Sequence Analysis of 16S rRNA and secA Genes Confirms the Association of 16SrI‐B Subgroup Phytoplasma with Oil Palm (Elaeis guineensis Jacq.) Stunting Disease in India

During January 2010, severe stunting symptoms were observed in clonally propagated oil palm (Elaeis guineensis Jacq.) in West Godavari district, Andhra Pradesh, India. Leaf samples of symptomatic oil palms were collected, and the presence of phytoplasma was confirmed by nested polymerase chain reaction (PCR) using universal phytoplasma‐specific primer pairs P1/P7 followed by R16F2n/R16R2 for amplification of the 16S rRNA gene and semi‐nested PCR using universal phytoplasma‐specific primer pairs SecAfor1/SecArev3 followed by SecAfor2/SecArev3 for amplification of a part of the secA gene. Sequencing and BLAST analysis of the ～1.25 kb and ～480 bp of 16S rDNA and secA gene fragments indicated that the phytoplasma associated with oil palm stunting (OPS) disease was identical to 16SrI aster yellows group phytoplasma. Further characterization of the phytoplasma by in silico restriction enzyme digestion of 16S rDNA and virtual gel plotting of sequenced 16S rDNA of ～1.25 kb using iPhyClassifier online tool indicated that OPS phytoplasma is a member of 16SrI‐B subgroup and is a ‘Candidatus Phytoplasma asteris’‐related strain. Phylogenetic analysis of 16S rDNA and secA of OPS phytoplasma also grouped it with 16SrI‐B. This is the first report of association of phytoplasma of the 16SrI‐B subgroup phytoplasma with oil palm in the world.     

Multiplex PCR assay for malaria vector Anopheles minimus and four related species in the Myzomyia Series from Southeast Asia

Abstract. Mosquitoes (Diptera: Culicidae) of the Anopheles (Cellia) Myzomyia Series are important malaria vectors in Africa, India and Southeast Asia. Among 10 named species of Myzomyia known from the Oriental Region, seven form the An. minimus group. Even for expert taxonomists, the adults of these species remain difficult to identify morphologically. For technical staff of malaria control programmes, confusion may extend to misidentification of species that are not formally within the minimus group. For identification of specimens from Indochina (Cambodia, Laos, Vietnam), we describe a multiplex polymerase chain reaction (PCR) assay, based on rDNA internal transcribed spacer 2 (ITS2) sequences, that employs a cocktail of primers to identify An. minimus Theobald sibling species A and C (sensu; Green et al., 1990) and three other species in the An. minimus group (An. aconitus Dönitz, An. pampanai Büttiker & Beales, An. varuna Iyengar), as well as An. jeyporiensis James, also belonging to the Myzomyia Series. As the test is DNA‐based, it can be applied to all life stages of these mosquitoes for ecological investigations and vector incrimination studies. This PCR assay is simpler, quicker, cheaper and more readily interpreted than previous assays.     

Detection of Rhopalosiphum insertum (apple-grass aphid) predation by the predatory mite Anystis baccarum using molecular gut analysis

Abstract 1 A simple, yet sensitive polymerase chain reaction based technique was developed for the detection of the apple‐grass aphid Rhopalosiphum insertum in the gut of Anystis baccarum, a predatory mite. 2 A range of conserved polymerase chain reaction primers for insect mitochondrial and ribosomal DNA were tested in order to amplify R. insertum DNA. The mitochondrial DNA primers LrRNAR2 + N1F1, amplified a region between the ND1 and large subunit RNA genes. 3 DNA sequencing of the R. insertum ND1‐LRNA polymerase chain reaction product allowed aphid‐specific polymerase chain reaction primers to be designed. These amplified a 283‐bp product from individual aphids. No polymerase chain reaction product was amplified from individual A. baccarum. 4 Using the aphid‐specific primers against A. baccarum fed on R. insertum, the diagnostic 283‐bp product was amplified. 5 Two restriction enzymes (RsaI and AluI) produced patterns that allowed unambiguous identification of R. insertum DNA from that of Macrosiphum euphorbiae and Myzus persicae.     

