase chain reaction (ISSR-PCR) and an enrichment protocol (fast isolation by amplified fragment length polymorphism of sequences containing repeats [FIASCO]) were both used to identify 28 unique microsatellite regions in the genome. From 22 primer pairs designed, 12 were polymorphic. These markers, screened on two populations representing 42 isolates, produced 40 alleles across all loci with an allelic diversity of 0.09-0.76 per locus. Cross-species amplification showed variable success with Dothistroma rhabdoclinis and Mycosphaerella dearnessi and some sequence variation within isolates of Dothistroma pini. These markers will be used to further study the population structure and diversity of D. septosporum.

Molecular characterization ofGonatocerus tuberculifemur(Ogloblin) (Hymenoptera: Mymaridae), a prospectiveHomalodisca vitripennis(Germar) (Hemiptera: Cicadellidae) biological control candidate agent from South America: divergent clades

We genetically characterized the prospective South American egg parasitoid candidate, Gonatocerus tuberculifemur, of the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, for a neoclassical biological control program in California. Two molecular methods, inter-simple sequence repeat-polymerase chain reaction DNA fingerprinting and a phylogeographic approach inferred from the mitochondrial cytochrome oxidase subunit I gene (COI), were utilized. Five geographic populations from South America were analyzed; in addition, a phylogenetic analysis was performed with several named and one unnamed Gonatocerus species using the COI gene. DNA fingerprinting demonstrated a fixed geographic banding pattern difference in the population from San Rafael, Mendoza Province, Argentina. The COI analysis uncovered haplotype or geographic structure in G. tuberculifemur. A neighbour-joining distance (NJ) and a single most parsimonious tree (MP) clustered the populations into two well-supported distinct clades with strong bootstrap values (97-99% and 92-99%, respectively) with populations from San Rafael clustering into clade 2 and the rest of the populations clustering into clade 1. No haplotype sharing was observed between individuals from the two clades. Phylogenetic analyses performed by NJ and MP methods with 15 Gonatocerus species confirmed species boundaries and again uncovered two distinct clades in G. tuberculifemur with strong bootstrap support (95-100% and 68-100%, respectively). However, the NJ tree supported the morphologically defined relationships better than the MP tree. The molecular evidence in the present study is suggestive of a species level divergence. Because G. tuberculifemur is under consideration as a potential biological control agent for GWSS in California, understanding cryptic variation in this species is critical.

Evaluation of Molecular Markers for Discriminating <I>Gonatocerus morrilli</I> (Hymenoptera: Mymaridae): A Biological Control Agent for <I>Homalodisca vitripennis</I>

Abstract We tested the utility of molecular markers for distinguishing between two closely related species, Gonatocerus morrilli (Howard) and Gonatocerus walkerjonesi S. Triapitsyn (Hymenoptera: Mymaridae), to evaluate whether postrelease G. morrilli specimens could be discriminated in the field. Initially, postrelease specimens from California collected in 2002 and 2003 were analyzed. Amplification size of the internal transcribed spacer (ITS) region 2 demonstrated that all of the specimens were of the G. walkerjonesi ITS2 genotype. Inter-simple sequence repeat-polymerase chain reaction DNA fingerprinting of specimens from the original G. morrilli“release” colony demonstrated that the DNA banding pattern was superimposable to that of G. walkerjonesi, confirming that the G. morrilli colony was contaminated. A new G. morrilli colony was initiated in spring 2005, and we continued to survey random postrelease specimens from the 2004–2006 collections. As expected, from 2004 and most of 2005, only the G. walke...