Characterisation of phytoplasma (16SrVI) associated with little leaf disease of Portulaca grandiflora – An in silico analysis

A new severe little leaf disease was observed on P. grandiflora, popular as Moss-rose Purslane, widely grown in temperate zones. Characteristic symptoms, ultrastructural studies, antibiotic response and amplification of 16S ribosomal DNA fragments (about 1.5 kb) by polymerase chain reaction (PCR) from infected samples, suspect the involvement of phytoplasma as a pathogen. Nested PCR product, 1.2 kb, with primer pairs R16F2n/R16R2 used for cloning and sequencing. Comparision of the 16S rRNA gene sequences showed that the causal, PLL phytoplasma, is very close (98%) to Indian brinjal little leaf (EF186820) and “Candidatus Phytoplasma trifolii” (AY390261), 16SrVI group phytoplasmas, previously reported from India and Canada respectively. Here, the status of PLL (EF651786) is verified by computer-simulated restriction fragment length polymorphism analysis of 16S rRNA genes of the F2n/R2 sequences of closely related strains of the 16SrVI group using 17 restriction enzymes.     

Identification of a DNA marker linked to sex determination in <Emphasis Type="Italic">Calamus tenuis</Emphasis> Roxb., an economically important rattan species in northeast India

Calamus tenuis (Roxb.), a versatile, dioecious rattan species predominant in northeast India, has emerged as an economical material for light furniture and cottage industries. For the genetic improvement of the species, it is essential to be able to recognize male and female plants at the seedling stage. Screening of genomic DNA with inter-simple sequence repeat (ISSR) primers was used to discover sex-specific polymerase chain reaction (PCR) amplification products. Thirty ISSR primers were screened on female and male C. tenuis plants from five different provinces of Assam, India. A putative female-specific marker was identified. The applicability of ISSR-PCR analysis for development of sex-linked molecular markers in Calamus is discussed.     

Identification of plant-derived genetically modified organisms in food and feed using a hydrogel oligonucleotide microchip

A method of multiplex polymerase chain reaction (PCR) followed by hybridization on a hydrogel oligonucleotide biochip was developed for simultaneous identification of ten different transgenic elements of plant DNA in food and feed products. The biochip contained 22 immobilized oligonucleotide probes that were intended for (1) detection of plant DNA, (2) determination of plant species (soybean, maize, potato, and rice), and (3) identification of transgenic elements, including sequences of 35S CaMV, 35S FMV, rice actin gene promoters, nos, 35S CaMV, ocs, pea rbcS1 gene terminators, and bar, gus, and nptII marker genes. The limit of detection was 0.5% for genetically modified (GM) soybean and maize in the analyzed samples. The tests on food and feed products using the developed approach and real-time PCR showed full agreement in determination of transgenic DNA in the samples. The proposed assay can be used for selection of GM samples by screening food and feed products for subsequent quantitative determination of GM component based on the identified transgene.     

Developmental and pathogen-induced activation of an msr gene, str246C, from tobacco involves multiple regulatory elements

A family of genes, the so-called msr genes (multiple stimulus response), has recently been identified on the basis of sequence homology in various plant species. Members of this gene family are thought to be regulated by a number of environmental or developmental stimuli, although it is not known whether any one member responds more specifically to one stimulus, or whether each gene member responds to various environmental stimuli. In this report, we address this question by studying the tobacco msr gene str246C. Using transgenic tobacco plants containing 2.1 kb of 5 flanking DNA sequence from the str246C gene fused to the -glucuronidase (GUS) coding region, the complex expression pattern of the str246C promoter has been characterized. Expression of the str246C promoter is strongly and rapidly induced by bacterial, fungal and viral infection and this induction is systemic. Elicitor preparations from phytopathogenic bacteria and fungi activate the str246C promoter to high levels, as do wounding, the application of auxin, auxin and cytokinin, salicylic acid or copper sulfate, indicating the absence of gene specialization within the msr gene family, at least for str246C. In addition, GUS activity was visualized. histochemically in root meristematic tissues of tobacco seedlings and is restricted to roots and sepals of mature plants. Finally, analysis of a series of 5 deletions of the str246C promoter-GUS gene fusion in transgenic tobacco plants confirms the involvement of multiple regulatory elements. A region of 83 by was found to be necessary for induction of promoter activity in response to Pseudomonas solanacearum, while auxin inducibility and root expression are apparently not controlled by this element, since its removal does not abolish either response. An element of the promoter with a negative effect on promoter activation by P. solanacearum was also identified.Joint first authors     

An allele-specific polymerase chain reaction assay for the differentiation of members of the<Emphasis Type="Italic">Anopheles culicifacies</Emphasis> complex

Anopheles culicifacies, the principal vector of malaria in India, is a complex of five cryptic species which are morphologically indistinguishable at any stage of life. In view of the practical difficulties associated with classical cytotaxonomic method for the identification of members of the complex, an allele-specific polymerase chain reaction (ASPCR) assay targeted to the D3 domain of 28S ribosomal DNA was developed. The assay discriminatesAn. culicifacies species A and D from species B, C and E. The assay was validated using chromosomally-identified specimens ofAn. culicifacies from different geographical regions of India representing different sympatric associations. The assay correctly differentiates species A and D from species B, C and E. The possible use of this diagnostic assay in disease vector control programmes is discussed.     

Polymorphism of the ITS-2 region of the ribosomal DNA of the Triatominae Rhodnius domesticus, R. pictipes, R. prolixus and R. stali

Abstract The polymerase chain reaction?restriction fragment length polymorphism technique (PCR?RFLP) was used to compare Rhodnius domesticus (Neiva & Pinto), R. pictipes (Stal), R. prolixus (Stal) and R. stali (Lent, Jurberg & Galvão) (Hemiptera: Reduviidae). The enzyme BstUI differentiated R. domesticus, R. pictipes and R. prolixus, and HhaI differentiated R. domesticus, R. pictipes and R. stali. With the fingerprinting analysis generated by these two enzymes, it was possible to clearly identify all four species in the study.     

Isolation and Characterization of a Species-Specific DNA Probe for the Detection of Candida krusei

In an attempt to design species-specific primers for the detection of Candida krusei by polymerase chain reaction, a partial genomic DNA library from Candida krusei was screened for hybridization with radiolabeled genomic probes from a broad variety of fungal and bacterial species and from human. Species-specific candidate DNA inserts were then tested for hybridization with dot blots of DNA from various organisms. One 570-basepair insert from Candida krusei DNA that hybridized under stringent conditions only with DNA from Candida krusei and human was sequenced. It revealed considerable homology with the gene for the mitochondrial inner membrane protease I of Saccharomyces cerevisiae, and the 147 amino acid residues deduced from an open reading frame showed considerable homology with the N-termial portion of the enzyme from Saccharomyces cerevisiae. From the sequence of the Candida krusei DNA fragment, a pair of 21-base oligonucleotide primers enclosing a 501-basepair sequence was designed for polymerase chain reaction. When these primers were tested with a broad range of genomic DNAs, the expected amplification was obtained only with Candida krusei DNA and not with DNA from any other source, including human. Experiments with DNA from mixed cultures of Candida krusei and other yeasts and bacteria showed that the polymerase chain reaction was specific for Candida krusei and that as few as ten cells could be detected. Received: 8 November 1995/Accepted: 12 February 1996     

Development of Streptococcus gordonii‐specific quantitative real‐time polymerase chain reaction primers based on the nucleotide sequence of rpoB

In this study, Streptococcus gordonii‐specific quantitative real‐time polymerase chain reaction (qPCR) primers, RTSgo‐F2/RTSgo‐R2, were developed based on the nucleotide sequences of RNA polymerase β‐subunit gene (rpoB). The specificity of the RTSgo‐F2/RTSgo‐R2 primers was assessed by conventional PCR on 99 strains comprising 63 oral bacterial species, including the type strain and eight clinical isolates of S. gordonii. PCR products were amplified from the genomic DNAs of only S. gordonii strains. The qPCR primers were able to detect as little as 40 fg of S. gordonii genomic DNA at a cycle threshold value of 33. These findings suggest that these qPCR primers detect S. gordonii with high specificity and sensitivity.     

Genetic Variation Among Rhynchosporium secalis Populations of West Asia and North Africa as Revealed by RAPD and AFLP Analysis

In order to study genetic variation among populations of Rhynchosporium secalis, 65 isolates were sampled from the West Asian and North African regions and used for polymerase chain reaction (PCR)‐based DNA marker analyses [namely random amplified polymorphism DNAs (RAPDs) and amplified fragment length polymorphisms (AFLPs)]. The study revealed that genetic diversity among and within populations accounted for 80 and 20%, respectively, of the total genetic diversity, indicating that the local field populations of R. secalis in West Asia and North Africa originated from genetically diverse source populations. Furthermore, high genetic similarity among isolates from the same location suggests that scald populations originated from a local founder population, possibly through rain‐splash‐dispersed conidia.     