Comparative Genomic Instabilities of Thyroid and Colon Cancers

To assess the forms and extent of genomic instability in thyroid cancers and colorectal neoplasms and to determine if such measurements could explain the generally excellent prognosis of thyroid malignant neoplasms compared with colon carcinoma. Tumor genome analyses. Genomic instability was measured by the following 4 methods, listed in ascending order based on the size of events detected: inter-simple sequence repeat polymerase chain reaction (ISSR-PCR), fractional allelic loss (FAL) analysis, array-based comparative genomic hybridization (aCGH), and spectral karyotyping (SKY). The genomic instability index of 32 thyroid carcinomas, 59 colon carcinomas, and 11 colon polyps was determined by ISSR-PCR; no difference was seen among the 3 groups by this method. Fractional allelic loss rates were comparable in thyroid cancers and colon polyps and lower than FAL rates in colorectal cancers. Indolent papillary thyroid carcinomas were essentially diploid with no large-scale alterations in chromosome number or structure when evaluated by aCGH or SKY. In anaplastic thyroid cancers, aCGH revealed abundant chromosome alterations. Colorectal carcinomas showed extensive copy number changes and chromosomal rearrangements when analyzed by aCGH and SKY. Genomic alterations in papillary thyroid carcinoma, such as in benign colon polyps, are principally smaller events detected by ISSR-PCR. With the more aggressive tumor types (ie, anaplastic thyroid and colorectal carcinomas), larger events detected by FAL analysis, aCGH, and SKY were revealed. We hypothesize that mutations caused by smaller genomic alterations enable thyroid cells to achieve a minimal malignant phenotype. Mutations for aggressive biological behavior appear with larger genomic events.

MOLECULAR CHARACTERIZATION OF TRICHINELLA GENOTYPES BY INTER-SIMPLE SEQUENCE REPEAT POLYMERASE CHAIN REACTION (ISSR-PCR)

A bulk analysis of inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) provides a quick, reliable, and highly informative system for DNA banding patterns that permit species identification. The present study evaluates the applicability of this system to Trichinella species identification. After a single amplification carried out on a single larva with the primer 816([CA]nRY) under high stringency conditions, which provide high reproducibility, we were able to identify by consistent banding patterns 5 sibling species: Trichinella spiralis (ISS48), 2 Trichinella britovi isolates (ISS11 and ISS86), Trichinella murrelli (ISS35), Trichinella nativa (ISS71), Trichinella nelsoni (ISS29); 3 additional Trichinella genotypes: T8 (ISS149), T9 (ISS408 and ISS409), and T6 (ISS34); and the nonencapsulated species Trichinella pseudospiralis (ISS13). Moreover, 33 new Trichinella isolates from 2 zoogeographical regions were unequivocally identified. All Trichinella isolates have shown an identical pattern with those produced by the reference strain. According to these data, we have demonstrated that ISSR-PCR is a robust technique that emerges as a useful new application for the molecular identification of Trichinella isolates in epidemiological studies.

Molecular Prognosticators and Genomic Instability in Papillary Thyroid Cancer

Tumor progression has been attributed to the accumulation of DNA damage concurrent with selection of advantageous mutations; this DNA damage may result from failure to maintain genomic integrity or from susceptibility to carcinogens. Glutathione S-transferases (GSTs), enzymes that metabolize many carcinogens, may play a role in preserving genome integrity. The objectives of this study are to assess the relationship of GST genotypes with prognosis, clinicopathologic parameters, and genomic instability in papillary thyroid cancer. Prospective analysis. GSTM1 and GSTT1 genotypes of 35 matched normal and papillary thyroid cancer specimens were determined by polymerase chain reaction (PCR) using primers specific for the coding sequences of each gene. Genomic instability was measured by intersimple sequence repeat PCR for each tumor/normal pair and compared with the GAMES prognostic scoring system and clinicopathologic parameters including age, extrathyroidal extension, tumor grade, size, stage metastasis, sex, and smoking history. GSTM1 and GSTT1 null genotypes were found in the normal tissues of 46% and 45%, respectively. No gene losses were detected in the tumor specimens. A significant association between the GSTM1 null genotype and increased risk of recurrence and death was observed. Elevated GII correlated with smoking and tumor stage but not with GST genotype. The association of GSTM1 null genotype with intermediate and high risk GAMES categories suggests that GSTM1 provides some protection against disease progression. However, this protection does not confer resistance to disease onset. GST genotyping may be a useful adjunct prognosticator with GAMES.