Synechocystis sp. PCC6803 fusB gene,located outside of the str operon,encodes a polypeptide related to protein synthesis factor EF-G

Synechocystis sp. PCC6803, a cyanobacterium, possesses an unusual gene (fusB) which encodes a protein with strong homology to protein synthesis elongation factor G (EF-G), although it is not linked to the classical str operon. The fusB gene is redundant, since a Synechocystis gene similar to str operon-encoded fusA genes of other bacteria is also present (based on PCR and hybridization results). There is no evidence for the presence of a fusB homologue in other bacteria. The Synechocystis fusB gene encodes unusual amino acids at some positions that are highly conserved in fusA genes of other prokaryotes.     

Oligonucleotide fingerprinting and RAPD analysis of Achillea species: Characterization and long-term monitoring of micropropagated clones

Two different DNA fingerprinting techniques were applied to a set of Achillea samples (Asteraceae), comprising ten taxa of the medicinally important A. millefolium group and six related species. Field-grown as well as in vitro-micropropagated plants were individually screened for abundance and polymorphism of target sequences recognized by oligonucleotide fingerprinting with 13 different microsatellite-complementary probes. While most probes revealed a high level of intra- and interspecific variability, fingerprints proved to be somatically stable in vegetatively propagated plant material. Analysis of the same samples by polymerase chain reaction with arbitrary 10-mer primers yielded less polymorphic patterns. Because of its higher discriminatory ability, oligonucleotide fingerprinting offers itself as the method of choice for the identification and discrimination of A. asplenifolia and A. roseoalba clones, as well as for monitoring their stability during micropropagation.Abbreviations BPA N-benzyl-9-[2-tetrahydropyranyl]-adenine - PCR polymerase chain reaction - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism - TAE buffer 40 mM Tris acetate, 20 mM sodium acetate, 1 mM EDTA, pH 7.8 Part of the Ph.D. Thesis     

Sex Determination in 58 Bird Species and Evaluation of CHD Gene as a Universal Molecular Marker in Bird Sexing

The aim of this research was to test the CHD gene (Chromo Helicase DNA‐binding gene) as a universal molecular marker for sexing birds of relatively distant species. The CHD gene corresponds to the aim because of its high degree of conservation and different lengths in Z and W chromosomes due to different intron sizes. DNA was isolated from feathers and the amplification of the CHD gene was performed with the following sets of polymerase chain reaction (PCR) primers: 2550F/2718R and P2/P8. Sex determination was attempted in 284 samples of 58 bird species. It was successful in 50 bird species; in 16 of those (Alopochen aegyptiacus, Ara severus, Aratinga acuticaudata, Bucorvus leadbeateri, Cereopsis novaehollandiae, Columba arquatrix, Corvus corax, C. frugilegus, Cyanoliseus patagonus, Guttera plumifera, Lamprotornis superbus, Milvus milvus, Neophron percnopterus, Ocyphaps lophotes, Podiceps cristatus, and Poicephalus senegalus), it was carried out for the first time using molecular markers and PCR. It is reasonable to assume that extensive research is necessary to define the CHD gene as a universal molecular marker for successful sex determination in all bird species (with exception of ratites). The results of this study may largely contribute to the aim. Zoo Biol 32:269–276, 2013. © 2012 Wiley Periodicals, Inc.     

VARIATION IN THE CHLOROPLAST GENES RPOC1 AND RPOC2 OF THE GENUS ASTRAGALUS (FABACEAE): EVIDENCE FROM RESTRICTION SITE MAPPING OF A PCR-AMPLIFIED FRAGMENT

A 4,100-base pair (bp) region of the chloroplast genome, amplified via the polymerase chain reaction, was obtained from 14 species of the genus Astragalus and mapped with 23 restriction enzymes. The amplified region encompassed the chloroplast genes RNA polymerase Cl (rpoCl; 90.8% of the gene) and RNA polymerase C2 (rpoC2; 32.7% of the gene) including the intron in rpoC1 and the intergenic spacer between the two genes. Approximately 144 sites (615 bp) were identified; 37 restriction site mutations and one 10-bp length mutation were detected. Estimated interspecific sequence divergence values ranged from 0.00% to 3.92%. Phylogenetic analysis with Wagner and Dollo parsimony both resulted in a single 41-step tree with a consistency index of 0.951. The relative positions of 115 restriction sites were mapped. The insertion and ten of the restriction site mutations mapped to the intron in rpoC1, 18 site mutations mapped to the rpoC1 exons, three site mutations mapped to rpoC2, three site changes mapped to the intergenic spacer, and four site changes were not mapped. This study demonstrates the utility of restriction site analysis of PCR-amplified chloroplast DNA to the study of plant phylogenetic relationships and molecular evolution.