Genetic differentiation among geographic populations of Gonatocerus ashmeadi, the predominant egg parasitoid of the glassy-winged sharpshooter, Homalodisca coagulata

The aim of genetically comparing different populations of the same species of natural enemies is to identify the strain that is most adapted to the environment where it will be released. In the present study, Inter-Simple Sequence Repeat-Polymerase Chain Reaction (ISSR–PCR) was utilized to estimate the population genetic structure of Gonatocerus ashmeadi (Girault) (Hymenoptera: Mymaridae), the predominant egg parasitoid of Homalodisca coagulata (Say) (Homoptera:Cicadellidae), the glassy-winged sharpshooter. Six populations from throughout the U.S. and a population from Argentina identified as near G. ashmeadi were analyzed. Four populations (California; San Antonio, Texas; Weslaco, Texas [WTX-2]; and Florida) were field collected and two (Louisiana and Weslaco, Texas [WTX-1]) were reared. Three ISSR–PCR reactions were pooled to generate 41 polymorphic markers among the six U.S. populations. Nei's expected heterozygosity values (h), including the reared population from Louisiana, were high (9.01–14.3%) for all populations, except for a reared population from WTX-1 (2.9%). The total genetic diversity value (Ht) for the field populations was high (23%). Interestingly, the Florida population that was collected from one egg mass (siblings) generated the greatest number of polymorphic markers (20) and was observed with the highest gene diversity value (14.3%). All populations, except WTX-2 generated population-specific markers. Comparison of genetic differentiation estimates, which evaluate the degree of genetic subdivision, demonstrated good agreement between GST and θ values, 0.38 and 0.50, respectively for field populations, and 0.44 and 0.50, respectively for all populations. Genetic divergence (D) indicated that the WTX-1 population was the most differentiated. Average D results from the Argentina population support the taxonomic data that it is a different species. The present results estimate the population genetic structure of G. ashmeadi, demonstrating genetic divergence and restricted gene flow (Nm = 0.83) among populations. These results are of interest to the Pierce's disease/glassy-winged sharpshooter biological control program because the key to successful biological control may not be in another species, but instead in different geographic races or biotypes.

Genetic differentiation among geographic populations of Gonatocerus ashmeadi, the predominant egg parasitoid of the glassy-winged sharpshooter, Homalodisca coagulata.

The aim of genetically comparing different populations of the same species of natural enemies is to identify the strain that is most adapted to the environment where it will be released. In the present study, Inter-Simple Sequence Repeat-Polymerase Chain Reaction (ISSR-PCR) was utilized to estimate the population genetic structure of Gonatocerus ashmeadi (Girault) (Hymenoptera: Mymaridae), the predominant egg parasitoid of Homalodisca coagulata (Say) (Homoptera:Cicadellidae), the glassy-winged sharpshooter. Six populations from throughout the U.S. and a population from Argentina identified as near G. ashmeadi were analyzed. Four populations (California; San Antonio, Texas; Weslaco, Texas [WTX-2]; and Florida) were field collected and two (Louisiana and Weslaco, Texas [WTX-1]) were reared. Three ISSR-PCR reactions were pooled to generate 41 polymorphic markers among the six U.S. populations. Nei's expected heterozygosity values (h), including the reared population from Louisiana, were high (9.01-14.3%) for all populations, except for a reared population from WTX-1 (2.9%). The total genetic diversity value (Ht) for the field populations was high (23%). Interestingly, the Florida population that was collected from one egg mass (siblings) generated the greatest number of polymorphic markers (20) and was observed with the highest gene diversity value (14.3%). All populations, except WTX-2 generated population-specific markers. Comparison of genetic differentiation estimates, which evaluate the degree of genetic subdivision, demonstrated good agreement between G(ST) and theta values, 0.38 and 0.50, respectively for field populations, and 0.44 and 0.50, respectively for all populations. Genetic divergence (D) indicated that the WTX-1 population was the most differentiated. Average D results from the Argentina population support the taxonomic data that it is a different species. The present results estimate the population genetic structure of G. ashmeadi, demonstrating genetic divergence and restricted gene flow (Nm = 0.83) among populations. These results are of interest to the Pierce's disease/glassy-winged sharpshooter biological control program because the key to successful biological control may not be in another species, but instead in different geographic races or biotypes.

Molecular distinction between populations of Gonatocerus morrilli, egg parasitoids of the glassy-winged sharpshooter from Texas and California: Do cryptic species exist?

Two molecular methods were utilized to distinguish geographic populations of Gonatocerus morrilli (Howard) from Texas and California and to test the possibility that this species could exist as a species-complex. Inter-Simple Sequence Repeat–Polymerase Chain Reactions (ISSR–PCR) were performed with a 5′-anchored ISSR primer. Twenty-five markers were generated with four populations (40 individuals) of G. morrilli. Twenty-three were polymorphic and the percentage of polymorphic loci was 92%. Most markers could be considered diagnostic since there was no band sharing between the Texas and California populations. Such differences typically are not found unless the populations are reproductively isolated. Exact tests for population differentiation indicated significant differences in marker frequencies among the populations. Comparison of other genetic differentiation estimates, which evaluate the degree of genetic subdivision, demonstrated excellent agreement between GST and θ values, 0.92 and 0.94, respectively, indicating that about 92 to 94% of the variance was distributed among populations. The average genetic divergence (D), as measured by genetic distance, was extremely high (Nei = 0.82 and Reynolds = 2.79). A dendrogram based on Nei's genetic distance separated the Texas and California populations into two clusters, respectively. Amplification of the Internal Transcribed Spacer-1 (ITS-1) region showed no size differences, whereas the ITS-2 DNA fragment varied in size between the two geographic populations. The ITS-2 fragment sizes were about 865 and 1099 base pairs for the California and Texas populations, respectively. The present study using the two molecular methods provides novel data critical to the glassy-winged sharpshooter/Pierce's disease biological control program in California.ISSR–PCR Inter-Simple Sequence Repeat–Polymerase Chain Reaction ITS Internal Transcribed Spacer

Genetic Analyses of Casuarinas Using ISSR and FISSR Markers

Inter simple sequence repeat polymerase chain reaction (ISSR-PCR) was used for the genetic analysis of the six species of Allocasuarina, five species of Casuarina and 12 superior performing selections of C. equisetifolia L. We also fingerprinted C. equisetifolia L. selections using Fluorescent-ISSR-PCR (FISSR-PCR), an improvised ISSR-PCR assay. The ISSR analysis provided information on the frequency of various simple sequence repeats in the casuarina genome. The di-nucleotide repeats were more common, among which (CA)n and its complementary nucleotide (GT),, repeat motifs amplified relatively higher number of bands with an average of 6.0+/-3.5 and 6.3+/-1.8 respectively. Eleven species of casuarinas were amplified with 10 primers anchored either at 5' or 3' end. A total of 253 PCR products were obtained and all were polymorphic, out of which 48 were specific to Allocasuarina and 36 were specific to Casuarina genus. Genetic similarity among the species was 0.251. A UPGMA dendrogram grouped all the Casuarina species together. The 12 superior performing selections of C. equisetifolia L. produced 57 polymorphic ISSR markers while the FISSR assay revealed 105 polymorphic markers. The primer CRR(ATT)4 distinguished all the selections. DNA profiles obtained with ISSR and FISSR assays would serve as a reference library for the establishment of clonal identity in casuarinas.

Detection of DNA Polymorphisms in <I>Homalodisca coagulata</I> (Homoptera: Cicadellidae) by Polymerase Chain Reaction-Based DNA Fingerprinting Methods

DNA polymorphisms were detected in Homalodisca coagulata (Say) (Homoptera: Cicadellidae) with the following DNA fingerprinting methods: inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) and primer pair-ISSR-PCR (pp-ISSR-PCR), randomly amplified microsatellite polymophisms (RAMP), selective amplification of microsatellite polymorphic loci (SAMPL), and primer pair-random amplification of polymorphic DNA-polymerase chain reaction (pp-RAPD-PCR). But first, a small-scale DNA fingerprinting screening procedure was initiated with these methods with a few individual insects to estimate the most sensitive and efficient method(s). In total, 205 polymorphic markers were generated with the four methods. The efficiency ratio estimated the following order for each method: 1) pp-ISSR-PCR and ISSR-PCR, 2) RAMP, 3) pp-RAPD-PCR, and 4) SAMPL. The screening efficiency ratio estimated that pp-ISSR-PCR and ISSR-PCR were the most efficient methods. DNA polymorphisms were detected in a natural population of 10-30 insects. The number of polymorphic loci ranged from five (pp-RAPD-PCR reaction 6) to 32 (ISSR-PCR primer 13), and the percentage of polymorphic loci was 100% for most primers tested. DNA fingerprinting methods tested were able to detect geographic variation in populations of H. coagulata from Bakersfield and Riverside, CA, and Weslaco, TX. Dendrograms based on Nei's genetic distance showed that H. coagulata from Bakersfield and Riverside formed a cluster separate from Weslaco in three DNA fingerprinting reactions tested incorporating simple sequence repeats. DNA fingerprinting methods tested were also able to distinguish between three Homalodisca sharpshooters: H. coagulata, Homalodisca insolita (Walker), and Homalodisca liturata (Ball). The present results confirmed the utility of the DNA fingerprinting screening procedure and demonstrated, for the first time, genetic variation in natural populations of glassy-winged sharpshooters by PCR-based DNA fingerprinting methods.

DNA Polymorphism Among Blackgram (Vigna Mungo L Hepper) Genotypes Using Inter-Simple Sequence Repeat (ISSR) Markers

AbstractThe technique of inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) was used to fingerprint blackgram (Vigna mungo) genotypes in order to differentiate them at a molecular level. Twenty-four primers were used to analyse 11 blackgram genotypes. The genotypes used in the study could be differentiated based on combined amplification profiles generated by two primers (UBC primer numbers 808 (AG)8C and 826 (AC)8C). It was observed that 3′-anchored repeat primers based on (AG) and (GA) repeats amplified a greater number of bands than 3′-anchored repeat primers of (CA) and (AC) repeats. This result perhaps indicates that GA/AG repeats are more frequent than CA/AC repeats in the blackgram genome.

Development and partial characterization of heliothine cell lines from embryonic and differentiated tissues.

The goal of this study was to generate cell lines from a variety of insect tissues that could be useful for developing in vitro assays with tissue-specific properties. In this article, we describe the establishment of new cell cultures from differentiated (primarily neural) and undifferentiated tissues (primarily embryonic) and their initial characterization. Cell lines were established from the following tissues of the budworm, Heliothis virescens, and the bollworm, Helicoverpa zea: larval ventral nerve cords (4 lines), larval midguts (1 line), adult ovaries (1 line), and embryonic tissues (11 lines). Cell lines were primarily characterized by morphological examination and polymerase chain reaction (PCR) (both deoxyribonucleic acid amplification fingerprinting and inter-simple sequence repeats PCR).

ASSESSMENT OF DNA VARIATIONS OF THE NOBLE CRAYFISH (ASTACUS ASTACUS L.) IN GERMANY AND POLAND USING INTER-SIMPLE SEQUENCE REPEATS (ISSRS)

Conservation of the endangered noble crayfish (Astacus astacus) requires preservation of existing populations and reintroduction into suitable habitats, as currently underway in several regional projects in Germany and Poland. Therefore, knowledge about the genetic variation between populations is vital but, unfortunately, still restricted. By means of ISSR-PCR (Inter-Simple Sequence Repeats - Polymerase Chain Reaction) eight stocks in Germany and Poland were analysed. These semi-arbitrary multiloci markers had not previously been used in population studies of the noble crayfish. A total of 22 unambiguous and polymorphic markers were detected to use in the subsequent statistical analysis. The number of polymorphic loci in one population ranged from 4 to 19. Therefore the ISSR markers proved suitable for assessing within- and between-population DNA variations and establishing significant separation of most of the stocks. Shannon´s Index, a relative estimate of genetic diversity within populations, ranged from 0.06 to 0.51. UPGMA cluster analysis based on pairwise ROGERS’ (1972) genetic distance revealed two main clusters corresponding to populations sampled in the western and eastern part of the study area. Results are relevant on the local, regional and supra-regional (landscape) level. The implications for conservational management and restocking programs are presented.